Flatworms are remarkable creatures, some of which have almost unbelievable abilities of tissue regeneration and growth. One particular species, Girardia dorotocephala, has been shown to be able to take this ability one bizarre step further: Researchers at Tufts University have successfully induced it to grow heads and certain brain structures of several other flatworm species on a temporary basis, all without altering its genetic sequence. Their findings are reported in the International Journal of Molecular Sciences. 
  
 The shape of an animal's body is largely determined by its genome. However, bioelectric networks of nerves and their connections (synapses) have their own way of forming constructs, and the careful manipulation of these can cause unique features to form in certain organisms, overriding the genetic material’s instructions. 
  
 With this in mind, the team of researchers decided to modify the synaptic connections in the head of their chosen flatworm to see if they could force it to produce new shapes. After decapitating them, the electrical connectivity between cells in its upper body were altered using a form of alcohol (octanol). 
  
 Incredibly, this caused the flatworms to grow not just new tissue, but entirely new heads – heads belonging to completely different species of flatworm: S. mediterranea, D. japonica, and P. felina. Not only were the head shapes replicated, but segments and characteristics of their brains were also seemingly copied, including the distribution of adult stem cells. The closer the two species were related, the easier it was for the researchers to induce the changes. 
  
 These changes, oddly, weren’t permanent: Within a few weeks, the genetic coding overcame the bioelectrically-induced head change, and their heads gradually returned to their original form. At present, the researchers aren’t entirely sure how this remodeling occurs. 
  
 Regardless, the fact that this all occurred without changing the DNA of the original flatworm is the study’s most significant discovery. This means that changes in a flatworm’s ability to “build” itself can sometimes originate from outside the instructions coded in its genetic material. This type of externally-induced alteration is known as epigenetics – literally meaning “extra growth.” 
  
 Transferring a selection of genes from two parents to their offspring isn’t the only way the building instructions for an organism are inherited. Along with an additional process called horizontal gene transfer employed by certain microscopic organisms, changes in the way genes are expressed can also be inherited. Several environmental or otherwise external factors can change the way cells read genes, meaning that the physical attributes of the offspring may be different – all without changing the DNA sequence. 
  
 Although it has yet to be shown whether or not these unusual flatworm changes can be transferred to their offspring, they do fit within the definition of epigenetics – although as some point out, without the inheritance being demonstrated, applying this definition can be somewhat controversial. 
  
 Nevertheless, the study has implications outside of just flatworm biology. Maya Emmons-Bell, a Tufts University biology undergraduate and first author on the paper, said in a statement: “This kind of information will be crucial for advances in regenerative medicine, as well as a better understanding of evolutionary biology.” 
  
 Image in text: G. dorotocephala (top left) was able to grow the heads of other species (top row) with no changes to its genome. Center for Regenerative and Developmental Biology, School of Arts and Sciences, Tufts University ||||| Call them Franken flatworms. Scientists have created worms with the heads and brains of other species just by manipulating cell communication. 
  
 The research is an example of how development is controlled by more than genetics alone. The researchers did not alter the flatworms' DNA in any way, but instead manipulated proteins that control conversations between cells. 
  
 "It is commonly thought that the sequence and structure of chromatin — the material that makes up chromosomes — determine the shape of an organism, but these results show that the function of physiological networks can override the species-specific default anatomy," study researcher Michael Levin, a biologist at Tufts University, said in a statement. [See Photos of the Franken Flatworms with Different Heads, Brains] 
  
 The changes are temporary for the worms, whose heads begin to revert back to their original shapes in weeks. But researchers hope the findings will lead to treatments for birth defects and even regenerative medicine, which aims to replace or rebuild damaged tissues and organs. 
  
 The researchers studied a small freshwater flatworm, Girardia dorotocephala, which is known for being able to regenerate lost tissues. These flatworms retain a large number of cells called neoblasts, which are totipotent stem cells, meaning they can become any cell type in the body. In humans, cells are totipotent only in the first days of embryonic development. 
  
 First, the scientists cut off the heads of their lab specimens. Next, to alter the flatworm's regenerating head, Levin and his colleagues interrupted protein channels called gap junctions. Cells send electrical impulses through these junctions in order to communicate. The researchers found that they could easily nudge the worm to develop head and brain shapes similar to those of other closely related flatworm species. 
  
 Normally, G. dorotocephala sports a pointy head with two elongated, earlike projections (called auricles) next to the eyes. After treatment, some of the worms developed their normal heads, while others grew rounded heads like those of S. mediterranea; heads with thick necks and pointy, catlike "ears" like those of P. felina; or triangular heads like those of D. japonica. 
  
 The brains seemed to follow suit with the altered head shape, so that flatworms that regenerated a head shaped like that of D. japonica, for instance, also showed brain morphologies that were shorter and wider than those of G. dorotocephala and more characteristic of D. japonica. 
  
 The farther two species were from each other on the evolutionary family tree, the harder it was to induce this mix-and-match effect, the researchers reported online Nov. 24 in the International Journal of Molecular Sciences. 
  
 "These findings raise significant questions about how genes and bioelectric networks interact to build complex body structures," Levin said. If genes provide a blueprint for an organism's body, cells are like the construction workers required to turn the plan into a structure — and gap junctions are the walkie-talkies these workers use to communicate. Disrupt the communications, and you can disrupt the building process. 
  
 G. dorotocephala remained altered for only a limited amount of time before its neoblasts took over and reasserted the old head and brain shapes. However, Levin's lab previously engineered another species of flatworm to grow two heads and stay that way. 
  
 Follow Stephanie Pappas on Twitter and Google+. Follow us @livescience, Facebook & Google+. Original article on Live Science. ||||| Importantly, different species of planaria have characteristic head shapes that are readily distinguished by anatomical inspection. Thus, we examined the consequences of disrupting the body-wide gap junction communication network for head shape in planaria. Remarkably, we observed a stochastic phenotype in which the regenerating heads of a genomically-normal G. dorotocephala flatworm acquired a head morphology appropriate to other extant planarian species. The effect was observed in the head, as well as in the brain morphology. Unlike our recent demonstration of a permanent (stable) change of target morphology in D. japonica [ 20 ], this effect was temporary, and the worms remodeled back to their native state within 30 days. The ability to stochastically select one of several discrete head shapes appropriate to a different species, simply by altering a physiological network, suggests that quantitative models of bioelectric network modes will be an important part of understanding evolutionary change and the role of genomic vs. physiological circuits in establishing anatomical structure. 
  
 Planaria are free-living flatworms with impressive regenerative abilities [ 55 , 56 , 57 ]. After traumatic injury such as amputation, planarians regenerate all missing tissues, reproducing their target morphology perfectly in a relatively short time span of about two weeks. This morphological remodeling is not restricted to trauma; after periods of starvation, planarians shrink themselves allometrically, and grow in the same, scaled manner after food has been reintroduced [ 58 ]. This remarkable example of spatial and temporal cellular organization requires the storage and sharing of morphological and physiological information across a diverse and widely dispersed population of cells. Such morphological robustness provides a unique system in which to study complex traits such as the plasticity of morphology. While recent work has made remarkable progress on the molecular details of pathways regulating adult stem cell behavior in planaria [ 59 , 60 , 61 ], very little information is available on how specific head shape is controlled. The vast majority of the functional literature reports phenotypes of either failure to regenerate or switching of head/tail identity; the field currently has no mechanistic understanding of how a species-specific head shape is established or regulated [ 62 ], although voltage-based signaling has been shown to be involved in head shape/size in at least one species [ 25 ]. 
  
 Global bioelectric network dynamics are regulated in part by gap junctions—electrical synapses between cells that facilitate direct ion exchange, thereby allowing cells to compare their V mem with those of their neighbors, and establish isopotential cell fields or boundaries between compartments [ 44 , 45 ]. Because gap junctions can be themselves voltage-sensitive [ 46 , 47 ], they in effect can function as transistors—enabling complex information processing and feedback loops. Gap junctions also provide rich opportunities for selective gating of small molecule signals in addition to ion current [ 48 ]. Unsurprisingly, because of these features, this versatile signaling element is crucial for development [ 49 , 50 , 51 ] and also for the plasticity of cognitive memory in the brain [ 52 , 53 , 54 ]. Thus, we investigated here the role of gap junction-mediated physiological networks in regulating pattern formation. 
  
 Three aspects of bioelectric signaling make them particularly relevant to the origin of large-scale shape and the role of the genome. First, bioelectric patterns specify shape in a distributed (non-local) manner: several studies have shown that the size, shape, and identity of specific structures integrates bioelectrical information from remote regions [ 32 , 35 , 36 , 37 , 38 , 39 ], making bioelectric signaling an ideal modality for coordinating individual cell behaviors towards a specific anatomical outcome. Second, bioelectric patterns can override default genetic/biochemical information: specific V mem changes can prevent mesenchymal stem cell differentiation despite the presence of chemical inducers [ 40 , 41 ], can induce an eye to form in a tissue (e.g., mesoderm or endoderm) that is otherwise not competent to become eye when misexpressing a “master” eye inducer like Pax6 [ 31 ], can induce metastatic melanoma in the absence of genetic damage [ 36 , 37 ], can prevent the formation of tumors in the presence of otherwise-sufficient oncogenes [ 42 , 43 ], and can rescue brain defects caused by mutations in powerful regulators of neurogenesis such as Notch [ 32 ]. Thus, bioelectric signaling is a good explanatory candidate in instances of epi-genetic influences over pattern formation. Finally, bioelectric properties seem to directly encode outcomes at the level of organs, inducing whole appendages [ 17 , 23 , 24 ] or complex eyes [ 31 ]. This ability to trigger downstream developmental modules, without having to specify individual cell positions (micromanage the process), makes bioelectric signals not only attractive control knobs for biomedical intervention, but also reveals how bioelectric network states can be seen as attractors instructing complex patterning outcomes. 
  
 Most of the field today is focused on gene-regulatory networks [ 8 , 9 ] and physical forces [ 10 , 11 ] in an effort to understand how final patterning outcomes arise and are maintained during development and regeneration. However, another fascinating layer of biological regulation has recently been implicated in the control of morphogenesis: endogenous bioelectrical signaling [ 12 , 13 , 14 ]. Spatio-temporal gradients of resting potential among all cell types (not just excitable nerve and muscle) can regulate cell proliferation, migration, shape, and apoptosis [ 15 , 16 ]. Even more importantly, they can function as instructive cues for large-scale morphogenesis, regulating positional information, organ identity, size, and axial polarity [ 1 , 17 , 18 ]. Recent work has implicated these voltage gradients in the regulation of anterior-posterior polarity [ 19 , 20 ], appendage regeneration [ 21 , 22 , 23 , 24 ], craniofacial patterning [ 25 ], left-right asymmetry [ 26 , 27 , 28 , 29 ], eye development [ 30 , 31 ], and brain patterning [ 32 ]. Numerous studies have now identified transduction mechanisms linking bioelectric properties with downstream transcriptional and epigenetic targets [ 13 , 18 , 33 , 34 ], thus revealing how these physical properties integrate with genetic information during patterning. 
  
 Whether via nucleotide sequences or chromatin modifications, the genome does not specify organismal 3-dimensional shape directly. Instead, large-scale morphology is an emergent feature of the dynamics of complex networks of activity carried out by cells. Thus, pattern formation is the outcome of a rich layer of chemical and physical processes occurring between DNA and anatomy. Indeed, distinct morphologies can arise from a single genotype [ 4 , 5 ]. Understanding how cells communicate and coordinate their functions in vivo to reliably form complex body plans and organs is of fundamental importance not only for evolutionary developmental biology, but also for biomedicine [ 6 ]. Transformative advances in regenerative medicine and synthetic bioengineering require us to know which inputs can be provided to a cellular system to induce specific morphological outcomes—rational control of growth and form. This is a truly difficult problem because of the complex nonlinearity of biological regulation [ 7 ]. Hence, step 1 is uncovering processes that provide instructive control over the determination of large-scale shape. 
  
 Development, cancer suppression, large-scale remodeling, and regeneration all hinge on an organism’s ability to store and process information about its correct anatomical structure, and correct any deviations from that structure that may occur during injury or other environmental impacts [ 1 , 2 ]. It is commonly assumed that species-specific anatomical shapes are encoded in the genome, although data from environmental epigenetics have long suggested that morphological outcomes are a function of not only inheritance but also of environment and life history inputs [ 3 ]. 
  
 Computational model reproduces the four discrete outcomes observed experimentally. () Four types of planarian heads obtained from the computational model. Different shapes result due to deactivation of different cells. Red lines show the trajectories of the red cells; () Morphometric measurement of different planarian heads, for comparison with real heads () measured (see Table 2 ). Scale bar 0.5mm. Cells of type A are shown in black, deactivated cells of type A white, cells of type B are red. The outer boundary has two parts differing by its rigidity (soft part of the boundary is shown in red, more rigid part in green). Thus, the red cells represent migrating neoblasts (or their progeny) while the black cells represent somatic cells interconnected by GJs. () Definitions of lengths of measurements L1 and L2, used in comparison of real worms to those produced by the model in Table 2 
  
 Computational model reproduces the four discrete outcomes observed experimentally. () Four types of planarian heads obtained from the computational model. Different shapes result due to deactivation of different cells. Red lines show the trajectories of the red cells; () Morphometric measurement of different planarian heads, for comparison with real heads () measured (see Table 2 ). Scale bar 0.5mm. Cells of type A are shown in black, deactivated cells of type A white, cells of type B are red. The outer boundary has two parts differing by its rigidity (soft part of the boundary is shown in red, more rigid part in green). Thus, the red cells represent migrating neoblasts (or their progeny) while the black cells represent somatic cells interconnected by GJs. () Definitions of lengths of measurements L1 and L2, used in comparison of real worms to those produced by the model in Table 2 
  
 Note that the shape of the auricles in Figure 8 D is more pronounced than in cases Figure 8 B,C. This corresponds to the experimentally observed shapes. In modeling, this difference is achieved with different properties of the outer boundary, being softer in the lower part (red in the figure) and resulting in sharper auricles. This is consistent with prior work showing that integument integrity (and thus stiffness) is dependent on the correct expression and function of gap junction channels [ 100 ]. Figure 8 shows the final positions of cells B and their trajectories after simulating four different deactivation patterns. In order to compare real worm morphological data to the results of our modeling, we carried out morphometric measurements as shown in Figure 8 E–H on the outcomes of our in silico simulations. We measured the length of the horizontal interval L1 and of the vertical interval L2 (in the case 1b and 2b these intervals necessarily had to not be straight). The results are seen in Table 2 , and provide a good concordance with the observed outcomes in real worms ( Figure 1 ). Let us note that the final morphologies of planarian’s head in computer simulations are determined by the initial cell configuration. They differ by locations of deactivated cells (white cells in Figure 8 ). If these cells are close, then the final head forms are also close. If we compare DJ ( Figure 8 C) and GD ( Figure 8 D), then the initial cell configuration and the final morphologies are more close to each other than these two compared with SM ( Figure 8 A). Moreover, DJ seems to be in between the SM and GD models. It is interesting to note that this ordering corresponds to their genetic distance ( Figure 3 ). We conclude that a small set of cell-level behaviors, such as migration and cell-cell signaling that are known to be regulated by GJC and thus are perturbed by octanol exposure [ 35 , 39 , 49 , 98 , 101 , 102 ], can quantitatively explain the morphologies we observed in planaria. 
  
 When simulated in silico, this model produced planarian head shapes observed in the experiments. These different shapes were achieved from the same initial cell configuration but different deactivation pattern of cells A and different elastic properties of the boundary. These deactivation patterns correspond to octanol treatments that reduces cell-cell communication among a subset of cells. We hypothesize that this results in deactivation of some of the cells’ signaling. The initial cells location configuration and its resulting equilibrium configuration are shown in Figure S2 H,I. Cells of type B remain surrounded by A cells. This configuration is chosen in such a way that after deactivation of some of cells A, cells B either remain inside or they migrate outside and produce one of the required four configurations ( Figure S2 A–D). Specific head shape emerges because cells B are pushed away by cells A and by other cells B. They escape through the lower concentration levels of substance C left by deactivated cells A, arriving at the outer membrane and changing its shape. 
  
 We considered two cell types, A and B. Cells of type A have fixed positions—they cannot move. They produce a substance whose concentration C spreads in space. Its distribution can be described by a reaction-diffusion equation or by some other models. Cells of the type B can move. They do not produce any substance but they receive the substance C produced by cells A and their motion is determined by its concentration distribution. We modeled the planarian head with the following elements: cells of type A, deactivated cells of type A, cells of type B, and a surface boundary. Deactivated A cells are fixed but they do not produce substance C. If there is more than one cell B, then they repulse each other in the same way as they are repulsed by cells A. We choose deactivated cells A symmetrically with respect to the anterior-posterior axis, in order to preserve symmetry of outcome (which implies that B cells move in a similar way on the left and right sides). The outer boundary is composed of points and elastic “springs” connecting them. When a cell B approaches the boundary, a repulsive force acts on it from the boundary. This force is proportional to the distance from the particles and from the intervals of the boundary. Full details of the modeling are given in Figure S2 . In this model, we hypothesize that instructive (GJ-dependent) signaling occurs from the somatic tissues to the neoblasts or their progeny, to guide the new tissue generation and shaping during regeneration. Thus, the red cells are migrating neoblasts (or their progeny) while the black cells are somatic cells interconnected by GJs ( Figure 8 ). In the model, octanol disruption of cell:cell communication is thus modeled by the deactivation of signaling from a specific cell type. 
  
 One of the key challenges facing developmental biology and regenerative medicine is linking large-scale patterning outcomes to the individual activity of cells guided by genetic networks and signaling pathways [ 2 , 85 , 86 , 87 , 88 , 89 ]. Most of the work in the planarian field deals with anterior-posterior fragment polarity [ 60 , 62 , 90 ] or stem cell differentiation [ 57 , 59 , 91 ], and does not address the actual morphology of the head or the rest of the body. Recent quantitative, genetically-grounded models of regeneration [ 92 , 93 , 94 ] likewise use anterior-posterior identity as a binary readout, which does not address or explain changes to the shape of these anatomical regions. Our study of the bioelectrical control of regeneration reported alterations of head remodeling [ 25 ], but was limited to scaling and did not model the detailed shape of the head. To begin to mechanistically link individual cell behaviors (such as those regulated by GJ-mediated signals) to large-scale anatomical outcomes, we next constructed an agent-based model of cell signaling and morphogenesis. Our model focused on cell migration and cell-cell signaling, as these are clearly important for implementing different morphogenetic outcomes [ 95 , 96 , 97 ], and also known to be regulated by GJ connectivity [ 98 ] and bioelectric properties of neighboring cells [ 99 ]. 
  
 In D. japonica, physiological perturbations can stably change the basic architecture of the planarian body-plan, producing two-headed worms that continue to regenerate as two-headed in perpetuity across future rounds of regeneration in plain water [ 20 ]. Thus, we next asked whether our observed head shape changes in G. dorotocephala were permanent. Photographs of worms after treatment with 8-OH were taken daily, from day 10 (when the morphologies of the worms were scored), through day 30 (the time necessary for complete cellular turnover in the planarian flatworm). We found that the induced morphologies were remodeled, long after regenerative processes had ended, to produce morphologies that bore closer resemblance to wild-type G. dorotocephala. Interestingly, the time course and result of this non-regenerative remodeling differed depending on the starting head shapes. Regenerates that had rounded heads, which resemble S. mediterranea, began to develop a more triangular head shape by day 17, and by day 30, bore more of a resemblance to D. japonica than S. mediterranea. Over the same time course, regenerates that had triangular heads, resembling D. japonica, developed pronounced auricles, and by day 30 were indistinguishable from wild-type G. dorotocephala ( Figure 7 A,B). These findings are fascinating in two respects. First, the change in morphology in absence of a trauma highlights dynamic and robust mechanisms underlying morphological homeostasis—the anatomical state is remodeled over the long-term from an abnormal configuration existing after regenerative repair was complete. Secondly, the remodeling occurs via “paths” through the shape space illustrated by canonical variate analysis ( Figure 7 C). DJ morphology lies between GD and SM morphologies in this space, and we observe remodeling that moves from SM morphology to DJ morphology to GD morphology. From these data, we conclude that morphology is both plastic and robust, restoration of the “target” morphology can occur without trauma to the organism, and that the CV shape space is informative in illustrating parameters and boundaries to morphological variation. 
  
 Figure 6. Membrane voltage reporter assay demonstrates long-term change of bioelectrical connectivity in octanol-exposed planaria. ( A – D ) Domains of relative membrane potential visualized using DiBAC 4 (3) in wild-type G. dorotocephala , wild-type S. mediterranea , wild-type D. japonica , and wild-type P. feline ; ( E – H ) Domains of relative membrane potential visualized using DiBAC(3) in pseudo G. dorotocephala (GDs), pseudo S. mediterranea (SMs), pseudo D. japonica (DJs), and pseudo P. felina (PFs), respectively. Scale bar 0.5 mm; ( I ) Number of isopotential regions in wild-type GD worms (control), all pseudo morphologies, and pseudo morphologies that have remodeled back to WT (wild-type) GD morphology after 30 days. After octanol treatment, the number of isopotential regions in pseudo morphologies is increased, but decreases to WT levels after remodeling. Black dots indicate worms with GD morphologies, blue dots indicate DJ morphologies, red dots indicate PF morphologies, green dots indicate SM morphologies, and pink dots indicate pseudo worms that have remodeled their morphologies to resemble wild-type GDs. n = 7 wild-type GD, n = 5 pseudo GD, n = 5 pseudo SM, n = 5 pseudo DJ, n = 2 pseudo PF, and n = 6 remodeled worms. Error bars are standard deviations. Non-parametric statistical analysis was done using a post-hoc comparison of all groups by Kruskal-Wallis test ( p = 0.0021), and then between groups using a Dunn’s Multiple Comparison test, which showed differences in voltage domain number between remodeled GDs and pseudo morphologies are statistically significant at p < 0.05. 
  
 Figure 6. Membrane voltage reporter assay demonstrates long-term change of bioelectrical connectivity in octanol-exposed planaria. ( A – D ) Domains of relative membrane potential visualized using DiBAC 4 (3) in wild-type G. dorotocephala , wild-type S. mediterranea , wild-type D. japonica , and wild-type P. feline ; ( E – H ) Domains of relative membrane potential visualized using DiBAC(3) in pseudo G. dorotocephala (GDs), pseudo S. mediterranea (SMs), pseudo D. japonica (DJs), and pseudo P. felina (PFs), respectively. Scale bar 0.5 mm; ( I ) Number of isopotential regions in wild-type GD worms (control), all pseudo morphologies, and pseudo morphologies that have remodeled back to WT (wild-type) GD morphology after 30 days. After octanol treatment, the number of isopotential regions in pseudo morphologies is increased, but decreases to WT levels after remodeling. Black dots indicate worms with GD morphologies, blue dots indicate DJ morphologies, red dots indicate PF morphologies, green dots indicate SM morphologies, and pink dots indicate pseudo worms that have remodeled their morphologies to resemble wild-type GDs. n = 7 wild-type GD, n = 5 pseudo GD, n = 5 pseudo SM, n = 5 pseudo DJ, n = 2 pseudo PF, and n = 6 remodeled worms. Error bars are standard deviations. Non-parametric statistical analysis was done using a post-hoc comparison of all groups by Kruskal-Wallis test ( p = 0.0021), and then between groups using a Dunn’s Multiple Comparison test, which showed differences in voltage domain number between remodeled GDs and pseudo morphologies are statistically significant at p < 0.05. 
  
 Figure 5. Neoblast (red dots) distribution is altered after octanol treatment. ( A – C ) Analysis of neoblast distribution by staining of phosphorylated histone H3 in adult, wild-type G. dorotocephala ( n = 9), D. japonica ( n = 9), and S. mediterranea ( n = 9) planarians. Dotted lines indicate edges of worm anatomy, as well as 1/6th of the length of the worm body from the anterior tip of the worm. This distance was used to define the posterior boundary of the head. Arrows indicate the region in which neoblasts were counted; ( D – F ) Analysis of neoblast distribution in G. dorotocephala regenerates treated in 8-OH that resembled G. dorotocephala heads ( n = 7), D. japonica heads ( n = 8), and S. mediterranea heads ( n = 7), by anti-phosphorylated histone H3 staining. Arrows indicate region in which neoblasts were counted. Scale bar 0.5 mm; ( G ) Average number of neoblasts in the anterior 1/6th of wild-type, and 8-OH treated worms (ANOVA p < 0.05). Error bars are standard deviations. 
  
 Figure 5. Neoblast (red dots) distribution is altered after octanol treatment. ( A – C ) Analysis of neoblast distribution by staining of phosphorylated histone H3 in adult, wild-type G. dorotocephala ( n = 9), D. japonica ( n = 9), and S. mediterranea ( n = 9) planarians. Dotted lines indicate edges of worm anatomy, as well as 1/6th of the length of the worm body from the anterior tip of the worm. This distance was used to define the posterior boundary of the head. Arrows indicate the region in which neoblasts were counted; ( D – F ) Analysis of neoblast distribution in G. dorotocephala regenerates treated in 8-OH that resembled G. dorotocephala heads ( n = 7), D. japonica heads ( n = 8), and S. mediterranea heads ( n = 7), by anti-phosphorylated histone H3 staining. Arrows indicate region in which neoblasts were counted. Scale bar 0.5 mm; ( G ) Average number of neoblasts in the anterior 1/6th of wild-type, and 8-OH treated worms (ANOVA p < 0.05). Error bars are standard deviations. 
  
 One of the physiological signals propagated within GJ-mediated cell networks is electric current: patterns of GJ-dependent connectivity can determine isopotential cell fields [ 19 , 25 , 79 ], and we observed that octanol indeed increased the number of regions with distinct V mem patterns ( Figure 6 ). Because analytical pipelines to read out encoded pattern states from bioelectrical measurements (as has been done for the human brain [ 80 ]) do not yet exist, we sought to begin to establish physiological metrics that could reveal permanent changes of the somatic bioelectric network induced by GJ blockade and could distinguish pseudo worms from those with the original (wild-type) morphology. Thus, we next examined the distribution of isopotential cell groups among the different worm shape outcomes, as such groups are established by the function of gap junctions and are a readout of the topology (connectivity) of developmental bioelectrical networks [ 20 , 35 , 39 , 51 , 81 , 82 ]. Patterns of endogenous relative membrane depolarization and hyperpolarization were visualized using a DiBAC (bis-(1,3-dibarbituric acid)-trimethine oxonol) dye [ 79 , 83 ]. DiBAC is anionic, so dye enters cell membranes based on relative degrees of depolarization [ 84 ]. Therefore, increased fluorescence indicates regions of depolarization, and decreased fluorescence indicates relative hyperpolarization. Wild-type worms of the species G. dorotocephala, D. japonica, S. mediterranea, and P. felina, and GJ-perturbed pseudo worms were imaged with DiBAC dye in order to assess potential differences in relative membrane potential. Images were analyzed with a custom image analysis program (as described in Methods) to determine the number of distinct isopotential regions present in the entire worm. Analysis of worms 10 days after 8-OH treatment (after regeneration was complete, Figure 6 A–H) revealed that transient perturbation of gap junction communication alters body-wide patterns of voltage distribution for many days after the end of 8-OH treatment. We detected an increased number of isopotential regions in the pseudo worms compared to the states of control worms; interestingly, the numbers of isopotential regions return to those of a wild-type state after 30 days of morphological remodeling ( Figure 6 I). Although we cannot be certain that every single cell had been penetrated by the dye, the pattern of isopotential regions and variability among animals suggests that octanol action is stochastic and not 100% effective, only partially disrupting electrical coupling (revealed as regions that differ in V mem ) throughout animals treated in drug. We conclude that octanol exposure alters the normal pattern of G. dorotocephala resting potential toward a pattern that persists even after 8-OH is withdrawn and regenerative repair occurs, producing an increased concentration of isopotential regions throughout the worm. 
  
 In wild-type G. dorotocephala, very few neoblasts reach into the most anterior 1/6th of the worm ( Figure 5 A). In wild-type D. japonica, the number of neoblasts in the anterior portion of the body is increased in comparison to G. dorotocephala, however it is still relatively low ( Figure 5 B). Wild-type S. mediterranea planarians have an abundant neoblast population in the anterior-most region ( Figure 5 C). All neoblasts in the anterior 1/6th of the worm’s anatomy were counted by hand ( Figure 5 G). Remarkably, the distribution of neoblasts in pseudo worms mirrored precisely the distribution of neoblasts in the wild-type species that they resembled ( Figure 5 D‒F) (ANOVA p < 0.05). We conclude that the transformation of G. dorotocephala worms to resemble other species also extends to the species-specific, characteristic internal distribution of their stem cells, and that the patterning processes that are disrupted after GJC perturbation are also responsible for organizing the distribution of mitotically active cells. 
  
 Planarians derive much of their remarkable regenerative power from a population of heterogeneous adult stem cells, called neoblasts, which comprise the only mitotically active cell population in the body of the flatworm [ 56 , 70 , 77 ]. We next investigated whether or not the spatial distribution of neoblasts was appreciably different between species of planarians, and whether GJ-inhibited worms acquired the neoblast distribution characteristic of the species whose morphology they had taken on. As neoblasts are the only mitotically active cells within the planarian body, we performed immunostaining of phosphorylated histone H3, a standard neoblast marker in planaria [ 78 ], in order to visualize neoblast populations. 
  
 Figure 4. Brain morphology is altered after 8-OH treatment. ( A – C ) Brain morphology visualized by anti-synapsin staining of wild-type G. dorotocephala ( n = 10), D. japonica ( n = 15), and S. mediterranea ( n = 6) planarians. Arrows indicate brain morphologies, and dotted lines indicate measurements used for calculation of length/width ratio; ( D – F ) Brain morphologies by anti-synapsin staining of G. dorotocephala regenerates treated in 8-OH that resembled G. dorotocephala heads ( n = 4), D. japonica heads ( n = 4), and S. mediterranea heads ( n = 6). Arrows indicate brain morphologies, and dotted lines indicated measurements used for calculation of length/width ratio. Scale bar 0.5 mm; ( G ) Average brain length/width ratios of wild-type, and 8-OH treated worms (ANOVA p < 4.9 × 10 −14 ). Error bars are standard deviations. 
  
 Figure 4. Brain morphology is altered after 8-OH treatment. ( A – C ) Brain morphology visualized by anti-synapsin staining of wild-type G. dorotocephala ( n = 10), D. japonica ( n = 15), and S. mediterranea ( n = 6) planarians. Arrows indicate brain morphologies, and dotted lines indicate measurements used for calculation of length/width ratio; ( D – F ) Brain morphologies by anti-synapsin staining of G. dorotocephala regenerates treated in 8-OH that resembled G. dorotocephala heads ( n = 4), D. japonica heads ( n = 4), and S. mediterranea heads ( n = 6). Arrows indicate brain morphologies, and dotted lines indicated measurements used for calculation of length/width ratio. Scale bar 0.5 mm; ( G ) Average brain length/width ratios of wild-type, and 8-OH treated worms (ANOVA p < 4.9 × 10 −14 ). Error bars are standard deviations. 
  
 We next asked whether internal structures were likewise converted to a different shape, as was external morphology. Few aspects of the planarian internal anatomy differ appreciably between species; however, brain size and shape offer an interesting exception to this rule. The brains of wild-type G. dorotocephala are elongated and narrow, while D. japonica and S. mediterranea have appreciably shorter and wider brain morphologies ( Figure 4 A–C). No living wild-type P. felina could be obtained for this work, and the low frequency of pseudo PF occurrence limited the number available for analysis. Thus, we focused on G. dorotocephala, D. japonica, and S. mediterranea. We performed immunostaining using an anti-synapsin antibody, in order to visualize both the brain, and ventral nerve cords of pseudo and wild-type worms. As recapitulation of wild-type head shape after ten days of regeneration in water had been confirmed by geometric morphometrics, we chose to compare pseudo morphologies to adult worms of other species, in order to minimize confounding data due to variability of regeneration time between species. Overall shape differences were captured by measurement of the brain length/width ratio. These calculations were used to quantify shape differences between species, and to quantify brain remodeling in “pseudo” worms ( Figure 4 G). Strikingly, we found that pseudo worms possessed brain morphologies that look like the brain morphologies of wild-type worms whose head shapes they resembled ( Figure 4 D–F) (ANOVA, p < 0.001). We conclude that the patterning processes that are disrupted after gap junction communication perturbation are also responsible for producing the morphology of the brain, and that the altered shapes are not limited to the overall head geometry but include the patterning of the central nervous system within. 
  
 Figure 3. Percentage of head shape outcomes correlates with evolutionary distance. ( A ) Evolutionary tree, constructed from rRNA data, showing relationships between species of interest. Species names in red are those that were analyzed in this work; ( B ) Frequency of head shapes obtained in the octanol exposure experiments ( n > 243). Failure to regenerate is defined as the loss of anterior-posterior polarity, and the failure to regenerate any head at all after octanol treatment. Error bars are standard deviations. 
  
 Figure 3. Percentage of head shape outcomes correlates with evolutionary distance. ( A ) Evolutionary tree, constructed from rRNA data, showing relationships between species of interest. Species names in red are those that were analyzed in this work; ( B ) Frequency of head shapes obtained in the octanol exposure experiments ( n > 243). Failure to regenerate is defined as the loss of anterior-posterior polarity, and the failure to regenerate any head at all after octanol treatment. Error bars are standard deviations. 
  
 Obtaining distinct regenerated morphologies, at different frequencies, despite the same treatment conditions, led us to explore the evolutionary relationship between the four species represented. We mapped out an evolutionary tree based on rRNA homology [ 75 , 76 ], and compared this tree ( Figure 3 A) to frequencies of the different species’ heads arising from 8-OH treatment ( Figure 3 B). Interestingly, morphologies corresponding to species that are most closely related to G. dorotocephala (SM and DJ, which are removed from GDs by at least 100 million years of evolutionary distance) occur with a much higher frequency than morphologies corresponding to less-related species (PF). We conclude that not only are phenotypic outcomes from physiological network perturbation stochastic (as the same treatment leads to one of several discrete shapes among individuals), but the frequencies are not equal and correlate roughly with evolutionary distance between the worm species these heads resemble. 
  
 Figure 2. Canonical variate analysis of head shape. ( A ) Graphical output, showing confidence ellipses for means, at a 0.9 probability, of shape data from wild-type and experimentally derived morphologies. Ellipses are colored to correspond with phenotype and treatment. n = 10 WT G. dorotocephala , n = 8 WT G. dorotocephala 10 days after amputation and regenerated in water, n = 9 WT D. japonica , n = 6 WT P. felina , n = 8 WT S. mediterranea , n = 7 pseudo G. dorotocephala , n = 13 pseudo D. japonica , n = 5 pseudo P. felina , and n = 6 S. mediterranea flatworms were measured; ( B ) Legend of landmark placement on a wild-type G. dorotocephala head shape (see Materials and Methods); ( C ) Schematic demonstrating alteration of morphology to better resemble another species after 8-OH treatments. Procrustes distances between wild-type G. dorotocephala , 8-OH treated G. dorotocephala with D. japonica head shape, and wild-type D. japonica show objective alteration of morphology to be more similar to the non-native species. 
  
 Figure 2. Canonical variate analysis of head shape. ( A ) Graphical output, showing confidence ellipses for means, at a 0.9 probability, of shape data from wild-type and experimentally derived morphologies. Ellipses are colored to correspond with phenotype and treatment. n = 10 WT G. dorotocephala , n = 8 WT G. dorotocephala 10 days after amputation and regenerated in water, n = 9 WT D. japonica , n = 6 WT P. felina , n = 8 WT S. mediterranea , n = 7 pseudo G. dorotocephala , n = 13 pseudo D. japonica , n = 5 pseudo P. felina , and n = 6 S. mediterranea flatworms were measured; ( B ) Legend of landmark placement on a wild-type G. dorotocephala head shape (see Materials and Methods); ( C ) Schematic demonstrating alteration of morphology to better resemble another species after 8-OH treatments. Procrustes distances between wild-type G. dorotocephala , 8-OH treated G. dorotocephala with D. japonica head shape, and wild-type D. japonica show objective alteration of morphology to be more similar to the non-native species. 
  
 The quantification was used to determine whether the shapes that looked like other species objectively resembled those species, and to suggest a continuous morphospace within which octanol-induced shape change can be visualized along a continuum (from normal to that of a different species). Canonical variate analysis supported the statistical significance of the given pre-defined morphological groupings (in this case, groupings were based on experimental treatment and morphology) ( Figure 2 A). Comparisons of the Procrustes distances between shape groups ( Figure 2 A, Table 1 ) showed that the experimentally derived morphologies were closer in shape to the wild-type morphologies they resembled than the wild-type Girardia dorotocephala head morphology. Analysis of variance (ANOVA) of both centroid size and shape between wild-type morphology and pseudo morphology groups also confirmed significant differences between groupings (F = 7.94, p < 0.0001, and F = 7.40, p < 0.0001, respectively). We conclude that amputation and treatment in 8-OH can produce regenerated worms whose morphology has changed to become significantly more like that of another species of planarian. 
  
 Geometric morphometrics [ 73 ] was used to quantify similarities and differences between head shapes of true species, as well as the experimentally derived pseudo morphologies. In brief, geometric morphometric analysis involves placement of a series of landmarks, which are both biologically significant and reproducible across all samples, removal of non-shape variation (size, rotation, etc.), and performance of a set of statistical analysis [ 74 ]. Landmarks were chosen based on the common biological landmarks that existed across samples, and semi-landmarks were placed with prescribed relations to these landmarks ( Figure 2 B). Landmark data was recorded for n > 60 worms, including GD worms whose head shape had been experimentally perturbed by 8-OH, control GD worms who had regenerated in water for 10 days, and adult wild-type worms from each of the three species. Principal components analyses (data not shown) and canonical variate analyses were run on the data set. This enabled visualization of mean shape changes between wild-type species morphologies, and between experimentally derived head shapes. Both analyses resulted in the separation of pseudo morphologies from the wild-type G. dorotocephala morphology. Procrustes distances between each of the groups were calculated, in order to produce a quantified metric for comparison of shape differences. 
  
 After 3 days of octanol exposure, worms were allowed to complete regeneration (10 days total). Remarkably, this process resulted in worms that exhibited head shapes highly similar to the heads appropriate to other species of planarians ( Figure 1 E–H). 8-OH treated fragments regenerated both head and tail correctly, but in many cases, head shape was drastically altered. Wild-type G. dorotocephala (GD) possess a very pointed head shape, with two elongated auricles at the plane of the eyes ( Figure 1 A). Fragments subjected to the same 8-OH treatment scheme regenerated one of: entirely rounded heads like the planarian S. mediterranea (SM) ( Figure 1 H), heads with thick necks and “cat-like” auricles like those of the planarian P. felina (PF) ( Figure 1 G), heads that are triangular like the planarian D. japonica (DJ) ( Figure 1 F), or heads that resemble wild-type G. dorotocephala ( Figure 1 E). We will refer to the head shapes of regenerates as “pseudo” of the morphologically most similar species. Variant, other species-specific head morphologies were never observed after amputation or during regeneration in water (the control condition)—the normal process of head regeneration has 100% fidelity to the species-specific shape ( Figure S1 A). The same schedule of exposure to hexanol (6-OH), a closely-related compound to octanol which does not effectively block GJs and thus can be used as a control [ 69 ], had no effect ( Figure S1 B). Likewise, intact worms soaked in 8-OH for >3 days did not exhibit any unusual morphological outcomes. Based on these results, we conclude that discrete, species-specific head shapes can be achieved by manipulating the connectivity of physiological networks in the planarian flatworm during head regeneration. 
  
 Figure 1. Characterization of varied head morphologies produced by octanol treatment. ( A – D ) Wild-type morphologies of four species of planaria flatworm. Arrows indicate auricle placement and general head shape; ( E – H ) pre-tail (PT) fragments of G. dorotocephala , treated in 8-OH for three days, and then moved into water for the remainder of regeneration ( n > 243). Arrows indicate auricle placement and general head shape. Scale bar 0.5 mm; ( I ) Experimental scheme of octanol treatment. PT fragments are amputated from G. dorotocephala worms. Fragments are treated in octanol (8-OH) for three days, and allowed to regenerate in water for seven days. 
  
 Figure 1. Characterization of varied head morphologies produced by octanol treatment. ( A – D ) Wild-type morphologies of four species of planaria flatworm. Arrows indicate auricle placement and general head shape; ( E – H ) pre-tail (PT) fragments of G. dorotocephala , treated in 8-OH for three days, and then moved into water for the remainder of regeneration ( n > 243). Arrows indicate auricle placement and general head shape. Scale bar 0.5 mm; ( I ) Experimental scheme of octanol treatment. PT fragments are amputated from G. dorotocephala worms. Fragments are treated in octanol (8-OH) for three days, and allowed to regenerate in water for seven days. 
  
 Planarian flatworm species display a broad range of head shapes, from the very rounded to the almost triangular, with varied shapes of auricles ( Figure 1 A–D). To interrogate the mechanisms responsible for regeneration and maintenance of head shape, G. dorotocephala planarians were amputated along a plane positioned posterior to the pharynx but anterior to the tail, to produce a pre-tail (PT) fragment. PT fragments were then treated with the gap junction (GJ) communication blocker octanol (8-OH). 8-OH is a commonly-used pan-GJ blocker [ 20 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 ], altering the physiological connectivity between populations of cells, and thereby perturbing the rate and pattern of transmission of bioelectrical and other small molecule signals. Dosage was titrated to a level low enough to enable interference with regenerative signaling without organismic toxicity. 8-OH exposure has been validated to be transient by GC-MS: drug levels are undetectable after a few hours of worm wash-out in water, and octanol does not alter genetic sequences in the worm [ 20 , 72 ]. 
  
 3. Discussion 
  
 3.1. Perturbation of Bioelectric Networks Produces Inter-Species Morphological Change Our data show that in the planarian flatworm G. dorotocephala, outcomes of morphogenesis can be modified by the perturbation of physiological networks. Brief exposure to a well-characterized disruptor of electrical synapses (gap junction blocker) resulted in regenerated head shapes that quantitatively resembled those of another planarian species. We observed head shapes (and internal structures) resembling D. japonica, S. mediterranea, and P. felina. Note that other species exist with head shapes similar to each (for example, 2 other species are known that exhibit P. felina-like flattened heads); because these three types are good representatives of each shape class and are available as living specimens for analysis (unlike other members of the same morphological head types), we refer to our phenotypes as pseudo japonica, pseudo mediterranea, and pseudo felina respectively. Future analysis of other planarian species will reveal if these phenotypes are in fact more closely resembling one of the other members in each shape class. The head morphology alteration effect was instructive, and not merely one of toxicity or a house-keeping defect, because the outcome was not merely disruption of regenerative ability or a generalized mis-patterning (e.g., tumor or other disorganized growth), but rather resulted in one of several coherent (and ecologically-valid) morphologies. No intermediate morphologies were observed, highlighting the discrete nature of the complex patterning outcomes following GJ blocker exposure. The same 3-day treatment with the closely related alcohol, hexanol (6-OH), which does not efficiently block gap junction channels, and subsequent regeneration in water for 7 days, did not produce altered morphologies, consistent with the importance of the GJ-blocking activity of octanol, and not some other generalized effect of long-chain alcohols. Dramatically-different morphologies can be derived from the same genome [103,104,105]. Mules and hinnies, which differ only in the sex of the respective donkey and horse parents, are quite morphologically distinct, despite almost identical DNA sequences. Toadflax (Linaria vulgaris) has a morphological variant produced solely by variations in DNA methylation patterns, and numerous reptilian species will develop morphologically distinct sexual organs in a temperature-dependent manner [106]. Moreover, recent data revealed how alterations in biochemical signaling can select among different species-specific morphologies [103,107]. Here, we identify a novel physiological input into the process by which distinct morphologies can be derived from the same genome: physiological bioelectric networks. The compatibility of multiple morphological outcomes with a single genomic sequence underlies the importance of learning to understand and control the instructive patterning information that is generated by chemical, physical, and bioelectrical processes beyond hardwired gene-regulatory networks. 
  
 3.2. Geometric Morphometrics Quantifies Planarian Shape Change Geometric morphometrics is a technique commonly used to quantify and compare biological shapes. We employed landmark-based analysis, which involves the recording of landmark data, the transformation of resulting shapes to remove scaling and rotational noise, and the performance of statistical analyses. Canonical variate analysis (CVA) was employed to compare wild-type morphologies of four species of worm with the corresponding pseudo morphologies, as well as compare wild-type G. dorotocephala morphology to all pseudo morphologies. CVA determines the statistical significance of variation between given groupings of samples [74,108]. In this case the groupings corresponded to observed phenotype and experimental treatment. We chose to employ Procrustes distances to quantify “distance” between shapes, as it is a measure of the square root of the summed squared distances between the corresponding landmarks in two shapes, and is commonly used in morphometric studies comparing differing morphologies. The Procrustes distances between pseudo morphologies and the species that they resembled are smaller than the Procrustes distances between the pseudo morphologies and wild-type G. dorotocephala. The observation was supplemented with both Procrustes ANOVA of centroid size and shape (p < 0.001, and p < 0.001, respectively). This demonstrates a significant alteration of morphology, and quantitatively confirms the apparent similarity between the pseudo phenotypes and their wild-type counterpart species. Interestingly, the centroids of wild-type S. mediterranea, D. japonica, and G. dorotocephala morphology groups (the three most closely related species) all vary along a linear axis through the CV space. This reveals the morphological consequences of variation along a canonical variate. Moving from negative to positive values of canonical variate one correlates with the elongation of the auricles, if they exist (with SMs being at the negative end of the spectrum, and GDs and PFs being at the positive end). Moving from negative to positive values of canonical variate two correlates with movement of the auricles, if they exist, from a very anterior position, to a more posterior one (with PFs being at the negative end of the spectrum, and GDs being at the negative end). 
  
 3.3. Morphological Change Is Not Just Skin-Deep Importantly, the patterning changes induced by electric synapse blockade affect the shape of the brain (Figure 4), not only the overall head shape. Worms with pseudo heads that resemble a given species possess brains that resemble that same species, revealing that the functions of this regulatory pathway extends beyond setting the external outline of the head but instead controls the shape of the brain within. These data also suggest a coupling of brain morphology to head shape. Whether brain morphology drives head shape, or whether both are parallel consequences of the same GJC-dependent upstream control mechanism, this finding demonstrates the depth of re-organization of body plan after perturbation of GJC connectivity. Coupling or involvement of the central nervous system in morphogenesis would not be entirely surprising, given past work demonstrating the necessity of long-range neural inputs for correct polarity patterning during regeneration in planaria [20] and vertebrates [109,110]. In addition, perturbation of gap junction communication reorganizes the distribution of the planarian adult stem cells (neoblasts) to spatial patterns appropriate to other species (Figure 5). Together with the change in brain shape, it is clear that the morphological alteration into pseudo morphologies of other species are thorough, involving not only external head shape but also internal organ scaling and positioning of key cell groups. These data reveal the coupling between proliferative stem cells and large-scale morphology, its variability throughout phylogeny, and its control by GJ-dependent signaling. It is possible that control of neoblast distribution is what drives large-scale morphology. Conversely, their spatial distribution may be one of several downstream consequences of the dynamics of a GJC-mediated cellular network. 
  
 3.4. Bioelectric State of Tissues and Its Stable Alteration in Species-Specific Shape Change Our results include the first analysis of the effects of GJ modulation on the long-term bioelectric profile of patterning tissues in vivo. While large-scale bioelectric gradients have been shown to functionally instruct pattern formation in a number of models [12], including determination of morphollaxis/scaling in S. mediterranea [19] and anterior-posterior polarity in D. japonica [19], no studies have yet examined patterns of V mem distribution in the context of changes in the electrical connectivity of tissues. We found that brief exposure to a GJ blocker, which is known to be washed out of worm tissues within 24 h [20], results in a greater degree of bioelectrical isolation (ability to maintain numerous smaller regions of isopotential V mem ) as late as 10 days after exposure (Figure 6). Although these long-lasting alterations in bioelectrical patterning are not directly caused by octanol remaining within the body of the worm (since 8-OH is known to depart planarian tissues within 24 h [20]), they are initiated by the octanol-mediated disruption of GJC, which alters bioelectric network topology, and results in persistent perturbation of bioelectric state. In this, the effect resembles the known properties of electrical synapses (GJs) in the nervous system, which help implement a kind of Hebbian plasticity that stably alters ionic conductivity among cells after transient electrical modulation [44,111,112,113,114]. Quantitative physiological models of bioelectrical dynamics will need to be developed in order to understand bioelectric state memory [115] and determine why different types of worms have such distinct profiles of regions of isopotential V mem . The variability and overall magnitude of the effect imply that the result of 8-OH exposure is only partially effective in shutting down GJC (affecting some percentage of GJs in any animal); this is consistent with the stochasticity of the morphological phenotype (Figure 3). While 8-OH is a commonly-used method for blocking GJC (and in D. japonica, produces the same effect as RNAi targeting innexins genes [20]), we cannot conclusively rule out additional targets besides GJs also being perturbed either directly or secondarily following 8-OH exposure. One difficulty with a molecular (gene-specific) approach to GJ shutdown, is that not only would ~13 innexins need to be cloned out of the GD genome (none have yet been characterized), but their RNAi targeting would have to be tested combinatorially. The space of all possible combinations of different innexin-RNAi’s that would have to be tried simultaneously is enormous, because GJ proteins can often compensate for one another. Nevertheless, our past work tested some such combinations (in D. japonica [20]), and showed that a simultaneous knockdown of three Innexins by RNAi reproduces the 8-OH phenotype, strengthening the conclusion that phenotypes are indeed resulting from 8-OH’s effect on gap junctions. A recent study [20] showing that transient gap junctional perturbation induces permanent changes in planarian target morphology raised the question of whether this pattern memory was established by alterations of GJC that remain long after the blocking reagent leaves the tissues, or by GJC states that alter biochemical or transcriptional cell states which are stable after GJC returns to normal. While we cannot rule out additional epigenetic changes that might occur, our data show that even 10 days later, the effects of brief 8-OH exposure are preserved as decreased electrical connectivity in the somatic tissues. The ability of GJC networks to stably perpetuate induced changes in coupling are consistent with the known roles of GJs in memory and synaptic plasticity [44,52,114,116,117,118]. Our analysis did not reveal a simple correspondence between the specific distribution of voltage gradients and morphological outcomes of pseudo worm regeneration, which could have several explanations. First, it is possible that not voltage but a different molecular signal is what traverses the GJ network to determine planarian head shape. GJ-permeant molecules with signaling roles include serotonin [119,120] and other neurotransmitters [121], histamine [122], calcium [123], and others, which will be tested in future studies, especially as fluorescent probes for some of these physiological signals come on-line [124,125,126]. Another possibility is that each morphological state is achieved by multiple possible distributions of voltage; in this case, the apparent variability would mask our ability to detect a specific mapping from distribution to head pattern outcome. The most likely explanation however is that shape outcomes may not be encoded via steady (single-image) distributions but in time-dependent activity, as occurs during information encoding in the central nervous system. In this case, movies of voltage change across time in vivo will have to be acquired and analyzed—a significant challenge in the planaria model system. Our analysis of isopotential regions is only a first attempt to mine the information encoded in bioelectric states. Overall, we believe it is very likely that a much more sophisticated analysis method will have to be developed, perhaps akin to what is being used to infer content of neural networks from brain scans [80,127], and applied to a large sample of worm imaging data, to truly crack the bioelectric code in this system. Functional studies in planaria and Xenopus [19,25,128] have demonstrated the relevance of bioelectrical gradients for anterior patterning in general, but new computational approaches will need to be undertaken to demonstrate what spatio-temporal pattern of bioelectric activity is required for each of the patterning outcomes we observed. Uncovering this mapping will be a focus of our efforts for forthcoming studies. 
  
 3.5. Morphological Remodeling in 2 Phases: Head Shape Change without Traumatic Injury The pseudo morphologies we observed are not permanent: they eventually remodel, after the first regeneration process is complete, to once again resemble G. dorotocephala. In this, they are distinct from the permanent double-headed phenotype induced in D. japonica, which represents a stable alteration of the worms’ target morphology following physiological perturbation [20,129]. Thus, in G. dorotocephala, repair occurs in two phases: head regeneration following amputation, which completes in the normal timeframe (<10 days) and results in different species’ shapes, and a subsequent much longer remodeling back to the genome-specific G. dorotocephala head shape. This latter process occurs in the absence of injury, within a complete head, over several weeks, illustrating the ability of cellular networks to detect deviations from correct shape, even in the absence of trauma or wounding, and to effect remodeling. Interestingly, the same biphasic process occurs in vertebrates: when a salamander tail blastema is transplanted onto a flank, a tail forms at first, but later remodels into a limb in a much slower process [130,131]. Morphology appears to be a homeostatic parameter, one that is consistently assessed and adjusted in order to maintain some “target” morphology. Morphology must first be decided within days after amputation, as it is possible to observe head shape as early as day six post-amputation. In 8-OH treated worms, this mechanism produces incorrect, pseudo morphologies. However, morphology must then be reassessed again after this first decision. When the current morphology does not agree with the target morphology, remodeling is initiated sometime between day 10 and day 17. The activity of these remodeling processes after regeneration has been completed is fascinating, and implies that morphology is consistently reassessed and edited, even after large-scale re-organization events have ended. The nature of the processes that drive cell behavior towards a specific, stable end-state (when remodeling ceases) is almost entirely obscure. At this time, it is not possible in any model system to derive specific shape information (to know precisely which anatomical pattern will be a sufficient end-goal state to cause remodeling to stop) from genomic data. 
  
 3.6. Computational Modeling of Head Shape Formation The different head morphologies we observed, which are distinct in outward anatomy, distribution of the stem cells, and shape of the brain, must result from differential cell migration and cell-cell communication. To link cell-level behaviors with the discrete large-scale morphologies and explore the consequences of reduced GJ communication (induced by octanol exposure) on emergent cells signaling and morphogenesis, we analyzed an agent-based model. In this model (Figure S2), some cells actively migrate, and alter the shape of the resulting tissues. Their movements are guided by a chemical gradient produced by other static cells in the head, which pattern of activation is established through GJC, as is known to occur in the worm [20,70] and in many other organisms [51,132,133]. Our model shows the morphogenetic consequences of disruption of this communication, which parallels the interplay between signaling of planarian neoblasts (stem cells) and surrounding somatic tissues. In this model, we propose that significant instructive (GJ-dependent) signaling occurs from the somatic tissues to the neoblasts or their progeny, to guide the new tissue generation and shaping during regeneration. The computational simulations revealed how a small number of cell configurations could result in head shapes quite closely corresponding to the observed morphologies (Figure 9, and Table 2). Interestingly, when specific static cells (black) lose the ability to send their signal due to GJC blockage by octanol, the migrating cells’ movements (red) are altered, giving rise to one of the 4 outcomes. Thus, the model exhibits the two main properties of the experimental dataset: multiple possible outcomes (due to different patterns of GJC-blocked static cells, possibly from physiological noise in pharmacological drug-target interaction), and discrete specific outcomes (resulting from the deterministic signaling of static “black” cells and consequent behavior of migratory “red” cells). It should be noted that our model is also readily applied to an organ that grows from an initial cell position to its final position: if we allow division of cells B, cell migration proceeds in the same way as shown: they will leave behind them deactivated (differentiated) cells B that do not move and do not divide. Future work will test the applicability of this class of models to other regenerating systems. Figure 9. A conceptual model of shape change driven by physiological network dynamics. Planaria regeneration parallels classical neural network behavior; both can be described in terms of free energy landscapes with multiple attractor states. (A) Behavior of a classical Hopfield neural network trained to reproduce three types of patterns, in this case shapes of the letters “F”, “H”, or “G”, which are the three stable states of the network’s free energy landscape. The state of the Hopfield network’s nodes directly relate to the brightness of pixels on a display, generating output. Perturbation of the network from a stable state (red arrow) by deleting (damaging) part of the pattern is akin to moving a ball to an unstable point on the free energy landscape, and leads to regeneration of the closest learned attractor state (blue arrow). In this, such networks’ well-known ability to implement memory is analogous to regenerating organisms restoring a specific target morphology upon damage; (B) The parallel concept of planaria regeneration into head shapes of one of three different species, which are attractor states of the free energy landscape. Outcome morphology is driven by the dynamic outputs of physiological cellular network. Amputation (red arrow) is akin to moving the system to an unstable point on the free energy landscape. Normal regeneration would return the system to its main attractor, but altering cell network dynamics via gap junction blockade allows for regenerative transitions (blue arrow) to alternative local minima, corresponding to morphospace regions normally occupied by P. felina and S. mediterranea worms. In time, remodeling (green arrow) transfers these morphologies to the global minimum of the wild-type state ( G. dorotocephala ). Figure 9. A conceptual model of shape change driven by physiological network dynamics. Planaria regeneration parallels classical neural network behavior; both can be described in terms of free energy landscapes with multiple attractor states. (A) Behavior of a classical Hopfield neural network trained to reproduce three types of patterns, in this case shapes of the letters “F”, “H”, or “G”, which are the three stable states of the network’s free energy landscape. The state of the Hopfield network’s nodes directly relate to the brightness of pixels on a display, generating output. Perturbation of the network from a stable state (red arrow) by deleting (damaging) part of the pattern is akin to moving a ball to an unstable point on the free energy landscape, and leads to regeneration of the closest learned attractor state (blue arrow). In this, such networks’ well-known ability to implement memory is analogous to regenerating organisms restoring a specific target morphology upon damage; (B) The parallel concept of planaria regeneration into head shapes of one of three different species, which are attractor states of the free energy landscape. Outcome morphology is driven by the dynamic outputs of physiological cellular network. Amputation (red arrow) is akin to moving the system to an unstable point on the free energy landscape. Normal regeneration would return the system to its main attractor, but altering cell network dynamics via gap junction blockade allows for regenerative transitions (blue arrow) to alternative local minima, corresponding to morphospace regions normally occupied by P. felina and S. mediterranea worms. In time, remodeling (green arrow) transfers these morphologies to the global minimum of the wild-type state ( G. dorotocephala ). Our agent-based model includes physical parameters of cells [95,134], as well as traditional chemical gradient diffusion [135,136]. In the model, octanol disruption of cell:cell communication is modeled by the deactivation of signaling from a specific cell type; one candidate for this unique cell population are the rare but widely distributed cells that express the gap junction protein Innexin 5 [71]. Future experiments will test the specific predictions of this model and assign molecular-genetic identities by the several different cell types postulated in the model. An additional important line of investigation is to mechanistically link large-scale dynamics of physiological signaling to the specific behavior of cells modeled in our simulations. 
  
 3.7. A hypothesis: Physiological Network Attractors Underlie Regions of Anatomical Morphospace The outcome of regeneration in this model is a set of coherent, discrete head shapes—not a confusing amalgam of different patterns that would be expected if cells made purely local decisions and were merely dis-coordinated by octanol treatment. Currently, the field largely lacks conceptual models for thinking about these kinds of inputs to development and regeneration; it is not clear how best to represent or model discrete, yet stochastically-chosen, emergent large-scale patterns. Yet, this is an increasingly important goal as advances in regenerative medicine and synthetic bioengineering require the ability to predict and control global outcomes by tweaking cell-level parameters. The field of developmental bioelectricity is still searching for an appropriate formalism, but one obvious candidate is that of the neural network paradigm used by computational neuroscientists to understand emergent dynamics of decision-making in brains composed of electrically-coupled cells. We have previously argued that because somatic cells have many of the same components as nerves (ion channels, neurotransmitters, GJs functioning as electrical synapses, gene expression regulated by electrical activity), we may profitably use some of the concepts from neuroscience to understand similar phenomena in slower developmental networks, where output is not muscle-driven behavior but pattern formation [1,13,137]. Scientists in computational neuroscience have modeled the states of electrically-coupled networks in an energy space that reveals the landscape of trajectories that the networks undertake [138,139,140,141,142,143]. Such (neural) networks can stabilize in attractors in this virtual space, and their activity can be parsimoniously explained by networks’ efforts to minimize a global “energy” function [144,145,146,147,148]. Thus, we propose that one way to conceptualize the linkage between bioelectrical connectivity among worm cells (GJC) and discrete global behavioral outcomes achieved by regeneration is to view the worms’ cells as a neural-like network, processing developmental information more slowly but obeying the same statistical dynamics rules as are known to occur in the brain (Figure 9A). It is likely no accident that GJs are well known to be important for information processing in the brain [54,149,150]. We hypothesize that distinct shape outcomes may be attractors in a state space describing distinct modes of the bioelectric network of planarian body cells connected by gap junctions (electrical synapses); in essence, these attractors correspond to regions of a morphospace as classically understood by evolutionary theory [151]. In the experiments, octanol disrupts the normal dynamics of this network, destabilizing and knocking its stochastic dynamics into one of several nearby attractors, normally belonging to different species of flatworm (Figure 9B). Developing a quantitative, computational model of cell networks that have such modes (with electrical states impinging on cell proliferation, apoptosis, and migration behaviors that result in different head shapes) forms the next deep challenge in this area. Such dynamic networks underlie virtually every complex process known in the biological world, from metabolism to group behavior [152,153,154]. These richly interconnected nonlinear networks, made up of many simple units, have the ability to produce complex pattern in open and thermodynamically unstable systems, without any single governing body [155,156]. Within a given system, these networks converge onto one or more discrete, stable states [157]. Neural networks are just one example of complex dynamic networks; they are implemented via electrical signaling and gap junctions that demonstrate discrete attractors corresponding to global states [141,158], and stable re-occupation of large-scale states (memory) after perturbation [54,147]. Given that neural signaling evolved by specializing much more ancient somatic physiological communication mechanisms, it is reasonable to postulate fundamentally similar network dynamics for physiological networks during pattern formation [137,159]. On this view (Figure 9), the discrete head patterns are outcomes of bioelectric network dynamics with the lowest free-energy; cutting the network raises the free energy by deviating the system from its optimal morphology (damage), and initiates the regenerative process—a journey back down to a low-energy attractor. Perturbing the physiological bioelectric network with 8-OH results in shifting the system dynamics from this attractor to another attractor, and subsequent regeneration of an altered morphology. Apparently, these pseudo shapes are only local minima, since after about 3–4 weeks the pseudo worms return to the global minimum of the standard G. dorotocephala morphology. In this class of models, the output of the physiological network consists of signals directing cells to proliferate, apoptose, or migrate, so that head morphogenesis is achieved. The details of this process at the cellular level is beginning to be understood, both in the CNS (control of gene expression by brain activity [160]) and developmental bioelectricity [12,23,32]. Variations in membrane potential between cell groups may act as electrophoresing forces, pulling small signaling molecules, small RNAs, or charged transcription factors to specific regions of the planarian body, producing numerous possible long-term effects [47,161,162,163,164]. Additional mechanisms include voltage-sensitive phosphatases [165], and voltage-regulated action of chromatin modifying enzymes [34,43,166]. Clearly, this is just one way to think about these data, and others are possible. The advantage of this paradigm is the rich set of mathematical results available from studies of computational neuroscience that can constrain future model-building and help make testable predictions. 
  
 3.8. Stochastic Phenotypes and Evolutionary Implications One of the key aspects of these phenotypes is their stochastic nature: the same treatment performed on a cohort of clonal (isogenic) animals results in one of several patterns within any exposed population. Strikingly, the frequencies with which species-specific patterns are observed match the evolutionary distances estimated for these species of planaria (Figure 5). There are significantly more pseudo G. dorotocephala, D. japonica, and S. mediterranea morphologies, which correspond to a smaller evolutionary distance from G. dorotocephala, while P. felina pseudo morphologies remain relatively infrequent. One possibility is that a core network (composed of gene regulatory elements and physiological gap junction communication (GJC)—mediated signals) is at work among many planarians, with species-specific differences tweaking its key dynamics. The evolutionary distance among the planarian types may thus be expected to reflect the ease of shifting this network into its different potential outcome states (as occurs during GJC inhibition). It should be noted that wild-type P. felina have multiple eyes along the doso-ventral body boundary, a trait that our pseudo PFs do not display. Incomplete mimicry of the most distant species, P. felina, is consistent with the association between evolutionary distance and ease with which species-specific morphology can be fully recapitulated during perturbed regeneration. It should be noted that Phagocata gracilis and Phagocata velata planarians also have head morphologies quite similar to P. felina, and therefore also to pseudo PFs. These two species possess only two eyes, and both species are similarly evolutionarily removed from G. dorotocephala. However, no living wild-type worms of these two species could be procured for this work, and therefore our analysis focused on P. felina. Overall, the ability to generate distinct morphologies from the same genome likely represents an extremely flexible mechanism that could be exploited by evolution via many known environmental and genetically-encoded modulators of gap junctional state. An obvious absence of “intermediate” morphologies, or head shapes that resemble a blending of two or more species of planarian, could be the result of constraints that limit the morphological space available to the regenerating planarian. Within this framework, the constraints on phenotypic space become driving factors in morphogenesis. These constraints are likely an amalgam of developmental, environmental, and evolutionary factors, which serve to limit the stable modes of the patterning network, and thus the morphogenetic possibilities available to a regenerating flatworm. Efforts currently on-going in our lab are aimed at producing mechanistic bioelectrical models of single cells and cell networks to quantitatively explain the discrete stable modes of such networks, the stochastic nature of each “run” of such a network following amputation, and the molecular links to downstream cell behaviors [94]. ||||| Biologists at Tufts University have succeeded in inducing one species of flatworm to grow heads and brains characteristic of another species of flatworm without altering genomic sequence. The work reveals physiological circuits as a new kind of epigenetics -- information existing outside of genomic sequence -- that determines large-scale anatomy. 
  
 The finding that head shape is not hard-wired by the genome but can be overridden by manipulating electrical synapses in the body suggests that differences in species could be determined in part by the activity of bioelectrical networks. The discovery could help improve understanding of birth defects and regeneration by revealing a new pathway for controlling complex pattern formation. It has long been known that neural networks exploit bioelectric synapses to store and re-write information in the brain. 
  
 The findings are detailed in the cover story of the November 2015 edition of the International Journal of Molecular Sciences, appearing online Nov. 24. 
  
 "It is commonly thought that the sequence and structure of chromatin -- material that makes up chromosomes -- determine the shape of an organism, but these results show that the function of physiological networks can override the species-specific default anatomy," says the paper's senior and corresponding author Michael Levin, Ph.D., who holds the Vannevar Bush Chair in biology and directs the Center for Regenerative and Developmental Biology in the School of Arts and Sciences at Tufts. "By modulating the connectivity of cells via electrical synapses, we were able to derive head morphology and brain patterning belonging to a completely different species from an animal with a normal genome." 
  
 Knowing how shape is determined and how to influence it is important because biologists could use that knowledge, for example, to fix birth defects or cause new biological structures to grow after an injury, adds Levin. "These findings raise significant questions about how genes and bioelectric networks interact to build complex body structures," he says. 
  
 Working with Girardia dorotocephala -- free-living planarian flatworms, which have remarkable regenerative capacity -- researchers were able to induce the development of different species-specific head shapes by interrupting gap junctions, which are protein channels that enable cells to communicate with each other by passing electrical signals back and forth. The changes were more than skin deep; they included not only the overall shape of the head but also the shape of the brain and the distribution of the worm's adult stem cells. 
  
 The ease with which a particular shape could be coaxed from a G. dorotocephala worm was proportional to the proximity of the target worm on the evolutionary timeline. The closer the two species were related, the easier it was to effect the change. This observation strengthens the connection to evolutionary history, suggesting that modulation of physiological circuits may be one more tool exploited by evolution to alter animal body plans. 
  
 However, unlike the Levin lab's previous work, in which a different species of planaria could be permanently altered to a two-headed morphology, this shape change was only temporary. Weeks after the planaria completed regeneration to the other species' head shapes, the worms once again began remodeling and re-acquired their original head morphology. Additional research is needed to determine how this occurs. The authors also presented a computational model that explains how changes in cell-to-cell communication can give rise to the diverse shape types. 
  
 First author on the paper was Tufts undergraduate Maya Emmons-Bell, a senior majoring in biology. "We've demonstrated that the electrical connections between cells provide important information for species-specific patterning of the head during regeneration in planarian flatworms," she says. "This kind of information will be crucial for advances in regenerative medicine, as well as a better understanding of evolutionary biology. As an undergraduate, it's been an extraordinary experience to conduct and author important research with leading biologists." 
  
 The interdisciplinary research involved U.S.- and Canada-based biologists and European mathematicians. |||||
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– Worms can grow the head and brain of another species? Then maybe we can do better at regenerating our own organs and tissues. That's the thrust of a new study by researchers who got flatworms to grow the heads of other flatworm species without any alteration to the worms' DNA, LiveScience reports. The researchers just altered the way proteins communicated between the flatworms' cells. "It is commonly thought that the sequence and structure of chromatin—the material that makes up chromosomes—determine the shape of an organism," says study researcher Michael Levin in a press release. "But these results show that the function of physiological networks can override the species-specific default anatomy." The researchers began by decapitating freshwater flatworms called Girardia dorotocephala, which are are able to regrow severed tissues; they have stem cells that can turn into any other kind of cell (we have similar cells, but only as early embyros). Researchers then used a kind of alcohol to alter electrical impulses that cells send through protein channels in the worms, IFL Science reports. Amazingly, some of the worms grew heads that were rounded, triangular, or had pointy ears—and all from other species. The worms eventually grew back their own heads, with their DNA fully intact. "This kind of information will be crucial for advances in regenerative medicine, as well as a better understanding of evolutionary biology," says Maya Emmons-Bell, first author of the study.
Plattmaskar är anmärkningsvärda varelser, av vilka en del har nästan otroliga förmågor för vävnadsförnyelse och tillväxt. En särskild art, Girardia dorotocephala, har visat sig kunna ta denna förmåga ett bisarrt steg längre: Forskare vid Tufts University har framgångsrikt fått den att tillfälligt odla huvuden och vissa hjärnstrukturer hos flera andra plattmaskarter, allt utan att ändra dess genetiska sekvens. Deras fynd redovisas i International Journal of Molecular Sciences. Formen på ett djurs kropp bestäms till stor del av dess genom. Men bioelektriska nervnät och deras förbindelser (synapser) har sitt eget sätt att bilda konstruktioner, och den noggranna manipulationen av dessa kan få unika egenskaper att bildas i vissa organismer, vilket överskuggar det genetiska materialets instruktioner. Med detta i åtanke beslöt forskarna att modifiera synapsförbindelserna i huvudet på sin valda plattmask för att se om de kunde tvinga den att producera nya former. Efter att ha halshuggit dem förändrades den elektriska förbindelsen mellan cellerna i dess överkropp med hjälp av en form av alkohol (oktanol). Otroligt nog fick detta plattmaskarna att växa inte bara ny vävnad, utan helt nya huvuden – huvuden som tillhörde helt olika arter av plattmask: S. mediterranea, D. japonica, och P. felina. Det var inte bara huvudformerna som replikerades, utan segment och egenskaper hos deras hjärnor var också till synes kopierade, inklusive distributionen av vuxna stamceller. Ju närmare de båda arterna var besläktade, desto lättare var det för forskarna att framkalla förändringarna. Dessa förändringar, märkligt nog, var inte permanenta: Inom några veckor övervann den genetiska kodningen den bioelektriskt framkallade huvudförändringen, och deras huvuden återgick gradvis till sin ursprungliga form. För närvarande är forskarna inte helt säkra på hur denna ombyggnad sker. Oavsett, det faktum att allt detta skedde utan att ändra DNA från den ursprungliga plattmasken är studiens mest betydande upptäckt. Detta innebär att förändringar i en plattmasks förmåga att “bygga” själv ibland kan härröra från utanför instruktionerna kodade i sitt genetiska material. Denna typ av externt framkallad förändring är känd som epigenetik – bokstavligen betyder “extra tillväxt”. Att överföra ett urval av gener från två föräldrar till sin avkomma är inte det enda sättet bygginstruktionerna för en organism ärvs. Tillsammans med en ytterligare process som kallas horisontell genöverföring som används av vissa mikroskopiska organismer, kan förändringar i hur gener uttrycks också ärvas. Flera miljömässiga eller på annat sätt externa faktorer kan förändra hur celler läser gener, vilket innebär att avkommans fysiska attribut kan vara olika – allt utan att DNA-sekvensen förändras. Även om det ännu inte har visats om dessa ovanliga plattmaskförändringar kan överföras till deras avkomma, passar de in i definitionen av epigenetik – även om det som vissa påpekar, utan att arvet kan påvisas, kan vara något kontroversiellt att tillämpa denna definition. Ändå har studien konsekvenser utanför bara plattmaskbiologin. Maya Emmons-Bell, en Tufts University biologi grundutbildning och första författare på tidningen, sade i ett uttalande: “Denna typ av information kommer att vara avgörande för framsteg inom regenerativ medicin, samt en bättre förståelse för evolutionsbiologi.” Bild i text: G. dorotocephala (överst till vänster) kunde växa huvuden av andra arter (översta raden) utan några förändringar i dess genom. Centre for Regenerative and Developmental Biology, School of Arts and Sciences, Tufts University <unk> <unk> Kalla dem Franken plattmaskar. Forskare har skapat maskar med andra arters huvuden och hjärnor bara genom att manipulera cellkommunikationen. Forskningen är ett exempel på hur utveckling styrs av mer än enbart genetik. Forskarna ändrade inte plattmaskarnas DNA på något sätt, utan manipulerade istället proteiner som styr samtal mellan celler. "Det är vanligt att man tror att kromatinets sekvens och struktur – det material som utgör kromosomer – bestämmer formen på en organism, men dessa resultat visar att funktionen hos fysiologiska nätverk kan åsidosätta den artspecifika standardanatomin", säger forskaren Michael Levin, biolog vid Tufts University, i ett uttalande. [Se Bilder av Franken Flatworms med olika huvuden, Hjärnor] Förändringarna är tillfälliga för maskarna, vars huvuden börjar återgå till sina ursprungliga former i veckor. Men forskare hoppas att resultaten kommer att leda till behandlingar för fosterskador och även regenerativ medicin, som syftar till att ersätta eller återuppbygga skadade vävnader och organ. Forskarna studerade en liten sötvattensplattmask, Girardia dorotocephala, som är känd för att kunna regenerera förlorade vävnader. Dessa plattmaskar behåller ett stort antal celler som kallas neoblaster, vilka är totipotenta stamceller, vilket innebär att de kan bli vilken celltyp som helst i kroppen. Hos människor är cellerna totipotenta först under de första dagarna av embryonal utveckling. Först skar forskarna av huvudet på sina labbprover. För att ändra plattmaskens regenererande huvud avbröt Levin och hans kollegor proteinkanalerna. Cellerna skickar elektriska impulser genom dessa korsningar för att kunna kommunicera. Forskarna fann att de lätt kunde knuffa masken för att utveckla huvud- och hjärnformer liknande dem hos andra närbesläktade plattmaskarter. Normalt sportar G. dorotocephala ett spetsigt huvud med två långsträckta, öronliknande projektioner (kallade aurikler) bredvid ögonen. Efter behandling utvecklade några av maskarna sina normala huvuden, medan andra växte rundade huvuden som hos S. mediterranea; huvuden med tjock hals och spetsiga, kattliknande "öron" som hos P. felina; eller triangulära huvuden som hos D. japonica. Hjärnorna tycktes följa efter den förändrade huvudformen, så att plattmaskar som återbildade ett huvud format som D. japonica, till exempel, också visade hjärnmorfologier som var kortare och bredare än de hos G. dorotocephala och mer karakteristiska för D. japonica. Ju längre två arter var från varandra på det evolutionära släktträdet, desto svårare var det att framkalla denna mix-and-match-effekt rapporterade forskarna online 24 november i International Journal of Molecular Sciences. "Dessa fynd väcker betydande frågor om hur gener och bioelektriska nätverk interagerar för att bygga komplexa kroppsstrukturer", sade Levin. Om gener ger en ritning för en organisms kropp, celler är som de byggnadsarbetare som krävs för att förvandla planen till en struktur - och gap korsningar är walkie-talkies dessa arbetare använder för att kommunicera. Avbryt kommunikationen, så kan du störa byggprocessen. G. dorotocephala förblev förändrad under endast en begränsad tid innan dess neoblaster tog över och åter bekräftade de gamla huvud- och hjärnformerna. Levins labb har dock tidigare konstruerat en annan art av plattmask för att odla två huvuden och hålla sig på det sättet. Följ Stephanie Pappas på Twitter och Google+. Följ oss @livescience, Facebook & Google+. Originalartikel om Levande vetenskap. Det är viktigt att notera att olika arter av planaria har karakteristiska huvudformer som lätt utmärker sig genom anatomisk inspektion. Således undersökte vi konsekvenserna av att störa det kroppsomfattande gapkopplingskommunikationsnätet för huvudform i planaria. Anmärkningsvärt observerade vi en stokastisk fenotyp, i vilken de regenererande huvudena hos en genomiskt normal G. dorotocephala plattmask förvärvade en huvudmorfologi som var lämplig för andra extant planariska arter. Effekten observerades i huvudet, liksom i hjärnmorfologin. Till skillnad från vår senaste demonstration av en permanent (stabil) förändring av målmorfologin i D. japonica [ 20 ], var denna effekt tillfällig, och maskarna remodellerades tillbaka till sitt hemland inom 30 dagar. Förmågan att stochastiskt välja en av flera diskreta huvudformer som är lämpliga för en annan art, helt enkelt genom att förändra ett fysiologiskt nätverk, antyder att kvantitativa modeller av bioelektriska nätverkslägen kommer att vara en viktig del av förståelsen av evolutionär förändring och genomiska kontra fysiologiska kretsars roll i etablerandet av anatomisk struktur. Planaria är frilevande plattmaskar med imponerande regenerativa förmågor [ 55, 56 , 57 ]. Efter traumatiska skador såsom amputation, planarianer regenerera alla saknade vävnader, reproducerar sin målmorfologi perfekt i en relativt kort tidsperiod på cirka två veckor. Denna morfologiska omdaning är icke begränsad till trauma; efter perioder av svält krymper planarier sig allometriskt, och växer på samma, skalade sätt efter att födan återinförts [ 58 ]. Detta anmärkningsvärda exempel på rumslig och temporal cellulär organisation kräver lagring och utbyte av morfologisk och fysiologisk information över en varierad och vitt spridd cellpopulation. Sådan morfologisk robusthet ger ett unikt system där man kan studera komplexa egenskaper som morfologins plasticitet. Medan det senaste arbetet har gjort anmärkningsvärda framsteg på de molekylära detaljerna i banor som reglerar vuxna stamcellers beteende i planaria [ 59 , 60 , 61 ], finns det mycket lite information om hur specifik huvudform kontrolleras. Det stora flertalet av den funktionella litteraturen redovisar fenotyper av antingen underlåtenhet att återskapa eller byta huvud/svansidentitet; fältet har för närvarande ingen mekanistisk förståelse för hur en artspecifik huvudform är etablerad eller reglerad [ 62 ], även om spänningsbaserad signalering har visat sig vara inblandad i huvudform/storlek hos minst en art [ 25 ]. Global bioelektrisk nätverksdynamik regleras delvis av gapkorsningar – elektriska synapser mellan celler som underlättar direkt jonbyte, och därmed låter celler jämföra sina V mem med sina grannars, och etablera isopotentiella cellfält eller gränser mellan fack [ 44 , 45 ]. Eftersom gapkorsningar kan vara själva spänningskänsliga [ 46 , 47 ], kan de i själva verket fungera som transistorer – vilket möjliggör komplex informationsbearbetning och återkopplingsslingor. Gapkorsningar ger också rika möjligheter till selektiv gatbildning av små molekylsignaler utöver jonström [ 48 ]. Oerhört är på grund af dessa drag detta mångsidiga signalelement af avgörande betydelse för utvecklingen [ 49 , 50 , 51 ]och äfven för det kognitiva minnets plasticitet i hjärnan [ 52 , 53 , 54 ]. Således undersökte vi här vilken roll gapkopplingsmedierade fysiologiska nätverk har för att reglera mönsterbildning. Tre aspekter av bioelektrisk signalering gör dem särskilt relevanta för uppkomsten av storskalig form och genomets roll. För det första specificerar bioelektriska mönster formen på ett distribuerat (icke-lokalt) sätt: flera studier har visat att storleken, formen och identiteten hos specifika strukturer integrerar bioelektrisk information från avlägsna regioner [ 32, 35, 36, 37, 38, 39 ], vilket gör bioelektrisk signalering till en idealisk modalitet för att samordna individuella cellbeteenden mot ett specifikt anatomiskt utfall. För det andra kan bioelektriska mönster åsidosätta standard genetisk/biokemisk information: specifika V-mem-förändringar kan förhindra mesenkymala stamcellsdifferentiering trots förekomsten av kemiska inducerare [ 40 , 41 ], kan framkalla ett öga att bilda i en vävnad (t.ex. mesoderm eller endoderm) som annars inte är kompetent att bli öga när man missuttrycker en “master” ögoninducerare som Pax6 [ 31 ], kan inducera metastaserande melanom i avsaknad av genetiska skador [ 36 , 37 ], kan förhindra bildandet av tumörer i närvaro av annars tillräckliga onkogener [ 42 , 43 ], och kan rädda hjärnskador orsakade av mutationer i kraftfulla regulatorer av neurogenes som Notch [ 32 ]. Således är bioelektrisk signalering en bra förklarande kandidat i fall av epigenetisk påverkan över mönsterbildning. Slutligen tyckas bioelektriska egenskaper direkt koda utfall på organnivå, framkalla hela bihang [ 17 , 23 , 24 ]eller komplexa ögon [ 31 ]. Denna förmåga att utlösa nedströmsutvecklingsmoduler, utan att behöva specificera enskilda cellpositioner (mikromanage the process), gör bioelektriska signaler inte bara attraktiva styrknappar för biomedicinska ingrepp, utan avslöjar också hur bioelektriska nätverkstillstånd kan ses som attraktioner instruerar komplexa mönstrande utfall. Större delen av fältet är idag inriktat på genreglerande nätverk [ 8 , 9 ]och fysiska krafter [ 10 , 11 ]i ett försök att förstå hur slutliga mönstrande utfall uppstår och upprätthålls under utveckling och förnyelse. Ett annat fascinerande lager av biologisk reglering har emellertid nyligen varit inblandat i kontrollen av morfogenesen: endogen bioelektrisk signalering [ 12, 13 , 14 ]. Spatio-temporella gradienter av vilande potential bland alla celltyper (inte bara excitabel nerv och muskel) kan reglera cellproliferation, migration, form, och apoptos [ 15 , 16 ]. Ännu viktigare är att de kan fungera som lärorika ledtrådar för storskalig morpogenes, reglerande positionsinformation, organidentitet, storlek och axiell polaritet [ 1 , 17 , 18 ]. Det senaste arbetet har inbegripit dessa spänningsgradienter i regleringen av främre-bakre polaritet [ 19 , 20 ], bihangsregenerering [ 21 , 22 , 23 , 24 ], kraniofacial mönstrande [ 25 ], vänster-höger asymmetri [ 26 , 27 , 28 , 29 ], ögonutveckling [ 30 , 31 ], och hjärnmönstrande [ 32 ]. Talrika studier har nu identifierat transduktionsmekanismer som kopplar samman bioelektriska egenskaper med nedströms transkriptionella och epigenetiska mål [ 13 , 18 , 33 , 34 ], och därmed avslöja hur dessa fysikaliska egenskaper integreras med genetisk information under mönstrandet. Vare sig det är via nukleotidsekvenser eller kromatinmodifieringar, specificerar genomet inte direkt organismisk 3-dimensionell form. I stället är storskalig morfologi ett emergent drag i dynamiken i komplexa nätverk av aktivitet som utförs av celler. Således är mönsterbildning resultatet av ett rikt lager av kemiska och fysikaliska processer som sker mellan DNA och anatomi. I själva verket kan distinkta morfologier uppstå ur en enda genotyp [ 4 , 5 ]. Att förstå hur celler kommunicerar och koordinerar sina funktioner in vivo för att på ett tillförlitligt sätt bilda komplexa kroppsplaner och organ är av grundläggande betydelse inte bara för evolutionär utvecklingsbiologi, utan även för biomedicin [ 6 ]. Transformativa framsteg inom regenerativ medicin och syntetisk bioengineering kräver att vi vet vilka indata som kan ges till ett cellulärt system för att inducera specifika morfologiska resultat-rationell kontroll av tillväxt och form. Detta är ett verkligt svårt problem på grund av den biologiska regleringens komplexa ickelinjäritet [ 7 ]. Därför är steg 1 att avslöja processer som ger instruktiv kontroll över bestämningen av storskalig form. Utveckling, cancersuppression, storskalig ombyggnad och förnyelse är alla beroende av en organisms förmåga att lagra och bearbeta information om dess korrekta anatomiska struktur, och korrigera eventuella avvikelser från denna struktur som kan uppstå vid skada eller annan miljöpåverkan [ 1 , 2 ]. Det antas allmänt att artspecifika anatomiska former kodas i genomet, även om data från miljöepigenetiken länge har antytt att morfologiska utfall är en funktion av inte bara arv utan även av miljö- och livshistoriska inflöden [ 3 ]. Beräkningsmodellen återger de fyra diskreta utfall som observerats experimentellt. () Fyra typer av plana huvuden som erhålls från beräkningsmodellen. Olika former resulterar på grund av deaktivering av olika celler. Röda linjer visar de röda cellernas banor; () Morfometrisk mätning av olika plana huvuden, för jämförelse med verkliga huvuden () uppmätt (se tabell 2 ). Skalstång 0,5 mm. Celler av typ A visas i svarta, avaktiverade celler av typ A vita, celler av typ B är röda. Den yttre gränsen har två delar som skiljer sig åt genom sin styvhet (mjuk del av gränsen visas i rött, styvare del i grönt). De röda cellerna representerar alltså migrerande neoblaster (eller deras avkomma) medan de svarta cellerna representerar somatiska celler som sammankopplas med GJs. () Definitioner av längder på mätningarna L1 och L2 som används i jämförelse med verkliga maskar jämfört med dem som produceras av modellen i tabell 2 Beräkningsmodellen återger de fyra diskreta resultat som observerats experimentellt. () Fyra typer av plana huvuden som erhålls från beräkningsmodellen. Olika former resulterar på grund av deaktivering av olika celler. Röda linjer visar de röda cellernas banor; () Morfometrisk mätning av olika plana huvuden, för jämförelse med verkliga huvuden () uppmätt (se tabell 2 ). Skalstång 0,5 mm. Celler av typ A visas i svarta, avaktiverade celler av typ A vita, celler av typ B är röda. Den yttre gränsen har två delar som skiljer sig åt genom sin styvhet (mjuk del av gränsen visas i rött, styvare del i grönt). De röda cellerna representerar alltså migrerande neoblaster (eller deras avkomma) medan de svarta cellerna representerar somatiska celler som sammankopplas med GJs. () Definitioner av längder på måtten L1 och L2 som används i jämförelse med verkliga maskar jämfört med dem som produceras av modellen i tabell 2 Observera att formen på auriklarna i figur 8 D är mer uttalad än i fall figur 8 B,C. Detta motsvarar de experimentellt observerade formerna. Vid modellering uppnås denna skillnad med olika egenskaper hos den yttre gränsen, vara mjukare i den nedre delen (röd i figuren) och resultera i skarpare aurikler. Detta är förenligt med tidigare arbete som visar att integumentintegritet (och därmed styvhet) är beroende av det korrekta uttrycket och funktionen hos gapkopplingskanaler [ 100 ]. Figur 8 visar cellerna B:s slutpositioner och deras banor efter att ha simulerat fyra olika deaktiveringsmönster. För att jämföra verkliga maskmorfologiska data med resultaten av vår modellering utförde vi morfometriska mätningar som visas i figur 8 E–H om resultaten av våra in silico-simuleringar. Vi mätte längden på det horisontella intervallet L1 och det vertikala intervallet L2 (i fallet 1b och 2b måste dessa intervall nödvändigtvis inte vara raka). Resultaten ses i tabell 2, och ger en god överensstämmelse med de observerade resultaten i verkliga maskar (figur 1). Låt oss lägga märke till att de slutliga morfologierna i planarens huvud i datorsimuleringar bestäms av den ursprungliga cellkonfigurationen. De skiljer sig genom placeringar av avaktiverade celler (vita celler i figur 8). Om dessa celler är nära, då de slutliga huvudformer är också nära. Om vi jämför DJ (figur 8 C) och GD (figur 8 D), då är den ursprungliga cellkonfigurationen och de slutliga morfologierna närmare varandra än dessa två jämfört med SM (figur 8 A). Dessutom verkar DJ finnas mellan SM- och GD-modellerna. Det är intressant att notera att denna ordning motsvarar deras genetiska avstånd ( Figur 3). Vi drar slutsatsen att en liten uppsättning cellnivåbeteenden, såsom migration och cellcellssignalering som är kända för att regleras av GJC och därmed är perturberade av oktanolexponering [ 35 , 39 , 49 , 98, 101 , 102 ], kan kvantitativt förklara de morfologier vi observerade i planaria. När den här modellen simulerades i silico, producerade den plana huvudformer som observerades i experimenten. Dessa olika former uppnåddes från samma ursprungliga cellkonfiguration men olika deaktiveringsmönster för celler A och olika elastiska egenskaper för gränsen. Dessa deaktiveringsmönster motsvarar oktanolbehandlingar som minskar cell-cellkommunikationen bland en undergrupp av celler. Vi förmodar att detta resulterar i deaktivering av vissa av cellernas signalering. Den ursprungliga cellpositionskonfigurationen och dess resulterande jämviktskonfiguration visas i figur S2 H,I. Celler av typ B förblir omgivna av A-celler. Denna konfiguration väljs på ett sådant sätt att celler B antingen stannar inne eller migrerar utanför och producerar en av de fyra konfigurationer som krävs (figur S2 A–D) efter deaktivering av vissa celler A. Specifik huvudform uppstår eftersom celler B skjuts bort av celler A och av andra celler B. De flyr genom de lägre koncentrationsnivåerna av ämne C som lämnas av avaktiverade celler A, anländer till det yttre membranet och ändrar dess form. Vi övervägde två celltyper, A och B. Celler av typ A har fasta positioner – de kan inte röra sig. De producerar ett ämne vars koncentration C sprider sig i rymden. Dess fördelning kan beskrivas genom en reaktionsdiffusionsekvation eller av vissa andra modeller. Celler av typ B kan röra sig. De producerar inget ämne men de får ämnet C som produceras av celler A och deras rörelse bestäms av dess koncentrationsfördelning. Vi modellerade det plana huvudet med följande element: celler av typ A, avaktiverade celler av typ A, celler av typ B och en ytgräns. Deaktiverade A-celler fixeras men de producerar inte ämne C. Om det finns mer än en cell B, då stöter de bort varandra på samma sätt som de stöts bort av celler A. Vi väljer avaktiverade celler A symmetriskt med avseende på den främre-bakre axeln, för att bevara resultatets symmetri (vilket innebär att B-celler rör sig på ett liknande sätt på vänster och höger sida). Den yttre gränsen består av punkter och elastiska ”fjädrar” som förbinder dem. När en cell B närmar sig gränsen verkar en motbjudande kraft på den från gränsen. Denna kraft är proportionell mot avståndet från partiklarna och från gränsintervallen. Fullständiga uppgifter om modelleringen ges i figur S2. I denna modell, vi hypoteserar att instruktiv (GJ-beroende) signalering sker från de somatiska vävnaderna till neoblasterna eller deras avkomma, för att vägleda den nya vävnadsgenerationen och forma under regenerering. Således är de röda cellerna migrerande neoblaster (eller deras avkomma) medan de svarta cellerna är somatiska celler sammankopplade med GJs ( Figur 8). I modellen modelleras på så sätt oktanolstörning av cell:cellkommunikation genom deaktivering av signalgivning från en specifik celltyp. En av de viktigaste utmaningarna som utvecklingsbiologi och regenerativ medicin står inför är att koppla samman storskaliga mönstrande resultat med den individuella aktiviteten hos celler som styrs av genetiska nätverk och signalvägar [ 2 , 85 , 86 , 87 , 88 , 89 ]. Större delen av arbetet på planariska fältet behandlar främre-bakre fragmentpolaritet [ 60 , 62 , 90 ]eller stamcellsdifferentiering [ 57 , 59 , 91 ], och behandlar inte den egentliga morfologin hos huvudet eller resten av kroppen. Sentida kvantitativa, genetiskt grundlagda modeller av regenerering [ 92, 93 , 94 ]använder likaledes främre-bakre identitet som en binär avläsning, som icke adresserar eller förklarar förändringar i dessa anatomiska regioners form. Vår studie av den bioelektriska regleringen av regenereringen rapporterade förändringar av huvudrenoveringen [ 25 ], men var begränsad till skalning och modellerade inte huvudets detaljerade form. För att mekaniskt börja länka individuella cellbeteenden (såsom de som regleras av GJ-medierade signaler) till storskaliga anatomiska utfall konstruerade vi därefter en agentbaserad modell av cellsignalering och morfogenes. Vår modell fokuserade på cellmigration och cell-cellsignalering, då dessa äro klart viktiga för att genomföra olika morfogenetiska utfall [ 95 , 96 , 97 ], och även kända för att regleras av GJ-anslutning [ 98 ]och bioelektriska egenskaper hos grannceller [ 99 ]. I D. japonica kan fysiologiska perturbationer stabilt förändra den planariska kroppsplanens grundarkitektur, vilket ger upphov till tvåhövdade maskar som fortsätter att regenerera som tvåhövdade i evighet över framtida omgångar av regenerering i vanligt vatten [ 20 ]. Vi frågade därför därefter om våra observerade huvudformsförändringar i G. dorotocephala var bestående. Fotografier av maskar efter behandling med 8-OH togs dagligen, från dag 10 (då maskarnas morfologier gjordes), till dag 30 (den tid som krävs för fullständig cellomsättning hos planarian plattmasken). Vi fann att de inducerade morfologierna omarbetades, långt efter att regenerativa processer hade upphört, för att producera morfologier som bar närmare likhet med den vilda typen G. dorotocephala. Intressant, tidsförloppet och resultatet av denna icke-regenerativa remodeling skilde sig åt beroende på starthuvudets former. Regenerater som hade rundade huvuden, som påminner om S. mediterranea, började utveckla en mer triangulär huvudform dag 17, och på dag 30, bar mer av en likhet med D. japonica än S. mediterranea. Under samma tid, regenerationer som hade trekantiga huvuden, som liknar D. japonica, utvecklade uttalade aurikler, och på dag 30 var omöjlig att skilja från vildtyp G. dorotocephala (Figur 7 A,B). Dessa fynd är fascinerande i två avseenden. För det första, förändringen i morfologi i avsaknad av ett trauma belyser dynamiska och robusta mekanismer bakom morfologisk homeostas – det anatomiska tillståndet är ombyggt över lång tid från en onormal konfiguration som existerar efter regenerativ reparation var klar. För det andra sker ombyggnaden via ”vägar” genom det formutrymme som illustreras av kanonisk variabelanalys (figur 7 C). DJ morfologi ligger mellan GD och SM morfologier i detta utrymme, och vi observerar ombyggnad som går från SM morfologi till DJ morfologi till GD morfologi. Utifrån dessa data drar vi slutsatsen att morfologin är både plastisk och robust, återställande av ”mål”-morfologin kan ske utan trauma mot organismen, och att CV-formens utrymme är informativt när det gäller att illustrera parametrar och gränser till morfologisk variation. Figur 6. Membranspänningsreporter analys visar långsiktig förändring av bioelektrisk anslutning i oktanol-exponerad planaria. ( A – D ) Domäner av relativ membranpotential visualiseras med DiBAC 4 (3) i vildtyp G. dorotocephala , vildtyp S. mediterranea , vildtyp D. japonica , och vildtyp P. feline ; ( E – H ) Domäner av relativ membranpotential visualiseras med DiBAC(3) i pseudo G. dorotocephala (GDs), pseudo S. mediterranea (SMs), pseudo D. japonica (DJs), och pseudo P. felina (PFs), respektive. Skalbar 0,5 mm; ( I ) Antal isopotentiella regioner i GD-maskar av vildtyp (kontroll), alla pseudomorfologier och pseudomorfologier som har remodellerats tillbaka till WT (vildtyp) GD-morfologi efter 30 dagar. Efter oktanolbehandling ökar antalet isopotentiella regioner i pseudomorfologier, men minskar till WT-nivåer efter ombyggnad. Svarta prickar indikerar maskar med GD-morfologier, blå prickar anger DJ-morfologier, röda prickar anger PF-morfologier, gröna prickar anger SM-morfologier, och rosa prickar indikerar pseudomaskar som har omformat sina morfologier för att likna vildtyps GD:er. n = 7 GD av vildtyp, n = 5 GD av pseudotyp, n = 5 SM av pseudotyp, n = 5 DJ av pseudotyp, n = 2 PF av pseudotyp, och n = 6 maskar av ombyggda maskar. Felstaplar är standardavvikelser. Icke-parametrisk statistisk analys gjordes med hjälp av en post-hoc jämförelse av alla grupper med Kruskal-Wallis test (p = 0,0021), och sedan mellan grupper med hjälp av ett Dunns Multiple Comparison test, som visade skillnader i spänningsdomäntal mellan ombyggda GD och pseudomorfologier är statistiskt signifikanta vid p < 0,05. Figur 6. Membranspänningsreporter analys visar långsiktig förändring av bioelektrisk anslutning i oktanol-exponerad planaria. ( A – D ) Domäner av relativ membranpotential visualiseras med DiBAC 4 (3) i vildtyp G. dorotocephala , vildtyp S. mediterranea , vildtyp D. japonica , och vildtyp P. feline ; ( E – H ) Domäner av relativ membranpotential visualiseras med DiBAC(3) i pseudo G. dorotocephala (GDs), pseudo S. mediterranea (SMs), pseudo D. japonica (DJs), och pseudo P. felina (PFs), respektive. Skalbar 0,5 mm; ( I ) Antal isopotentiella regioner i GD-maskar av vildtyp (kontroll), alla pseudomorfologier och pseudomorfologier som har remodellerats tillbaka till WT (vildtyp) GD-morfologi efter 30 dagar. Efter oktanolbehandling ökar antalet isopotentiella regioner i pseudomorfologier, men minskar till WT-nivåer efter ombyggnad. Svarta prickar indikerar maskar med GD-morfologier, blå prickar anger DJ-morfologier, röda prickar anger PF-morfologier, gröna prickar anger SM-morfologier, och rosa prickar indikerar pseudomaskar som har omformat sina morfologier för att likna vildtyps GD:er. n = 7 GD av vildtyp, n = 5 GD av pseudotyp, n = 5 SM av pseudotyp, n = 5 DJ av pseudotyp, n = 2 PF av pseudotyp, och n = 6 maskar av ombyggda maskar. Felstaplar är standardavvikelser. Icke-parametrisk statistisk analys gjordes med hjälp av en post-hoc jämförelse av alla grupper med Kruskal-Wallis test (p = 0,0021), och sedan mellan grupper med hjälp av ett Dunns Multiple Comparison test, som visade skillnader i spänningsdomäntal mellan ombyggda GD och pseudomorfologier är statistiskt signifikanta vid p < 0,05. Figur 5. Distributionen av neoblaster (röda prickar) förändras efter oktanolbehandling. ( A – C ) Analys av neoblastfördelningen genom färgning av fosforylerad histon H3 hos vuxna, vildtyp G. dorotocephala (n = 9), D. japonica (n = 9) och S. mediterranea (n = 9) planarianer. Prickade linjer anger kanter av maskens anatomi, samt 1/6 av maskkroppens längd från maskens främre spets. Detta avstånd användes för att definiera huvudets bakre gräns. Pilar anger den region där neoblaster räknades; ( D – F ) Analys av neoblastfördelningen i G. dorotocephala regenererar behandlade i 8-OH som liknade G. dorotocephala huvuden ( n = 7), D. japonica huvuden ( n = 8), och S. mediterranea huvuden ( n = 7), genom fosfornedsättande histon H3-färgning. Pilar anger region där neoblaster räknades. Skalstången 0,5 mm; ( G ) Genomsnittligt antal neoblaster i den främre 1/6:e av vildtyp, och 8-OH behandlade maskar (ANOVA p < 0,05). Felstaplar är standardavvikelser. Figur 5. Distributionen av neoblaster (röda prickar) förändras efter oktanolbehandling. ( A – C ) Analys av neoblastfördelningen genom färgning av fosforylerad histon H3 hos vuxna, vildtyp G. dorotocephala (n = 9), D. japonica (n = 9) och S. mediterranea (n = 9) planarianer. Prickade linjer anger kanter av maskens anatomi, samt 1/6 av maskkroppens längd från maskens främre spets. Detta avstånd användes för att definiera huvudets bakre gräns. Pilar anger den region där neoblaster räknades; ( D – F ) Analys av neoblastfördelningen i G. dorotocephala regenererar behandlade i 8-OH som liknade G. dorotocephala huvuden ( n = 7), D. japonica huvuden ( n = 8), och S. mediterranea huvuden ( n = 7), genom fosfornedsättande histon H3-färgning. Pilar anger region där neoblaster räknades. Skalstången 0,5 mm; ( G ) Genomsnittligt antal neoblaster i den främre 1/6:e av vildtyp, och 8-OH behandlade maskar (ANOVA p < 0,05). Felstaplar är standardavvikelser. En av de fysiologiska signaler som förökas inom GJ-medierade cellnät är elektrisk ström: mönster av GJ-beroende anslutning kan bestämma isopotentiella cellfält [ 19 , 25 , 79 ], och vi observerade att oktanol verkligen ökade antalet regioner med distinkta V mem mönster ( Figur 6 ). Eftersom analytiska pipelines för att utläsa kodade mönstertillstånd från bioelektriska mätningar (som har gjorts för den mänskliga hjärnan [ 80 ]) ännu inte existerar, sökte vi börja fastställa fysiologiska mått som kunde avslöja permanenta förändringar av det somatiska bioelektriska nätverk som framkallats av GJ-blockaden och kunde skilja pseudomaskar från de med den ursprungliga (vilda-typen) morfologin. Således undersökte vi härnäst fördelningen av isopotentiella cellgrupper bland de olika maskformsutfallen, eftersom sådana grupper etableras genom funktionen av gapkorsningar och är en avläsning av topologin (sammankoppling) av utvecklingsbioelektriska nätverk [ 20 , 35 , 39 , 51 , 81 , 82 ]. Mönster av endogen relativ membrandepolarisering och hyperpolarisering visualiserades med hjälp av ett DiBAC (bis-(1,3-dibarbitursyra)-trimetinoxonol) färgämne [ 79 , 83 ]. DiBAC är anjoniskt, så färgämnet kommer in i cellmembranen baserat på relativa depolariseringsgrader [ 84 ]. Därför, ökad fluorescens indikerar regioner av depolarisering, och minskad fluorescens indikerar relativ hyperpolarisering. Maskar av vildtyp av arterna G. dorotocephala, D. japonica, S. mediterranea, P. felina, och GJ-perturberade pseudomaskar avbildades med DiBAC-färgämne för att kunna bedöma potentiella skillnader i relativ membranpotential. Bilder analyserades med ett anpassat bildanalysprogram (som beskrivs i Metoder) för att bestämma antalet distinkta isopotentiella regioner som finns i hela masken. Analys av maskar 10 dagar efter 8-OH-behandling (efter regenereringen var klar, figur 6 A–H) visade att övergående perturbation av gapkopplingskommunikation förändrar kroppsomfattande mönster för spänningsfördelning under många dagar efter avslutad 8-OH-behandling. Vi upptäckte ett ökat antal isopotentiella regioner i pseudomaskarna jämfört med kontrollmaskarnas tillstånd; intressant nog återgår antalet isopotentiella regioner till dem i ett tillstånd av vildtyp efter 30 dagars morfologisk ombyggnad (figur 6 I). Även om vi inte kan vara säkra på att varje enskild cell hade penetrerats av färgämnet, mönstret av isopotentiella regioner och variation bland djur tyder på att oktanol verkan är stokastisk och inte 100% effektiv, endast delvis stör elektrisk koppling (uppenbar som regioner som skiljer sig i V mem ) genom djur som behandlas i läkemedel. Vi drar slutsatsen att oktanolexponering förändrar det normala mönstret av G. dorotocephala vilande potential mot ett mönster som kvarstår även efter 8-OH dras tillbaka och regenerativ reparation sker, vilket ger en ökad koncentration av isopotentiella regioner i hela masken. I vildtyp G. dorotocephala når mycket få neoblaster in i maskens mest främre 1/6:e (figur 5 A). I vildtyp D. japonica ökar antalet neoblaster i den främre delen av kroppen i jämförelse med G. dorotocephala, dock är det fortfarande relativt lågt (figur 5 B). Vildtyp S. mediterranea planarianer har en riklig neoblastpopulation i den främre-mest regionen (figur 5 C). Alla neoblaster i främre 1/6:e av maskens anatomi räknades för hand (figur 5 G). Anmärkningsvärt är att fördelningen av neoblaster i pseudomaskar exakt återspeglade fördelningen av neoblaster hos de arter av vildtyp som de liknade (figur 5 D−F) (ANOVA p < 0,05). Vi drar slutsatsen att omvandlingen av G. dorotocephala-maskar till att likna andra arter också sträcker sig till den artspecifika, karakteristiska inre distributionen av deras stamceller, och att de mönstrande processer som störs efter GJC-perturbation också är ansvariga för att organisera fördelningen av mitotiskt aktiva celler. Planarianer härleda mycket af sin anmärkningsvärda regenerativa kraft från en population af heterogena vuxna stamceller, som kallas neoblaster, hvilka utgöra den enda mitotiskt verksamma cellpopulationen i plattmaskens kropp [ 56 , 70 , 77 ]. Vi undersökte härnäst om neoblasternas rumsliga fördelning var märkbart olika mellan arter av planarianer, och om GJ-hämnade maskar förvärvade den neoblastfördelning som var karakteristisk för den art vars morfologi de hade tagit på sig. Då neoblaster äro de enda mitotiskt verksamma cellerna inom den planariska kroppen, utförde vi immunofärgning af fosforylerad histon H3, en vanlig neoblastmarkör i planaria [ 78 ], för att visualisera neoblastpopulationer. Figur 4. Hjärnmorfologin förändras efter 8- OH behandling. ( A – C ) Hjärnmorfologi visualiseras genom antisynapsinfärgning av vildtyp G. dorotocephala (n = 10), D. japonica (n = 15), och S. mediterranea (n = 6) planarianer. Pilar indikerar hjärnmorfologier, och streckade linjer indikerar mätningar som används för beräkning av längd/breddförhållande; ( D – F ) Hjärnmorfologier genom antisynapsinfärgning av G. dorotocephala-regener som behandlas i 8-OH som liknar G. dorotocephala-huvuden (n = 4), D. japonica-huvuden (n = 4), och S. mediterranea-huvuden (n = 6). Pilar indikerar hjärnmorfologier, och streckade linjer indikerade mätningar som används för beräkning av längd/breddförhållande. Skala 0,5 mm; ( G ) Genomsnittligt förhållande mellan hjärnlängd och bredd hos vildtyp och 8-OH-behandlade maskar (ANOVA p < 4,9 × 10 −14 ). Felstaplar är standardavvikelser. Figur 4. Hjärnmorfologin förändras efter 8- OH behandling. ( A – C ) Hjärnmorfologi visualiseras genom antisynapsinfärgning av vildtyp G. dorotocephala (n = 10), D. japonica (n = 15), och S. mediterranea (n = 6) planarianer. Pilar indikerar hjärnmorfologier, och streckade linjer indikerar mätningar som används för beräkning av längd/breddförhållande; ( D – F ) Hjärnmorfologier genom antisynapsinfärgning av G. dorotocephala-regener som behandlas i 8-OH som liknar G. dorotocephala-huvuden (n = 4), D. japonica-huvuden (n = 4), och S. mediterranea-huvuden (n = 6). Pilar indikerar hjärnmorfologier, och streckade linjer indikerade mätningar som används för beräkning av längd/breddförhållande. Skala 0,5 mm; ( G ) Genomsnittligt förhållande mellan hjärnlängd och bredd hos vildtyp och 8-OH-behandlade maskar (ANOVA p < 4,9 × 10 −14 ). Felstaplar är standardavvikelser. Därefter frågade vi om inre strukturer på samma sätt omvandlades till en annan form, liksom yttre morfologi. Få aspekter av den planariska inre anatomin skiljer sig märkbart mellan arter; hjärnstorlek och hjärnform erbjuder dock ett intressant undantag från denna regel. Hjärnorna hos vildtyp G. dorotocephala är långsträckta och smala, medan D. japonica och S. mediterranea har märkbart kortare och bredare hjärnmorfologier (figur 4 A–C). Ingen levande vildtyp P. felina kunde erhållas för detta arbete, och den låga frekvensen av pseudo-PF förekomst begränsade antalet tillgängliga för analys. Därför fokuserade vi på G. dorotocephala, D. japonica och S. mediterranea. Vi utförde immunotaining med hjälp av en antisynapsin antikropp, för att visualisera både hjärnan, och ventrala nervsträngar av pseudo och vildtyp maskar. Som rekapitulation av vildtyp huvudform efter tio dagars regenerering i vatten hade bekräftats av geometrisk morfometri, valde vi att jämföra pseudomorfologier till vuxna maskar av andra arter, för att minimera förvirrande data på grund av variabilitet i regenereringstiden mellan arter. Totala formskillnader fångades upp genom mätning av hjärnlängd/bredd-förhållandet. Dessa beräkningar användes för att kvantifiera formskillnader mellan arter och för att kvantifiera hjärnans remodellering i ”pseudo”-maskar (figur 4 G). Vi fann att pseudomaskar hade hjärnmorfologier som liknar hjärnmorfologierna hos maskar av vildtyp vars huvudformer de liknade (figur 4 D–F) (ANOVA, p < 0,001). Vi drar slutsatsen att de mönstrande processer som störs efter gapkorsningskommunikations perturbation också är ansvariga för att producera hjärnans morfologi, och att de förändrade formerna inte är begränsade till den övergripande huvudgeometrin utan inkluderar mönstrandet av det centrala nervsystemet inom. Figur 3. Procentuell andel av huvudformens utfall korrelerar med evolutionärt avstånd. ( A ) Evolutionärt träd, konstruerat utifrån rRNA-data, som visar samband mellan arter av intresse. Artnamn i rött är de som analyserades i detta arbete; ( B ) Frekvens av huvudformer som erhållits i oktanolexponeringsexperimenten (n > 243). Underlåtenhet att regenerera definieras som förlust av främre-bakre polaritet, och underlåtenhet att regenerera något huvud alls efter oktanolbehandling. Felstaplar är standardavvikelser. Figur 3. Procentuell andel av huvudformens utfall korrelerar med evolutionärt avstånd. ( A ) Evolutionärt träd, konstruerat utifrån rRNA-data, som visar samband mellan arter av intresse. Artnamn i rött är de som analyserades i detta arbete; ( B ) Frekvens av huvudformer som erhållits i oktanolexponeringsexperimenten (n > 243). Underlåtenhet att regenerera definieras som förlust av främre-bakre polaritet, och underlåtenhet att regenerera något huvud alls efter oktanolbehandling. Felstaplar är standardavvikelser. Att få fram distinkta regenererade morfologier, vid olika frekvenser, trots samma behandlingsbetingelser, fick oss att utforska det evolutionära förhållandet mellan de fyra representerade arterna. Vi kartlade ett evolutionärt träd baserat på rRNA-homologi [ 75 , 76 ], och jämförde detta träd (figur 3 A) med frekvenser hos de olika arternas huvuden som uppstod vid 8-OH-behandling (figur 3 B). Intressant nog förekommer morfologier som motsvarar arter som är närmast släkt med G. dorotocephala (SM och DJ, som avlägsnas från GDs med minst 100 miljoner års evolutionärt avstånd) med mycket högre frekvens än morfologier som motsvarar mindre besläktade arter (PF). Vi drar slutsatsen att inte bara är fenotypiska utfall från fysiologiska nätverk perturbation stochastic (som samma behandling leder till en av flera diskreta former bland individer), men frekvenserna är inte lika och korrelerar ungefär med evolutionärt avstånd mellan maskarterna dessa huvuden liknar. Figur 2. Canonical variate analys av huvudets form. ( A ) Grafisk utmatning, som visar konfidensellipser för medel, med en 0,9 sannolikhet, av formdata från vildtyp och experimentellt härledda morfologier. Ellipser är färgade för att motsvara fenotyp och behandling. n = 10 WT G. dorotocephala , n = 8 WT G. dorotocephala 10 dagar efter amputation och regenererad i vatten, n = 9 WT D. japonica , n = 6 WT P. felina , n = 8 WT S. mediterranea , n = 7 pseudo G. dorotocephala , n = 13 pseudo D. japonica , n = 5 pseudo P. felina , och n = 6 S. mediterranea plattmaskar mättes; ( B ) Legend om landmärke placering på en vild typ G. dorotocephal huvudform (se Material och metoder); ( C ) Schematisk demonstration av förändring av morfologi för att bättre likna en annan art efter 8-OH behandlingar. Procrustes avstånd mellan vildtyp G. dorotocephala , 8-OH behandlad G. dorotocephala med D. japonica huvudform, och vildtyp D. japonica visar objektiv förändring av morfologin att vara mer lik de icke-infödda arterna. Figur 2. Canonical variate analys av huvudets form. ( A ) Grafisk utmatning, som visar konfidensellipser för medel, med en 0,9 sannolikhet, av formdata från vildtyp och experimentellt härledda morfologier. Ellipser är färgade för att motsvara fenotyp och behandling. n = 10 WT G. dorotocephala , n = 8 WT G. dorotocephala 10 dagar efter amputation och regenererad i vatten, n = 9 WT D. japonica , n = 6 WT P. felina , n = 8 WT S. mediterranea , n = 7 pseudo G. dorotocephala , n = 13 pseudo D. japonica , n = 5 pseudo P. felina , och n = 6 S. mediterranea plattmaskar mättes; ( B ) Legend om landmärke placering på en vild typ G. dorotocephal huvudform (se Material och metoder); ( C ) Schematisk demonstration av förändring av morfologi för att bättre likna en annan art efter 8-OH behandlingar. Procrustes avstånd mellan vildtyp G. dorotocephala , 8-OH behandlad G. dorotocephala med D. japonica huvudform, och vildtyp D. japonica visar objektiv förändring av morfologin att vara mer lik de icke-infödda arterna. Kvantifieringen användes för att avgöra om de former som såg ut som andra arter objektivt liknade dessa arter, och för att antyda en kontinuerlig morforymd inom vilken oktanolinducerad formförändring kan visualiseras längs ett kontinuum (från normalt till normalt hos en annan art). Canonical variate analys stödde den statistiska signifikansen av de givna fördefinierade morfologiska grupperingarna (i detta fall baserades grupperingar på experimentell behandling och morfologi) (figur 2 A). Jämförelser av Procrustes avstånd mellan formgrupper ( figur 2 A, tabell 1 ) visade att de experimentellt härledda morfologierna var närmare till formen de morfologier av vildtyp som de liknade än den vilda typen Girardia dorotocephala huvudmorfologi. Analys av varians (ANOVA) av både centroidstorlek och form mellan vildtypsmorfologi och pseudomorfologigrupper bekräftade också signifikanta skillnader mellan grupperingar (F = 7,94, p < 0,0001, respektive F = 7,40, p < 0,0001). Vi drar slutsatsen att amputation och behandling i 8-OH kan producera regenererade maskar vars morfologi har förändrats till att bli betydligt mer lik den hos en annan art av planarier. Geometrisk morfometri [ 73 ]användes för att kvantifiera likheter och olikheter mellan hufvudformer af sanna arter, samt de experimentellt härledda pseudomorfologierna. I korthet innebär geometrisk morfometrisk analys placering av en rad landmärken, som är både biologiskt signifikanta och reproducerbara över alla prover, avlägsnande av icke-formsvariation (storlek, rotation, etc.). ), och utförande av en uppsättning statistiska analyser [ 74 ]. Landmärken valdes utifrån de gemensamma biologiska landmärken som fanns över prover, och halvmarkmärken placerades med föreskrivna relationer till dessa landmärken (figur 2 B). Markdata registrerades för n > 60 maskar, inklusive GD-maskar vars huvudform hade experimenterats med att störas av 8-OH, kontrollera GD-maskar som hade regenererat i vatten i 10 dagar, och vuxna maskar av vildtyp från var och en av de tre arterna. Analyser av huvudkomponenter (uppgifter saknas) och analys av kanoniska variater utfördes på datamängden. Detta möjliggjorde visualisering av medelformsförändringar mellan vilda artmorfologier, och mellan experimentellt härledda huvudformer. Båda analyserna resulterade i att pseudomorfologierna separerades från den vilda typen G. dorotocephala-morfologin. Prokrustes avstånd mellan var och en av grupperna beräknades, i syfte att producera ett kvantifierat metriska för jämförelse av formskillnader. Efter 3 dagars exponering för oktanol tilläts maskarna att slutföra regenereringen (10 dagar totalt). Anmärkningsvärt är att denna process resulterade i maskar som uppvisade huvudformer som i hög grad liknade de huvuden som var lämpliga för andra arter av planarianer (figur 1 E–H). 8-OH behandlade fragment återbildade både huvud och svans korrekt, men i många fall förändrades huvudets form drastiskt. Vildtyp G. dorotocephala (GD) har en mycket spetsig huvudform, med två långsträckta auriklar vid ögonplanet (figur 1 A). Fragment som omfattas av samma 8-OH behandlingsschema regenererade ett av: helt rundade huvuden som planarianen S. mediterranea (SM) (figur 1 H), huvuden med tjock hals och ”kattliknande” auriklar som de hos planarianen P. felina (PF) (figur 1 G), huvuden som är triangulära som planarian D. japonica (DJ) (figur 1 F), eller huvuden som liknar vildtyp G. dorotocephala (figur 1 E). Vi kommer att hänvisa till huvudet former av regenererar som "pseudo" av de morfologiskt mest liknande arter. Variant, andra artspecifika huvudmorfologier observerades aldrig efter amputation eller under regenerering i vatten (kontrolltillståndet) – den normala processen för huvudförnyelse har 100% trohet till den artspecifika formen (figur S1 A). Samma schema för exponering för hexanol (6-OH), en närbesläktad förening mot oktanol som inte effektivt blockerar GJs och därmed kan användas som kontroll [ 69 ], hade ingen effekt (figur S1 B). På samma sätt uppvisade intakta maskar som blötlagts i 8-OH i > 3 dagar inga ovanliga morfologiska resultat. Baserat på dessa resultat, drar vi slutsatsen att diskreta, artspecifika huvudformer kan uppnås genom att manipulera anslutning av fysiologiska nätverk i den planariska plattmasken under huvudförnyelse. Figur 1. Karakterisering av varierande huvudmorfologier som framställs genom oktanolbehandling. ( A – D ) Vildtypsmorfologier av fyra arter av planariaplattmask. Pilarna anger auricle placering och allmän huvudform; ( E – H ) pre-tail (PT) fragment av G. dorotocephala , behandlas i 8-OH i tre dagar, och sedan flyttas i vatten för återstoden av regenerering ( n > 243). Pilar indikerar auricle placering och allmän huvudform. Skalstång 0,5 mm; ( I ) Experimentellt schema för oktanolbehandling. PT-fragment amputeras från G. dorotocephala-maskar. Fragment behandlas i oktanol (8-OH) i tre dagar, och får regenerera i vatten i sju dagar. Figur 1. Karakterisering av varierande huvudmorfologier som framställs genom oktanolbehandling. ( A – D ) Vildtypsmorfologier av fyra arter av planariaplattmask. Pilarna anger auricle placering och allmän huvudform; ( E – H ) pre-tail (PT) fragment av G. dorotocephala , behandlas i 8-OH i tre dagar, och sedan flyttas i vatten för återstoden av regenerering ( n > 243). Pilar indikerar auricle placering och allmän huvudform. Skalstång 0,5 mm; ( I ) Experimentellt schema för oktanolbehandling. PT-fragment amputeras från G. dorotocephala-maskar. Fragment behandlas i oktanol (8-OH) i tre dagar, och får regenerera i vatten i sju dagar. Planariska plattmaskarter uppvisar ett brett spektrum av huvudformer, från de mycket rundade till de nästan trekantiga, med varierande former av auriklar (figur 1 A–D). För att förhöra de mekanismer som var ansvariga för regenerering och underhåll av huvudets form amputerades G. dorotocephala-planarianer längs ett plan placerat posteriort mot svalget men främre mot svansen, för att producera ett fragment före svansen (PT). Därefter behandlades PT-fragment med gapkorsningen (GJ) kommunikationsblockeraren oktanol (8-OH). 8-OH är en allmänt använd pan-GJ-blockerare [ 20 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 ], som förändrar den fysiologiska förbindelsen mellan populationer av celler, och därmed stör hastigheten och mönstret för överföring av bioelektriska och andra små molekylsignaler. Doseringen titrerades till en nivå som var tillräckligt låg för att möjliggöra interferens med regenerativ signalering utan organismisk toxicitet. 8-OH-exponeringen har validerats för att vara övergående med GC-MS: läkemedelsnivåerna är ej detekterbara efter några timmars maskwash-out i vatten, och oktanol förändrar inte genetiska sekvenser i masken [ 20 , 72 ]. 3. Diskussion 3.1. Perturbation av bioelektriska nätverk Producerar inter-species Morfologisk förändring Våra data visar att i planarian plattmask G. dorotocephala, resultat av morphogenes kan modifieras av perturbation av fysiologiska nätverk. Kort exponering för en väl karakteriserad störare av elektriska synapser (gap knutpunkter blocker) resulterade i regenererade huvudformer som kvantitativt liknade de av en annan planariska arter. Vi observerade huvudformer (och inre strukturer) som liknar D. japonica, S. mediterranea och P. felina. Notera att andra arter existerar med huvudformer som liknar varje (till exempel är 2 andra arter kända som uppvisar P. felina-liknande tillplattade huvuden); eftersom dessa tre typer är bra representanter för varje formklass och finns som levande exemplar för analys (till skillnad från andra medlemmar av samma morfologiska huvudtyper), hänvisar vi till våra fenotyper som pseudojaponica, pseudomediterranea, respektive pseudofelina. Framtida analys av andra planariska arter kommer att avslöja om dessa fenotyper i själva verket påminner närmare om en av de andra medlemmarna i varje formklass. Huvudets morfologiförändringseffekt var lärorikt, och inte bara en av toxicitet eller en hushållningsdefekt, eftersom resultatet inte bara var en störning av regenerativ förmåga eller en generaliserad felpattering (t.ex. tumör eller annan oorganiserad tillväxt), utan snarare resulterade i en av flera sammanhängande (och ekologiskt giltiga) morfologier. Inga mellanliggande morfologier observerades, vilket framhäver den diskreta karaktären hos de komplexa mönstrande resultaten efter exponering för GJ-blockerare. Samma 3-dagars behandling med den närbesläktade alkoholen, hexanol (6-OH), som inte effektivt blockerar gapkopplingskanaler, och efterföljande regenerering i vatten under 7 dagar, gav inte förändrade morfologier, som överensstämmer med betydelsen av GJ-blockerande aktivitet av oktanol, och inte någon annan generaliserad effekt av långkedjiga alkoholer. Dramatiskt olika morfologier kan härledas från samma genom [103,104,105]. Muler och hinnies, som endast skiljer sig åt i könet hos respektive åsne- och hästföräldrar, är ganska morfologiskt distinkta, trots nästan identiska DNA-sekvenser. Toadflax (Linaria vulgaris) har en morfologisk variant som enbart framställs genom variationer i DNA-metyleringsmönster, och talrika kräldjursarter kommer att utveckla morfologiskt distinkta könsorgan på ett temperaturberoende sätt [106]. Dessutom visade färska uppgifter hur förändringar i biokemisk signalering kan väljas bland olika artspecifika morfologier [103,107]. Här identifierar vi en ny fysiologisk ingång i processen genom vilken distinkta morfologier kan härledas från samma genom: fysiologiska bioelektriska nätverk. Kompatibiliteten mellan flera morfologiska resultat med en enda genomisk sekvens ligger till grund för vikten av att lära sig förstå och styra den lärorika mönstrande information som genereras av kemiska, fysikaliska och bioelektriska processer bortom hårdkopplade genreglerande nätverk. 3.2. Geometriska Morfometri Kvantifierar Planarian Shape Change Geometriska Morphometrics är en teknik som vanligen används för att kvantifiera och jämföra biologiska former. Vi använde oss av landmärkesbaserad analys, som innebär registrering av landmärkesdata, omvandling av resulterande former för att ta bort skalning och rotationsbuller, samt utförande av statistiska analyser. Canonical variate analysis (CVA) användes för att jämföra morfologier av vildtyp av fyra arter av mask med motsvarande pseudomorfologier, samt jämföra vildtyp G. dorotocephala morfologi med alla pseudomorfologier. CVA bestämmer den statistiska signifikansen av variation mellan givna grupper av prover [74,108]. I detta fall motsvarade grupperingarna observerad fenotyp och experimentell behandling. Vi valde att använda Procrustes avstånd för att kvantifiera “avstånd” mellan former, eftersom det är ett mått på kvadratroten av de summerade kvadratavstånden mellan motsvarande landmärken i två former, och används ofta i morfometriska studier som jämför olika morfologier. Procrustes-avstånden mellan pseudomorfologierna och de arter som de liknade är mindre än Procrustes-avstånden mellan pseudomorfologierna och vildtyp G. dorotocephala. Observationen kompletterades med både Procrustes ANOVA av centroidstorlek och form (p < 0,001 respektive p < 0,001). Detta visar på en betydande förändring av morfologin, och bekräftar kvantitativt den skenbara likheten mellan pseudofenotyperna och deras motsvarigheter av vildtyp. Intressant nog varierar centroiderna i grupperna för vildtyp S. mediterranea, D. japonica och G. dorotocephala morfologi (de tre mest närbesläktade arterna) alla längs en linjär axel genom CV-utrymmet. Detta avslöjar de morfologiska konsekvenserna av variation längs en kanonisk variat. Att gå från negativa till positiva värden av kanonisk variat korrelerar med förlängningen av auriklarna, om de finns (med SM:er som befinner sig i den negativa änden av spektrumet, och GD:er och PF:er som befinner sig i den positiva änden). Att gå från negativa till positiva värden av kanonisk variate två korrelerar med rörelse av auricles, om de finns, från en mycket främre position, till en mer posterior en (med PFs finnas i den negativa änden av spektrumet, och GDs finnas i den negativa änden). 3.3. Morfologisk förändring är inte bara huddjup Viktigt, de mönstrande förändringar som orsakas av elektrisk synaps blockad påverkar formen på hjärnan (figur 4), inte bara den övergripande huvudformen. Maskar med pseudohuvuden som liknar en given art besitter hjärnor som liknar samma art, och avslöjar att funktionerna i denna regleringsväg sträcker sig bortom att ställa in huvudets yttre kontur men istället styr hjärnans form inom. Dessa data tyder också på en koppling av hjärnans morfologi till huvudets form. Oavsett om hjärnans morfologi driver huvudets form, eller om båda är parallella konsekvenser av samma GJC-beroende uppströms-kontrollmekanism, visar detta fynd djupet av omorganiseringen av kroppsplanen efter perturbation av GJC-anslutning. Koppling eller inblandning av det centrala nervsystemet i morfogenesen skulle inte vara helt förvånande, med tanke på tidigare arbete som visar att det är nödvändigt med långväga neurala ingångar för korrekt polaritetsmönsterering under regenerering i planaria [20] och ryggradsdjur [109,110]. Dessutom omorganiserar perturbation av gapkopplingskommunikationen fördelningen av planariska vuxna stamceller (neoblaster) till rumsliga mönster som är lämpliga för andra arter (figur 5). Tillsammans med förändringen i hjärnformen är det tydligt att den morfologiska förändringen till pseudomorfologier hos andra arter är grundlig, vilket inte bara innebär yttre huvudform utan även inre organskalning och positionering av nyckelcellsgrupper. Dessa data avslöjar kopplingen mellan proliferativa stamceller och storskalig morfologi, dess variabilitet genom hela fylogenin och dess kontroll genom GJ-beroende signalering. Det är möjligt att kontroll av neoblastdistribution är det som driver storskalig morfologi. Omvänt kan deras rumsliga fördelning vara en av flera nedströmseffekter av dynamiken i ett GJC-medierat cellulärt nätverk. 3.4. Bioelektriskt tillstånd hos vävnader och dess stabila förändring i artspecifik formförändring Våra resultat inkluderar den första analysen av effekterna av GJ-modulering på den långsiktiga bioelektriska profilen hos mönstrande vävnader in vivo. Även om storskaliga bioelektriska gradienter har visat sig funktionellt instruera mönsterbildning i ett antal modeller [12], inklusive bestämning av morphollaxis/skalning i S. mediterranea [19] och främre-bakre polaritet i D. japonica [19], har inga studier ännu undersökt mönster för V-memsfördelning i samband med förändringar i vävnaders elektriska anslutning. Vi fann att kort exponering för en GJ-blockerare, som är känd för att tvättas ut från maskvävnader inom 24 timmar [20], resulterar i en större grad av bioelektrisk isolering (förmåga att upprätthålla många mindre regioner av isopotentiella V mem ) så sent som 10 dagar efter exponering (figur 6). Även om dessa långvariga förändringar i bioelektrisk mönstrande inte direkt orsakas av oktanol som finns kvar i maskens kropp (eftersom 8-OH är känt för att avgå planariska vävnader inom 24 timmar [20], inleds de av den oktanolmedierade störningen av GJC, som förändrar bioelektrisk nätverkstopologi, och resulterar i en ihållande perturbation av bioelektriskt tillstånd. I detta liknar effekten de kända egenskaperna hos elektriska synapser (GJs) i nervsystemet, som hjälper till att genomföra ett slags hebbiansk plasticitet som stabiliserar den joniska konduktiviteten bland celler efter övergående elektrisk modulering [44,111,112.113,114]. Kvantitativa fysiologiska modeller av bioelektrisk dynamik kommer att behöva utvecklas för att förstå bioelektriskt tillståndsminne [115] och avgöra varför olika typer av maskar har så distinkta profiler av regioner av isopotential V mem . Variabiliteten och den totala omfattningen av effekten tyder på att resultatet av 8-OH-exponeringen endast delvis är effektivt när det gäller att stänga av GJC (som påverkar en viss procentandel av GJs hos något djur); detta är förenligt med morfologiska fenotypens stokasticitet (figur 3). Medan 8-OH är en vanlig metod för att blockera GJC (och i D. japonica, ger samma effekt som RNAi som innexins-gener [20]), kan vi inte slutgiltigt utesluta att ytterligare mål förutom GJs också blir perturberade antingen direkt eller sekundärt efter 8-OH exponering. En svårighet med ett molekylärt (genspecifikt) förhållningssätt till GJ-avstängning, är att inte bara skulle ~13 innexiner behöva klonas ut ur GD-genomet (ingen har ännu karakteriserats), utan deras RNAi-målsökning skulle behöva testas kombinatorialt. Utrymmet för alla möjliga kombinationer av olika innexin-RNAi som skulle behöva provas samtidigt är enormt, eftersom GJ-proteiner ofta kan kompensera för varandra. Trots detta testade vårt tidigare arbete några sådana kombinationer (i D. japonica [20]), och visade att en samtidig nedslagning av tre Innexins av RNAi återger 8-OH fenotypen, vilket stärker slutsatsen att fenotyper verkligen är resultatet av 8-OH:s effekt på gapkorsningar. En nyligen genomförd studie [20] som visade att övergående glappskopplingsförändringar framkallar permanenta förändringar i plana målmorfologin väckte frågan om detta mönsterminne fastställdes genom förändringar av GJC som kvarstår långt efter det blockerande reagenset lämnar vävnaderna, eller av GJC-stater som ändrar biokemiska eller transkriptionella celltillstånd som är stabila efter att GJC återgår till det normala. Även om vi inte kan utesluta ytterligare epigenetiska förändringar som kan inträffa, visar våra data att även 10 dagar senare, effekterna av kort 8-OH exponering bevaras som minskad elektrisk anslutning i de somatiska vävnaderna. GJC-nätverkens förmåga att stabilisera inducerade förändringar i kopplingen överensstämmer med GJs kända roller i minnet och synapsplasticitet [44.52.114.116.117.118]. Vår analys avslöjade inte en enkel överensstämmelse mellan den specifika fördelningen av spänningsgradienter och morfologiska resultat av pseudomaskens regenerering, vilket kunde ha flera förklaringar. För det första är det möjligt att inte spänning utan en annan molekylär signal är det som genomkorsar GJ-nätet för att bestämma plana huvudform. GJ-permeenta molekyler med signalerande roll är bland annat serotonin [119,120] och andra signalsubstanser [121], histamin [122], kalcium [123], med flera, som kommer att testas i framtida studier, särskilt som fluorescerande sonder för några av dessa fysiologiska signaler kommer på nätet [124,125,126]. En annan möjlighet är att varje morfologiskt tillstånd uppnås genom flera möjliga fördelningar av spänningen; i detta fall skulle den skenbara variabiliteten dölja vår förmåga att upptäcka en specifik kartläggning från distribution till huvudmönster utfall. Den troligaste förklaringen är dock att formutfall inte får kodas via stadiga (enkelbilds)distributioner utan i tidsberoende aktivitet, vilket sker under informationskodning i centrala nervsystemet. I detta fall, filmer av spänningsförändring över tid in vivo måste förvärvas och analyseras-en betydande utmaning i planaria modellsystemet. Vår analys av isopotentiella regioner är bara ett första försök att bryta informationen kodad i bioelektriska tillstånd. Sammantaget tror vi att det är mycket troligt att en mycket mer sofistikerad analysmetod måste utvecklas, kanske i likhet med vad som används för att härleda innehållet i neurala nätverk från hjärnskanningar [80, 127], och tillämpas på ett stort urval av maskavbildningsdata, för att verkligen knäcka den bioelektriska koden i detta system. Funktionella studier i planaria och Xenopus [19,25,128] har visat relevansen av bioelektriska gradienter för främre mönstrande i allmänhet, men nya beräkningsmetoder kommer att behöva vidtas för att visa vilket spatiotemporellt mönster av bioelektrisk aktivitet som krävs för vart och ett av de mönstrande resultat vi observerade. Att avslöja denna kartläggning kommer att stå i fokus för våra insatser för kommande studier. 3.5. Morfologisk remodeling i 2 faser: Huvudform Förändring utan Traumatisk skada De pseudomorfologier vi observerade är inte permanenta: de remodeformerar, efter att den första regenereringsprocessen är klar, för att återigen likna G. dorotocephala. I detta skiljer de sig från den permanenta dubbelhövdade fenotyp som induceras hos D. japonica, vilket representerar en stabil förändring av maskarnas målmorfologi efter fysiologisk perturbation [20,129]. Hos G. dorotocephala sker reparationen således i två faser: huvudregenerering efter amputation, som fullbordas inom den normala tidsramen (< 10 dagar) och resulterar i olika arters former, och en efterföljande mycket längre remodellering tillbaka till den genomspecifika G. dorotocephala-huvudformen. Denna senare process sker i avsaknad av skada, inom ett komplett huvud, under flera veckor, illustrerar förmågan hos cellulära nätverk att upptäcka avvikelser från korrekt form, även i avsaknad av trauma eller sår, och att effekt remodeling. Det är intressant att lägga märke till att samma bifasiska process förekommer hos ryggradsdjur: när ett salamanderstjärtsblastema transplanteras på en flank bildas först en svans, men senare omgestaltas den till en lem i en mycket långsammare process (130.131). Morfologin verkar vara en homeostatisk parameter, en parameter som bedöms och justeras konsekvent för att upprätthålla någon ”mål” morfologi. Morfologi måste först beslutas inom dagar efter amputation, eftersom det är möjligt att observera huvudform redan dag sex efter amputation. Hos 8-OH-behandlade maskar producerar denna mekanism felaktiga, pseudomorfologier. Dock måste morfologin sedan omprövas igen efter detta första beslut. När den aktuella morfologin inte stämmer överens med målmorfologin inleds ombyggnad någon gång mellan dag 10 och dag 17. Aktiviteten i dessa omdaningsprocesser efter regenereringen har slutförts är fascinerande, och innebär att morfologin konsekvent omvärderas och redigeras, även efter att storskaliga omorganiseringshändelser har avslutats. Naturen hos de processer som driver cellbeteendet mot ett specifikt, stabilt sluttillstånd (när ombyggnaden upphör) är nästan helt dunkel. Vid denna tid är det inte möjligt i något modellsystem att härleda specifik forminformation (att veta exakt vilket anatomiskt mönster som kommer att vara ett tillräckligt slutmålstillstånd för att få remodeling att upphöra) från genomiska data. 3.6. Beräkningsmodellering av huvudets formering De olika huvudmorfologier vi observerade, som är distinkta i yttre anatomi, distribution av stamcellerna, och hjärnans form, måste vara resultatet av differentialcellmigration och cell-cellkommunikation. För att länka cellnivåbeteenden till de diskreta storskaliga morfologierna och utforska konsekvenserna av minskad GJ-kommunikation (framkallad av oktanolexponering) på emergent cellsignalering och morpogenes analyserade vi en agentbaserad modell. I denna modell (figur S2), några celler migrerar aktivt, och ändra formen på de resulterande vävnaderna. Deras rörelser styrs av en kemisk gradient som bildas av andra statiska celler i huvudet, vilket aktiveringsmönster etableras genom GJC, vilket man vet förekommer i masken [20,70] och i många andra organismer [51,132.133]. Vår modell visar de morfogenetiska konsekvenserna av avbrott i denna kommunikation, som parallellerar samspelet mellan signalering av plana neoblaster (stamceller) och omgivande somatiska vävnader. I denna modell föreslår vi att signifikant instruktiv (GJ-beroende) signalering sker från de somatiska vävnaderna till neoblasterna eller deras avkomma, för att vägleda den nya vävnadsgenerationen och forma under regenerering. Beräkningssimuleringarna visade hur ett litet antal cellkonfigurationer kunde resultera i huvudformer som ganska nära motsvarar de observerade morfologierna (figur 9 och tabell 2). Intressant nog, när specifika statiska celler (svarta) förlorar förmågan att skicka sin signal på grund av GJC blockering av oktanol, ändras de flyttande cellernas rörelser (röda) vilket ger upphov till ett av de 4 utfallen. Modellen uppvisar således de två huvudsakliga egenskaperna hos den experimentella datauppsättningen: flera möjliga utfall (på grund av olika mönster av GJC-blockerade statiska celler, eventuellt från fysiologiskt brus i farmakologisk läkemedels-målinteraktion), och diskreta specifika utfall (som resultat av deterministisk signalering av statiska "svarta" celler och därav följande beteende av flyttande "röda" celler). Det bör noteras att vår modell också lätt tillämpas på ett organ som växer från en initial cellposition till dess slutposition: om vi tillåter delning av celler B, fortsätter cellmigrationen på samma sätt som visas: de kommer att lämna bakom sig avaktiverade (differentierade) celler B som inte rör sig och inte delar sig. I det framtida arbetet kommer man att testa denna modellklasss tillämplighet på andra regenereringssystem. Figur 9. En konceptuell modell av formförändring driven av fysiologisk nätverksdynamik. Planaria regenerering paralleller klassiskt neuralt nätverk beteende; båda kan beskrivas i termer av fri energi landskap med flera attraherande tillstånd. (A) Beteende av ett klassiskt Hopfield neurala nätverk tränas att reproducera tre typer av mönster, i detta fall former av bokstäverna “F”, “H” eller “G”, som är de tre stabila tillstånden i nätverkets fria energilandskap. Tillståndet i Hopfield nätverkets noder direkt relatera till ljusstyrkan hos pixlar på en display, generera utdata. Perturbation av nätverket från ett stabilt tillstånd (röd pil) genom att ta bort (skadlig) del av mönstret är liktydigt med att flytta en boll till en instabil punkt på det fria energilandskapet, och leder till regenerering av det närmaste lärda attraktionstillståndet (blå pil). I detta, är sådana nätverk välkända förmåga att genomföra minnet analogt med regenererande organismer återställa ett specifikt mål morfologi vid skador; (B) Parallellbegreppet planaria regenerering till huvudformer av en av tre olika arter, som är attraherande tillstånd av det fria energilandskapet. Resultatmorfologin drivs av de dynamiska utgångarna i fysiologiska cellulära nätverk. Amputation (röd pil) liknar att flytta systemet till en instabil punkt på det fria energilandskapet. Normal regenerering skulle återföra systemet till dess huvudattraktion, men att förändra cellnätsdynamiken via gapkorsningsblockad möjliggör regenerativa övergångar (blå pil) till alternativa lokala minima, motsvarande morforymdsregioner som normalt upptas av P. felina och S. mediterranea maskar. Med tiden, remodeling (grön pil) överför dessa morfologier till det globala minimum av vildtyp tillstånd ( G. dorotocephala ). Figur 9. En konceptuell modell av formförändring driven av fysiologisk nätverksdynamik. Planaria regenerering paralleller klassiskt neuralt nätverk beteende; båda kan beskrivas i termer av fri energi landskap med flera attraherande tillstånd. (A) Beteende av ett klassiskt Hopfield neurala nätverk tränas att reproducera tre typer av mönster, i detta fall former av bokstäverna “F”, “H” eller “G”, som är de tre stabila tillstånden i nätverkets fria energilandskap. Tillståndet i Hopfield nätverkets noder direkt relatera till ljusstyrkan hos pixlar på en display, generera utdata. Perturbation av nätverket från ett stabilt tillstånd (röd pil) genom att ta bort (skadlig) del av mönstret är liktydigt med att flytta en boll till en instabil punkt på det fria energilandskapet, och leder till regenerering av det närmaste lärda attraktionstillståndet (blå pil). I detta, är sådana nätverk välkända förmåga att genomföra minnet analogt med regenererande organismer återställa ett specifikt mål morfologi vid skador; (B) Parallellbegreppet planaria regenerering till huvudformer av en av tre olika arter, som är attraherande tillstånd av det fria energilandskapet. Resultatmorfologin drivs av de dynamiska utgångarna i fysiologiska cellulära nätverk. Amputation (röd pil) liknar att flytta systemet till en instabil punkt på det fria energilandskapet. Normal regenerering skulle återföra systemet till dess huvudattraktion, men att förändra cellnätsdynamiken via gapkorsningsblockad möjliggör regenerativa övergångar (blå pil) till alternativa lokala minima, motsvarande morforymdsregioner som normalt upptas av P. felina och S. mediterranea maskar. Med tiden, remodeling (grön pil) överför dessa morfologier till det globala minimum av vildtyp tillstånd ( G. dorotocephala ). Vår agentbaserade modell omfattar fysikaliska parametrar hos celler [95,134], liksom traditionell kemisk gradientdiffusion [135,136]. I modellen modelleras oktanolstörning av cell:cellkommunikation genom deaktivering av signalgivning från en specifik celltyp; en kandidat för denna unika cellpopulation är de sällsynta men vitt spridda celler som uttrycker gapkorsningsproteinet Innexin 5 [71]. Framtida experiment kommer att testa de specifika förutsägelserna av denna modell och tilldela molekylär-genetiska identiteter av de flera olika celltyper som postuleras i modellen. Ytterligare en viktig undersökningslinje är att mekaniskt koppla storskalig dynamik av fysiologisk signalgivning till det specifika beteendet hos celler som modelleras i våra simuleringar. 3.7. En hypotes: Fysiologiska nätverk Attractors Underlie Regioner av anatomiska Morphospace Utfallet av regenerering i denna modell är en uppsättning sammanhängande, diskret huvudformer-inte en förvirrande amalgam av olika mönster som skulle förväntas om celler gjorde rent lokala beslut och var bara dis-coordinated av oktanol behandling. För närvarande saknar området i stort sett konceptuella modeller för att tänka på denna typ av input till utveckling och förnyelse; det är inte klart hur man bäst ska representera eller modellera diskreta, men ändå stokastiskt valda, framväxande storskaliga mönster. Ändå är detta ett allt viktigare mål då framsteg inom regenerativ medicin och syntetisk bioteknik kräver förmågan att förutsäga och kontrollera globala resultat genom att justera cellnivåparametrar. Området utvecklingsbioelektricitet söker fortfarande efter en lämplig formalism, men en uppenbar kandidat är det av det neurala nätverksparadigmet som används av beräkningsneuroforskare för att förstå emergent dynamik av beslutsfattande i hjärnor som består av elektriskt kopplade celler. Vi har tidigare hävdat att eftersom somatiska celler har många av samma komponenter som nerver (jonkanaler, signalsubstanser, GJs fungerar som elektriska synapser, genuttryck regleras av elektrisk aktivitet), kan vi med fördel använda några av begreppen från neurovetenskapen för att förstå liknande fenomen i långsammare utvecklingsnätverk, där produktionen inte är muskeldrivet beteende utan mönsterbildning [1,13,137]. Forskare inom beräkningsneurovetenskap har modellerat tillstånden för elkopplade nätverk i ett energiområde som avslöjar det landskap av trajectorier som nätverken genomför [138,139.140,141,142,143]. Sådana (neurala) nätverk kan stabiliseras hos attraherare i detta virtuella rum, och deras verksamhet kan på ett par sätt förklaras av nätverkens ansträngningar att minimera en global ”energi”-funktion [144.145.146.148.148]. Således föreslår vi att ett sätt att konceptualisera kopplingen mellan bioelektrisk konnektivitet bland maskceller (GJC) och diskreta globala beteenden som uppnås genom regenerering är att betrakta maskarnas celler som ett neuralliknande nätverk, bearbeta utvecklingsinformation långsammare men lyda samma statistiska dynamikregler som är kända för att förekomma i hjärnan (figur 9A). Det är sannolikt ingen tillfällighet att GJs är väl kända för att vara viktiga för informationsbehandling i hjärnan [54,149.150]. Vi förmodar att distinkta formresultat kan vara attraktioner i ett tillståndsutrymme som beskriver distinkta lägen i det bioelektriska nätverket av planariska kroppsceller som är sammankopplade genom gapkorsningar (elektriska synapser); i huvudsak motsvarar dessa attraktioner regioner i en morforymd som klassiskt förstås av evolutionsteorin [151]. I experimenten stör oktanol den normala dynamiken i detta nätverk, destabiliserande och knackande dess stokastiska dynamik till en av flera närliggande attraktioner, normalt tillhörande olika arter av plattmask (Figur 9B). Utveckling av en kvantitativ, beräkningsmodell av cellnätverk som har sådana lägen (med elektriska tillstånd som hindrar cellproliferation, apoptos och migration beteenden som resulterar i olika huvudformer) bildar nästa djupa utmaning i detta område. Sådana dynamiska nätverk ligger till grund för i stort sett varje komplex process som är känd i den biologiska världen, från ämnesomsättning till gruppbeteende [152.153.154]. Dessa rikt sammankopplade ickelinjära nätverk, som består av många enkla enheter, har förmågan att producera komplexa mönster i öppna och termodynamiskt instabila system, utan någon enda styrande instans [155.156]. Inom ett givet system sammanstrålar dessa nätverk till ett eller flera diskreta, stabila tillstånd [157]. Neutrala nät är bara ett exempel på komplexa dynamiska nätverk; de genomförs via elektriska signalerings- och gapkorsningar som visar diskreta attraktioner som motsvarar globala tillstånd [141.158] och stabil återanställning av storskaliga tillstånd (minne) efter perturbation [54.147]. Med tanke på att neural signaling utvecklades genom att specialisera sig på mycket mer forntida somatiska fysiologiska kommunikationsmekanismer, är det rimligt att postulera i grunden liknande nätverksdynamik för fysiologiska nätverk under mönsterbildning [137,159. I denna vy (figur 9) är de diskreta huvudmönstren resultat av bioelektrisk nätverksdynamik med den lägsta fria energin; att skära av nätet höjer den fria energin genom att avvika systemet från dess optimala morfologi (skada), och initierar den regenerativa processen – en resa tillbaka ner till en lågenergi-attraktion. Att störa det fysiologiska bioelektriska nätverket med 8-OH resulterar i att systemdynamiken flyttas från denna attraherare till en annan attraherare, och efterföljande regenerering av en förändrad morfologi. Tydligen är dessa pseudoformer endast lokala minima, eftersom pseudomaskarna efter cirka 3–4 veckor återgår till det globala minimum som finns i standarden G. dorotocephala morfologi. I denna klass av modeller, utdata av det fysiologiska nätverket består av signaler som dirigerar celler att proliferera, apoptos, eller migrera, så att huvudet morphogenes uppnås. Detaljerna i denna process på cellnivå börjar förstås, både i CNS (kontroll av genuttryck genom hjärnaktivitet [160]) och utvecklingsbioelektricitet [12,23,32]. Variationer i membranpotential mellan cellgrupper kan fungera som elektroforeerande krafter, dra små signalerande molekyler, små RNA:er eller laddade transkriptionsfaktorer till specifika regioner i den planariska kroppen, vilket ger många möjliga långtidseffekter [47,161,162,163,164]. Ytterligare mekanismer inkluderar spänningskänsliga fosfataser [165] och spänningsreglerad verkan av kromatinmodifierande enzymer [34,43,166]. Det är uppenbart att detta bara är ett sätt att tänka på dessa data, och andra är möjliga. Fördelen med detta paradigm är den rika uppsättning matematiska resultat som finns tillgängliga från studier av beräkningsneurovetenskap som kan begränsa framtida modellbyggande och hjälpa till att göra testbara förutsägelser. 3.8. Stokastiska fenotyper och Evolutionära implikationer En av de viktigaste aspekterna av dessa fenotyper är deras stokastiska natur: samma behandling utförs på en kohort av kloniska (isogena) djur resulterar i ett av flera mönster inom någon exponerad population. De frekvenser med vilka artspecifika mönster observeras motsvarar de evolutionära avstånd som uppskattas för dessa arter av planaria (figur 5). Det finns betydligt mer pseudo G. dorotocephala, D. japonica, och S. mediterranea morfologier, som motsvarar ett mindre evolutionärt avstånd från G. dorotocephala, medan P. felina pseudomorfologier fortfarande är relativt sällsynta. En möjlighet är att ett stomnät (som består av genregulatoriska element och fysiologisk gapkopplingskommunikation (GJC)—medierade signaler) är i arbete bland många planarianer, med artspecifika skillnader som justerar dess nyckeldynamik. Det evolutionära avståndet mellan de planariska typerna kan därför förväntas återspegla hur lätt det är att överföra detta nätverk till dess olika potentiella utfallstillstånd (vilket sker under GJC-hämning). Det bör noteras att vildtyp P. felina har flera ögon längs med den doso-ventrala kroppsgränsen, ett drag som våra pseudo-PF inte uppvisar. Ofullständig mimikry av den mest avlägsna arten, P. felina, överensstämmer med sambandet mellan evolutionärt avstånd och lätthet med vilken artspecifik morfologi kan rekapituleras helt under perturbed regeneration. Det bör noteras att även Phagocata gracilis och Phagocata velata planarianer har huvudmorfologier som är ganska lika P. felina, och därför även pseudo-PF. Dessa två arter besitter endast två ögon, och båda arterna är på liknande sätt evolutionärt borttagna från G. dorotocephala. Emellertid kunde inga lefvande vildtypsmaskar af dessa båda arter anskaffas för detta arbete, och derföre fokuserade vår analys på P. felina. Sammantaget representerar förmågan att generera distinkta morfologier från samma genom sannolikt en extremt flexibel mekanism som skulle kunna utnyttjas genom evolution via många kända miljö- och genetiskt kodade modulerare av gapkopplingstillstånd. En uppenbar avsaknad av ”mellanliggande” morfologier, eller huvudformer som liknar en blandning av två eller flera arter av planarier, kan vara resultatet av begränsningar som begränsar det morfologiska utrymme som finns för den regenererande planarien. Inom denna ram blir begränsningarna av fenotypiskt utrymme drivande faktorer i morpogenesen. Dessa begränsningar är sannolikt en amalgam av utvecklingsmässiga, miljömässiga och evolutionära faktorer, som tjänar till att begränsa de stabila lägen i mönstrande nätverk, och därmed de morfogenetiska möjligheter som finns för en regenererande plattmask. Ansträngningar som för närvarande pågår i vårt labb syftar till att producera mekanistiska bioelektriska modeller av enskilda celler och cellnätverk för att kvantitativt förklara de diskreta stabila lägena i sådana nätverk, den stokastiska karaktären hos varje ”kör” av ett sådant nätverk efter amputation, och de molekylära kopplingarna till nedströms cellbeteenden [94]. Biologer vid Tufts University har lyckats få en art av plattmask att odla huvuden och hjärnor som är karakteristiska för en annan art av plattmask utan att ändra genomsekvensen. Verket avslöjar fysiologiska kretsar som en ny sorts epigenetik -- information som finns utanför genomisk sekvens -- som bestämmer storskalig anatomi. Fyndet att huvudets form inte är hårdtrådad av genomet utan kan övermannas genom att man manipulerar elektriska synapser i kroppen tyder på att skillnader i arter delvis skulle kunna bestämmas av aktiviteten i bioelektriska nätverk. Upptäckten skulle kunna bidra till att förbättra förståelsen av fosterskador och förnyelse genom att avslöja en ny väg för att kontrollera komplex mönsterbildning. Det har länge varit känt att neurala nätverk utnyttjar bioelektriska synapser för att lagra och skriva om information i hjärnan. Resultaten är detaljerade i omslaget till november 2015 års upplaga av International Journal of Molecular Sciences, som publiceras online 24 nov. "Det är allmänt tänkt att sekvensen och strukturen av kromatin -- material som utgör kromosomer -- bestämmer formen på en organism, men dessa resultat visar att funktionen av fysiologiska nätverk kan åsidosätta den artspecifika standardanatomin", säger tidningens senior och motsvarande författare Michael Levin, Ph.D., som innehar Vannevar Bush-stolen i biologi och leder Center for Regenerative and Developmental Biology i School of Arts and Sciences at Tufts. "Genom att modulera cellernas konnektivitet via elektriska synapser kunde vi härleda huvudmorfologi och hjärnmönstrande som tillhörde en helt annan art än ett djur med ett normalt genom." Att veta hur form bestäms och hur man ska påverka det är viktigt eftersom biologer skulle kunna använda den kunskapen till exempel för att åtgärda fosterskador eller få nya biologiska strukturer att växa efter en skada, tillägger Levin. "Dessa fynd väcker stora frågor om hur gener och bioelektriska nätverk samverkar för att bygga komplexa kroppsstrukturer", säger han. Att arbeta med Girardia dorotocephala -- frilevande planariska plattmaskar, som har anmärkningsvärd regenerativ kapacitet -- forskare kunde framkalla utvecklingen av olika artspecifika huvudformer genom att avbryta gapkorsningar, som är proteinkanaler som gör det möjligt för celler att kommunicera med varandra genom att sända elektriska signaler fram och tillbaka. Förändringarna var mer än huddjup; de inkluderade inte bara huvudets övergripande form utan även hjärnans form och fördelningen av maskens adulta stamceller. Den lätthet med vilken en viss form kunde coaxas från en G. dorotocephala-mask var proportionell mot närheten av målmasken på den evolutionära tidslinjen. Ju närmare de båda arterna var besläktade, desto lättare var det att åstadkomma förändringen. Denna iakttagelse stärker kopplingen till evolutionär historia, vilket antyder att modulering av fysiologiska kretsar kan vara ytterligare ett verktyg som utvecklats för att förändra djurens kroppsplaner. Till skillnad från Levinlabbets tidigare arbeten, i vilka en annan art av planaria permanent kunde ändras till en tvåhövdad morfologi, var dock denna formförändring endast tillfällig. Veckor efter att planaria fullbordat regenereringen till den andra artens huvudformer började maskarna återigen omdana och återförvärvade sin ursprungliga huvudmorfologi. Ytterligare forskning behövs för att avgöra hur detta sker. Författarna presenterade också en beräkningsmodell som förklarar hur förändringar i cell-till-cell-kommunikation kan ge upphov till de olika formtyperna. Första författaren på tidningen var Tufts grundexamen Maya Emmons-Bell, en senior med biologi som huvudämne. "Vi har visat att de elektriska kopplingarna mellan cellerna ger viktig information för artspecifik mönstersättning av huvudet under regenerering i plana plattmaskar", säger hon. "Denna typ av information kommer att vara avgörande för framsteg inom regenerativ medicin, samt en bättre förståelse för evolutionsbiologi. Som grundutbildning har det varit en fantastisk upplevelse att bedriva och författa viktig forskning med ledande biologer. Den tvärvetenskapliga forskningen involverade USA- och Kanadabaserade biologer och europeiska matematiker. Vad är en sammanfattning av ovanstående artikel?
Flatworms are remarkable creatures, some of whom have almost incredible abilities for tissue renewal and growth. A particular species, Girardia dorotocephala, has been shown to take this ability a bizarre step further: Scientists at Tufts University have successfully caused it to temporarily grow heads and certain brain structures of several other flatworm species, all without altering its genetic sequence. Their findings are reported in the International Journal of Molecular Sciences. The shape of an animal's body is largely determined by its genome. But bioelectric nerve networks and their connections (synapses) have their own way of forming structures, and the careful manipulation of these can have unique properties to form in certain organisms, overshadowing the instructions of the genetic material. With this in mind, the researchers decided to modify the synapse connections in the head of their chosen flat mask to see if they could force it to produce new forms. After decapitating them, the electrical connection between the cells in its upper body was altered using a form of alcohol (octanol). Amazingly, this caused the flatworms to grow not only new tissue, but completely new heads — heads belonging to completely different species of flatworm: S. meditranea, D. japonica, and P. felina. Not only were the main forms replicated, but segments and properties of their brains were also seemingly copied, including the distribution of adult stem cells. The closer the two species were related, the easier it was for scientists to induce the changes. These changes, strangely enough, were not permanent: Within weeks genetic coding overcame the bioelectrically induced main change, and their heads gradually returned to their original form. At present, scientists are not entirely sure how this reconstruction takes place. Regardless, the fact that all this happened without changing the DNA from the original flatworm is the study's most significant discovery. This means that changes in a flatworm’s ability to “build” itself may sometimes originate from outside the instructions encoded in its genetic material. This type of externally induced change is known as epigenetics – literally means “extra growth”. Transmitting a selection of genes from two parents to their offspring is not the only way the building instructions for an organism are inherited. Along with an additional process called horizontal gene transfer used by certain microscopic organisms, changes in how genes are expressed can also be inherited. Several environmental or otherwise external factors can change how cells read genes, meaning that the physical attributes of the offspring can be different – all without changing the DNA sequence. Although it has not yet been shown whether these unusual flatworm changes can be transmitted to their offspring, they fit into the definition of epigenetics—although, as some point out, without the heritage being demonstrated, it may be somewhat controversial to apply this definition. Yet the study has consequences outside of just flatworm biology. Maya Emmons-Bell, a Tufts University biology undergraduate and first author at the paper, said in a statement: “This type of information will be crucial for progress in regenerative medicine, as well as a better understanding of evolutionary biology.” Image in text: G. dorotocephala (top left) could grow heads of other species (top row) without any changes in its genome. Centre for Regenerative and Developmental Biology, School of Arts and Sciences, Tufts University <unk> <unk> Call them Franken flatworms. Scientists have created worms with the heads and brains of other species simply by manipulating cell communication. The research is an example of how development is governed by more than genetics alone. The researchers did not alter the DNA of the flatworms in any way, but instead manipulated proteins that control conversations between cells. "It is common to believe that the sequence and structure of chromatin – the material that constitutes chromosomes – determines the shape of an organism, but these results show that the function of physiological networks can override the species-specific standard anatomy," says researcher Michael Levin, biologist at Tufts University, in a statement. [See Pictures of Franken Flatworms with different heads, Brains] The changes are temporary for the worms, whose heads begin to return to their original forms for weeks. However, researchers hope that the results will lead to treatments for birth defects and also regenerative medicine, which aims to replace or rebuild damaged tissues and organs. The researchers studied a small freshwater flatworm, Girardia dorotocephala, which is known to regenerate lost tissues. These flatworms retain a large number of cells called neoblasts, which are totipotent stem cells, meaning they can become any cell type in the body. In humans, the cells are totipotent only during the first days of embryonic development. First, the scientists cut off the head of their lab samples. To change the regenerating head of the flatworm, Levin and his colleagues interrupted the protein channels. The cells send electrical impulses through these junctions to communicate. The researchers found that they could easily push the mask to develop head and brain forms similar to those of other closely related flatworm species. Normally, G. dorotocephala sports a pointed head with two elongated, ear-like projections (called auricles) next to the eyes. After treatment, some of the worms developed their normal heads, while others grew rounded heads as in S. meditranea; heads with thick neck and pointed, cat-like "ears" as in P. felina; or triangular heads as in D. japonica. The brains seemed to follow the changed head shape, so that flatworms that re-created a head format such as D. japonica, for example, also showed brain morphologies shorter and wider than those of G. dorotocephala and more characteristic of D. japonica. The longer two species were apart on the evolutionary family tree, the harder it was to elicit this mix-and-match effect, the researchers reported online November 24 in the International Journal of Molecular Sciences. "These findings raise significant questions about how genes and bioelectric networks interact to build complex body structures," Levin said. If genes provide a blueprint for an organism's body, cells are like the construction workers required to transform the plan into a structure - and gap crossings are walkie-talkies these workers use to communicate. Discontinue communication, and you can disrupt the construction process. G. dorotocephala remained altered for only a limited time before its neoblasts took over and reconfirmed the old head and brain forms. Levin's lab, however, has previously constructed another species of flatworm to grow two heads and stay that way. Follow Stephanie Pappas on Twitter and Google+. Follow us @livescience, Facebook & Google+. Original article on Living Science. It is important to note that different species of planaria have characteristic main forms that are easily distinguished by anatomical inspection. Thus, we investigated the consequences of disrupting the body-wide gap communication network for main form in planaria. Remarkably, we observed a stochastic phenotype, in which the regenerating heads of a genomic normal G. dorotocephala flatworm acquired a main morphology suitable for other extant planar species. The effect was observed in the head, as well as in brain morphology. Unlike our recent demonstration of a permanent (stable) change in the target morphology of D. japonica [20], this effect was temporary, and the worms were remodelled back to their homeland within 30 days. The ability to stochastically select one of several discrete main forms suitable for another species, simply by changing a physiological network, suggests that quantitative models of bioelectric network modes will be an important part of the understanding of evolutionary change and the role of genomic versus physiological circles in the establishment of anatomical structure. Planaria are free-living flatworms with impressive regenerative abilities [55, 56 , 57 ]. After traumatic injuries such as amputation, planarians regenerate all missing tissues, their target morphology perfectly reproduces in a relatively short time period of about two weeks. This morphological transformation is not limited to trauma; after periods of starvation, planaries shrink allometrically, growing in the same, scaled manner after the reintroduction of food [58]. This notable example of spatial and temporal cellular organization requires the storage and exchange of morphological and physiological information across a varied and widely spread cell population. Such morphological robustness provides a unique system where one can study complex properties such as the plasticity of morphology. While recent work has made remarkable progress on the molecular details of pathways regulating the behavior of adult stem cells in planaria [59 , 60 , 61 ], there is very little information on how specific headform is controlled. The vast majority of functional literature records phenotypes of either failure to recreate or change head/tail identity; the field currently has no mechanistic understanding of how a species-specific main form is established or regulated [62], although voltage-based signalling has been shown to be involved in the main form/size of at least one species [25]. Global bioelectric network dynamics are partially regulated by gap intersections—electric synapses between cells that facilitate direct ion exchange, thus allowing cells to compare their V mem with those of their neighbors, and establish isopotential cell fields or boundaries between compartments [44 , 45 ]. Since gap crossings can be voltage sensitive [46 , 47 ] themselves, they can actually function as transistors — enabling complex information processing and feedback loops. Gap crossings also provide rich opportunities for selective street formation of small molecular signals beyond ion current [48]. Due to these features, this versatile signal element is crucial to development [49 , 50 , 51 ] and also to the plasticity of cognitive memory in the brain [52 , 53 , 54 ]. Thus, we examined here the role of gap-mediated physiological networks in regulating pattern formation. Three aspects of bioelectric signalling make them particularly relevant to the emergence of large-scale form and the role of the genome. First, bioelectric patterns specify the shape in a distributed (non-local) manner: several studies have shown that the size, shape, and identity of specific structures integrate bioelectric information from remote regions [32, 35, 36, 37, 38, 39 ], making bioelectric signaling an ideal modality to coordinate individual cell behaviors towards a specific anatomical outcome. Second, bioelectric patterns may override standard genetic/biochemical information: specific V-mem changes may prevent mesenchymal stem cell differentiation despite the presence of chemical inducers [40 , 41 ], may induce an eye to form in a tissue (e.g. mesoderma or endoderm) otherwise unable to become an eye when misexpressing a “master” eye inducer such as Pax6 [31], may induce metastatic melanoma in the absence of genetic damage [36 , 37 ], may prevent the formation of tumors in the presence of otherwise sufficient oncogenes [42 , 43 ], and may save brain damage caused by mutations in powerful neurogenesis regulators such as Notch [32]. Thus, bioelectric signalling is a good explanatory candidate in cases of epigenetic influence over pattern formation. Finally, bioelectric properties seem to directly encode outcomes at organ level, eliciting whole appendages [17 , 23 , 24] or complex eyes [31]. This ability to trigger downstream development modules, without having to specify individual cell positions (micromanage the process), makes bioelectric signals not only attractive control buttons for biomedical interventions, but also reveals how bioelectric network states can be seen as attractions instruct complex patterning outcomes. Most of the field is today focused on gene-regulating networks [8 , 9 ] and physical forces [10 , 11 ] in an effort to understand how final patterning outcomes arise and are maintained during development and renewal. Another fascinating layer of biological regulation, however, has recently been involved in the control of morfogenesis: endogenous bioelectric signalling [12, 13 , 14 ]. Spatio-temporal gradients of dormant potential among all cell types (not only excitable nerve and muscle) can regulate cell proliferation, migration, form, and apoptosis [15 , 16 ]. More importantly, they can serve as instructive clues for large-scale morphogenes, regulating positional information, organ identity, size, and axial polarity [1, 17, 18 ]. Recent work has included these voltage gradients in the regulation of front-rear polarity [19 , 20 ], appendage regeneration [21 , 22 , 23 , 24 ], craniofacial patterning [25], left-right asymmetry [26 , 27 , 28 , 29 ], eye development [30 , 31], and brain patterning [32]. Numerous studies have now identified transduction mechanisms linking bioelectric properties to downstream transcriptional and epigenetic targets [13 , 18 , 33 , 34 ], thereby revealing how these physical properties are integrated with genetic information during patterning. Whether via nucleotide sequences or chromatin modifications, the genome does not directly specify the organismic 3-dimensional shape. Instead, large-scale morphology is an emergent feature of the dynamics of complex networks of activity performed by cells. Thus, pattern formation is the result of a rich layer of chemical and physical processes occurring between DNA and anatomy. In fact, distinct morphologies may arise from a single genotype [4, 5 ]. Understanding how cells communicate and coordinate their functions in vivo to reliably form complex body plans and organs is of fundamental importance not only to evolutionary developmental biology, but also to biomedicine [6]. Transformative advances in regenerative medicine and synthetic bioengineering require that we know what input data can be given to a cellular system to induce specific morphological result-rational control of growth and form. This is a really difficult problem because of the complex nonlinearity of biological regulation [7]. Therefore, step 1 is to reveal processes that provide instructive control over the determination of large-scale form. Development, cancer suppression, large-scale reconstruction and renewal all depend on an organism's ability to store and process information about its correct anatomical structure, and correct any deviations from this structure that may occur in the event of damage or other environmental impact [1 , 2 ]. It is generally assumed that species-specific anatomical forms are encoded in the genome, although data from environmental epigenetics have long indicated that morphological outcomes are a function not only of inheritance but also of environmental and life-historical inflows [3 ]. The calculation model reproduces the four discrete outcomes observed experimentally. () four types of flat heads obtained from the calculation model; Different forms result due to deactivation of different cells. Red lines show the paths of the red cells; () Morphometric measurement of different flat heads, for comparison to real heads () measured (see Table 2 ). Scale bar 0,5 mm. Type A cells are shown in black, deactivated Type A white cells, Type B cells are red. The outer boundary has two parts which differ by their stiffness (soft part of the boundary appears in red, stiffer part in green). The red cells thus represent migrant neoblasts (or their offspring) while the black cells represent somatic cells associated with GJs. () Definitions of lengths of measurements L1 and L2 used in comparison to actual worms compared to those produced by the model in Table 2 The calculation model reproduces the four discrete results observed experimentally. () four types of flat heads obtained from the calculation model; Different forms result due to deactivation of different cells. Red lines show the paths of the red cells; () Morphometric measurement of different flat heads, for comparison to real heads () measured (see Table 2 ). Scale bar 0,5 mm. Type A cells are shown in black, deactivated Type A white cells, Type B cells are red. The outer boundary has two parts which differ by their stiffness (soft part of the boundary appears in red, stiffer part in green). The red cells thus represent migrant neoblasts (or their offspring) while the black cells represent somatic cells associated with GJs. () Definitions of the lengths of the measurements L1 and L2 used in comparison to actual worms compared to those produced by the model in Table 2 Note that the shape of the auricles in Figure 8 D is more pronounced than in the case of Figure 8 B,C. This corresponds to the experimentally observed forms. When modeling, this difference is achieved with different properties of the outer boundary, being softer in the lower part (red in the figure) and resulting in sharper auricles. This is consistent with previous work showing that integrity integrity (and thus rigidity) depends on the correct expression and function of gap coupling channels [100]. Figure 8 shows the final positions of cells B and their orbits after simulated four different deactivation patterns. To compare real worm morphological data with the results of our modelling, we performed morphometric measurements as shown in Figure 8 E–H on the results of our in silico simulations. We measured the length of the horizontal range L1 and the vertical range L2 (in the case of 1b and 2b, these ranges must not necessarily be straight). The results are seen in Table 2, and give a good consistency with the observed results in real worms (Figure 1). Let us note that the final morphologies of the planner's head in computer simulations are determined by the original cell configuration. They differ by placing deactivated cells (white cells in Figure 8). If these cells are close, then the final main forms are also close. If we compare DJ (Figure 8 C) and GD (Figure 8 D), then the original cell configuration and final morphologies are closer than these two compared to SM (Figure 8 A). In addition, DJ appears to be between the SM and GD models. It is interesting to note that this order corresponds to their genetic distance (Figure 3). We conclude that a small set of cell-level behaviors, such as migration and cell cell signaling known to be regulated by GJC and thus are perturbated by octanol exposure [ 35 , 39 , 49 , 98, 101 , 102 ], can quantitatively explain the morphologies we observed in planaria. When simulated in silico, this model produced flat main forms observed in the experiments. These different forms were achieved from the same original cell configuration but different deactivation patterns for cells A and different elastic properties for the boundary. These deactivation patterns correspond to octanol treatments that reduce cell-cell communication among a subset of cells. We assume that this results in deactivation of some of the signaling of cells. The original cell position configuration and its resulting equilibrium configuration are shown in Figure S2 H,I. Type B cells remain surrounded by A cells. This configuration is chosen in such a way that cells B either stay inside or migrate outside and produce one of the four configurations required (Figure S2 A–D) after deactivation of certain cells A. Specific main form occurs because cells B are pushed away by cells A and by other cells B. They escape through the lower concentration levels of substance C left by deactivated cells A, arrive at the outer membrane and change its shape. We were considering two cell types, A and B. Type A cells have fixed positions – they cannot move. They produce a substance whose concentration C spreads into space. Its distribution can be described by a reaction diffusion equation or by some other models. Type B cells can move. They produce no substance but they get the substance C produced by cells A and their movement is determined by its concentration distribution. We modeled the flat head with the following elements: Type A cells, deactivated Type A cells, Type B cells and a surface boundary. Deactivated A cells are fixed but they do not produce substance C. If there is more than one cell B, then they reject each other in the same way as they are rejected by cells A. We select deactivated cells A symmetrically with respect to the front-rear axis, to preserve the symmetry of the result (meaning B cells moving similarly on the left and right sides). The external boundary consists of points and elastic ‘springs’ connecting them. As a cell B approaches the border, a repulsive force appears on it from the border. This force is proportional to the distance from the particles and from the boundary ranges. Full details of the modeling are given in Figure S2. In this model, we hypothesize that instructive (GJ-dependent) signaling takes place from the somatic tissues to the neoblasts or their offspring, to guide the new tissue generation and shape during regeneration. Thus, the red cells are migrating neoblasts (or their offspring) while the black cells are somatic cells connected with GJs (Figure 8). In the model, octanol interference is thus modelled by cell:cell communication by deactivation of signaling from a specific cell type. One of the most important challenges facing developmental biology and regenerative medicine is to link large-scale patterning results with the individual activity of cells controlled by genetic networks and signalling pathways [ 2 , 85 , 86 , 87 , 88 , 89 ]. Most of the work on the levelar field deals with front-rear fragment polarity [60 , 62 , 90 ] or stem cell differentiation [57 , 59 , 91 ], and does not deal with the actual morphology of the head or the rest of the body. Recent quantitative, genetically based models of regeneration [92, 93 , 94 ] likewise use front-back identity as a binary reading, which does not address or explain changes in the form of these anatomical regions. Our study of the bioelectric regulation of regeneration reported changes in the main renovation [25], but was limited to scaling and did not model the detailed shape of the head. To mechanically begin linking individual cell behaviors (such as those regulated by GJ-mediated signals) to large-scale anatomical outcomes, we subsequently constructed an agent-based model of cell signaling and morfogenesis. Our model focused on cell migration and cell-cell signaling, as these are clearly important in implementing various morphogenetic outcomes [95 , 96 , 97 ], and also known to be regulated by GJ connection [98] and bioelectric properties of neighboring cells [99]. In D. japonica, physiological perturbations can stableally change the basic architecture of the planear body plan, giving rise to two-headed worms that continue to regenerate as two-headed forever over future rounds of regeneration in ordinary water [20]. We then asked if our observed main shape changes in G. dorotocephala were permanent. Photographs of worms after treatment with 8-OH were taken daily, from Day 10 (when worms morphologies were made), to Day 30 (the time required for complete cell turnover in the planarian flatworm). We found that the induced morphologies were reworked, long after regenerative processes had ceased, to produce morphologies that bore closer resemblance to the wild type G. dorotocephala. Interesting, the course of time and the result of this non-regenerative remodeling differed depending on the shape of the start head. Regenates who had rounded heads, resembling S. meditranea, began to develop a more triangular headform on Day 17, and on Day 30, bore more of a resemblance to D. japonica than S. meditranea. During the same time, regenerations that had triangular heads, similar to D. japonica, developed pronounced auricles, and on Day 30 was indistinguishable from wild type G. dorotocephala (Figure 7 A,B). These findings are fascinating in two respects. First, the change in morphology in the absence of a trauma highlights dynamic and robust mechanisms behind morphological homeostasis – the anatomical state is rebuilt over a long time from an abnormal configuration existing after regenerative repair was completed. Second, the conversion is done via ‘roads’ through the form space illustrated by canonical variable analysis (Figure 7 C). DJ morphology lies between GD and SM morphologies in this space, and we observe conversion going from SM morphology to DJ morphology to GD morphology. Based on these data, we conclude that morphology is both plastic and robust, restoration of the “target” morphology can occur without trauma to the organism, and that the space of the CV form is informative in illustrating parameters and limits to morphological variation. Figure 6. Membrane voltage reporter analysis shows long-term change of bioelectric connection in octanol-exposed planaria. ( A – D ) Domains of relative membrane potential are visualized with DiBAC 4 (3) in wild type G. dorotocephala , wild type S. mediterranea , wild type D. japonica , and wild type P. feline ; ( E – H ) Domains of relative membrane potential are visualized with DiBAC(3) in pseudo G. dorotocephala (GDs), pseudo S. mediterranea (SMs), pseudo D. japonica (DJs), and pseudo P. felina (PFs), respectively. Scalable 0.5 mm; ( I ) Number of isopotential regions in wild-type GD worms (control), all pseudomorphologies and pseudomorphologies that have been remodelled back to WT (wild-type) GD morphology after 30 days. After octanol treatment, the number of isopotential regions increases in pseudomorphologies, but decreases to WT levels after conversion. Black dots indicate worms with GD morphologies, blue dots indicate DJ morphologies, red dots indicate PF morphologies, green dots indicate SM morphologies, and pink dots indicate pseudoworms that have reshaped their morphologies to resemble wild-type GDs. n = 7 wild-type GD, n = 5 pseudo-type GD, n = 5 pseudo-type SM, n = 5 pseudo-type DJ, n = 2 pseudo-type PF, and n = 6 masks of converted worms. Error bars are standard deviations. Non-parametric statistical analysis was made using a post-hoc comparison of all groups with the Kruskal-Wallis test (p = 0.0021), and then between groups using a Dunns Multiple Comparison test, showing differences in voltage domain numbers between converted GD and pseudomorphologies are statistically significant at p < 0.05. Figure 6. Membrane voltage reporter analysis shows long-term change of bioelectric connection in octanol-exposed planaria. ( A – D ) Domains of relative membrane potential are visualized with DiBAC 4 (3) in wild type G. dorotocephala , wild type S. mediterranea , wild type D. japonica , and wild type P. feline ; ( E – H ) Domains of relative membrane potential are visualized with DiBAC(3) in pseudo G. dorotocephala (GDs), pseudo S. mediterranea (SMs), pseudo D. japonica (DJs), and pseudo P. felina (PFs), respectively. Scalable 0.5 mm; ( I ) Number of isopotential regions in wild-type GD worms (control), all pseudomorphologies and pseudomorphologies that have been remodelled back to WT (wild-type) GD morphology after 30 days. After octanol treatment, the number of isopotential regions increases in pseudomorphologies, but decreases to WT levels after conversion. Black dots indicate worms with GD morphologies, blue dots indicate DJ morphologies, red dots indicate PF morphologies, green dots indicate SM morphologies, and pink dots indicate pseudoworms that have reshaped their morphologies to resemble wild-type GDs. n = 7 wild-type GD, n = 5 pseudo-type GD, n = 5 pseudo-type SM, n = 5 pseudo-type DJ, n = 2 pseudo-type PF, and n = 6 masks of converted worms. Error bars are standard deviations. Non-parametric statistical analysis was made using a post-hoc comparison of all groups with the Kruskal-Wallis test (p = 0.0021), and then between groups using a Dunns Multiple Comparison test, showing differences in voltage domain numbers between converted GD and pseudomorphologies are statistically significant at p < 0.05. Figure 5. The distribution of neoblasts (red dots) changes following octanol treatment. ( A – C ) Analysis of the neoblast distribution by staining phosphorylated histone H3 in adults, wild type G. dorotocephala (n = 9), D. japonica (n = 9) and S. meditranea (n = 9) planarians. Trayed lines indicate edges of the anatomy of the mask, as well as 1/6 of the length of the mesh body from the front tip of the mask. This distance was used to define the rear boundary of the head. Pilar indicates the region where neoblasts were counted; ( D – F ) Analysis of the neoblast distribution in G. dorotocephal regenerates treated in 8-OH similar to G. dorotocephal heads ( n = 7), D. japonica heads ( n = 8), and S. mediterranea heads ( n = 7), by phosphorus reducing histone H3 staining. Pilar indicates region where neoblasts were counted. Scale bar 0.5 mm; (G ) Average number of neoblasts in the anterior 1/6th of wild type, and 8-OH treated worms (ANOVA p < 0.05). Error bars are standard deviations. Figure 5. The distribution of neoblasts (red dots) changes following octanol treatment. ( A – C ) Analysis of the neoblast distribution by staining phosphorylated histone H3 in adults, wild type G. dorotocephala (n = 9), D. japonica (n = 9) and S. meditranea (n = 9) planarians. Trayed lines indicate edges of the anatomy of the mask, as well as 1/6 of the length of the mesh body from the front tip of the mask. This distance was used to define the rear boundary of the head. Pilar indicates the region where neoblasts were counted; ( D – F ) Analysis of the neoblast distribution in G. dorotocephal regenerates treated in 8-OH similar to G. dorotocephal heads ( n = 7), D. japonica heads ( n = 8), and S. mediterranea heads ( n = 7), by phosphorus reducing histone H3 staining. Pilar indicates region where neoblasts were counted. Scale bar 0.5 mm; (G ) Average number of neoblasts in the anterior 1/6th of wild type, and 8-OH treated worms (ANOVA p < 0.05). Error bars are standard deviations. One of the physiological signals propagated within GJ-mediated cell networks is electric current: patterns of GJ-dependent connection can determine isopotential cell fields [19 , 25 , 79 ], and we observed that octanol really increased the number of regions with distinct V mem patterns (Figure 6 ). Since analytical pipelines to detect encoded pattern states from bioelectric measurements (which have been made for the human brain [80]) do not yet exist, we sought to begin to determine physiological measures that could reveal permanent changes to the somatic bioelectric network developed by the GJ blockade and could distinguish pseudoworms from those with the original (wild-type) morphology. Thus we next examined the distribution of isopotential cell groups among the various worm form outcomes, since such groups are established by the function of gap intersections and are a reading of the topology (interconnection) of developmental bioelectric networks [ 20 , 35 , 39 , 51 , 81 , 82 ]. Patterns of endogenous relative membrane depolarization and hyperpolarization were visualized using a DiBAC (bis-(1,3-dibarbituric acid)-trimethinoxonol) dye [79 , 83 ]. DiBAC is anionic, so the dye enters the cell membranes based on relative depolarization degrees [84]. Therefore, increased fluorescence indicates regions of depolarization, and decreased fluorescence indicates relative hyperpolarization. Wild-type worms of the species G. dorotocephala, D. japonica, S. mediterranea, P. felina, and GJ-perturbated pseudoworms were depicted with DiBAC dye in order to assess potential differences in relative membrane potential. Images were analyzed with a custom image analysis program (as described in Methods) to determine the number of distinct isopotential regions found throughout the mask. Analysis of worms 10 days after 8-OH treatment (after regeneration was completed, Figure 6 A–H) showed that transient perturbation of gap coupling communication changes body-wide patterns of stress distribution for many days after completion of 8-OH treatment. We discovered an increased number of isopotential regions in the pseudomasks compared to the state of the controlworms; interestingly, the number of isopotential regions returns to them in a wild-type state after 30 days of morphological reconstruction (Figure 6 I). Although we cannot be sure that each individual cell had been penetrated by the dye, the pattern of isopotential regions and variation among animals suggests that octanol action is stochastic and not 100% effective, only partially disrupting electrical coupling (obviously as regions different in V mem ) through animals treated in drugs. We conclude that octanol exposure changes the normal pattern of G. dorotocephal dormant potential against a pattern that persists even after 8-OH is withdrawn and regenerative repair occurs, resulting in an increased concentration of isopotential regions throughout the mask. In wild type G. dorotocephala, very few neoblasts reach into the most anterior 1/6th of the mask (Figure 5 A). In wild type D. japonica, the number of neoblasts in the anterior part of the body increases in comparison to G. dorotocephala, however it is still relatively low (Figure 5 B). Wild type S. meditranea planarians have a abundant neoblast population in the anterior-most region (Figure 5 C). All neoblasts in the anterior 1/6th of the anatomy of the mask were counted by hand (Figure 5 G). Noteworthy is that the distribution of neoblasts in pseudoworms accurately reflected the distribution of neoblasts in the wild-type species they resembled (Figure 5 D−F) (ANOVA p < 0.05). We conclude that the transformation of G. dorotocephala worms into similar to other species also extends to the species-specific, characteristic internal distribution of their stem cells, and that the patterning processes disrupted after GJC perturbation are also responsible for organizing the distribution of mitotically active cells. Planarians derive much of their notable regenerative power from a population of heterogeneous adult stem cells, called neoblasts, which constitute the only mitotically active cell population in the body of the flatworm [56 , 70 , 77 ]. We next examined whether the spatial distribution of neoblasts was markedly different between species of planarians, and whether GJ-revenged worms acquired the neoblast distribution characteristic of the species whose morphology they had taken on. As neoblasts are the only mitotically active cells within the planar body, we performed immunocoloration of phosphorylated histone H3, a common neoblast marker in planaria [78], to visualize neoblast populations. Figure 4. Brain morphology changes after 8-OH treatment. ( A – C ) Brain morphology is visualized by wild-type antisynapsin dyeing G. dorotocephala (n = 10), D. japonica (n = 15), and S. meditranea (n = 6) planarians. Arrows indicate brain morphologies, and dotted lines indicate measurements used for the calculation of length/width ratio; ( D – F ) Brain morphologies by antisynapsin staining of G. dorotocephala regeners treated in 8-OH similar to G. dorotocephala heads (n = 4), D. japonica heads (n = 4), and S. mediterranea heads (n = 6). Arrows indicate brain morphologies, and dotted lines indicated measurements used for calculating length/width ratio. Scale 0.5 mm; (G ) Average ratio of brain length to width of wild type to 8-OH treated worms (ANOVA p < 4.9 × 10 −14 ). Error bars are standard deviations. Figure 4. Brain morphology changes after 8-OH treatment. ( A – C ) Brain morphology is visualized by wild-type antisynapsin dyeing G. dorotocephala (n = 10), D. japonica (n = 15), and S. meditranea (n = 6) planarians. Arrows indicate brain morphologies, and dotted lines indicate measurements used for the calculation of length/width ratio; ( D – F ) Brain morphologies by antisynapsin staining of G. dorotocephala regeners treated in 8-OH similar to G. dorotocephala heads (n = 4), D. japonica heads (n = 4), and S. mediterranea heads (n = 6). Arrows indicate brain morphologies, and dotted lines indicated measurements used for calculating length/width ratio. Scale 0.5 mm; (G ) Average ratio of brain length to width of wild type to 8-OH treated worms (ANOVA p < 4.9 × 10 −14 ). Error bars are standard deviations. Then we asked whether internal structures were similarly transformed into another form, as was external morphology. Few aspects of the planar internal anatomy differ noticeably between species; however, brain size and brain shape offer an interesting exception to this rule. The brains of wild type G. dorotocephala are elongated and narrow, while D. japonica and S. meditranea have noticeably shorter and wider brain morphologies (Figures 4 A–C). No live wild type P. felina could be obtained for this work, and the low frequency of pseudo-PF occurrence limited the number available for analysis. Therefore, we focused on G. dorotocephala, D. japonica and S. meditranea. We performed immunotainment using an antisynapsin antibody, to visualize both the brain, and ventral nerve strands of pseudo- and wild-type worms. As wild-type headform recapitulation after ten days of regeneration in water had been confirmed by geometric morphometrics, we chose to compare pseudomorphologies to adult worms of other species, to minimize confusing data due to variability in regeneration time between species. Total shape differences were captured by measuring the brain length/width ratio. These calculations were used to quantify shape differences between species and to quantify brain remodelling in ‘pseudo’ worms (Figure 4 G). We found that pseudoworms had brain morphologies similar to the brain morphologies of wild-type worms whose main forms they resembled (Figure 4 D–F) (ANOVA, p < 0.001). We conclude that the patterning processes disrupted after the perturbation of gap crossing communication are also responsible for producing the morphology of the brain, and that the altered forms are not limited to the overall main geometry but include the patterning of the central nervous system within. Figure 3. Percentage of headform outcomes correlate with evolutionary distance. ( A ) Evolutionary tree, constructed based on rRNA data, showing relationships between species of interest. Species names in red are those analysed in this work; ( B ) Frequency of main forms obtained in the octanol exposure experiments (n > 243). Failure to regenerate is defined as loss of anterior-rear polarity, and failure to regenerate any head at all after octanol treatment. Error bars are standard deviations. Figure 3. Percentage of headform outcomes correlate with evolutionary distance. ( A ) Evolutionary tree, constructed based on rRNA data, showing relationships between species of interest. Species names in red are those analysed in this work; ( B ) Frequency of main forms obtained in the octanol exposure experiments (n > 243). Failure to regenerate is defined as loss of anterior-rear polarity, and failure to regenerate any head at all after octanol treatment. Error bars are standard deviations. Obtaining distinct regenerated morphologies, at different frequencies, despite the same treatment conditions, led us to explore the evolutionary relationship between the four represented species. We mapped an evolutionary tree based on rRNA homology [75 , 76 ], and compared this tree (Figure 3 A) to frequencies of the heads of the different species that emerged during 8-OH treatment (Figure 3 B). Interestingly, there are morphologies corresponding to species most closely related to G. dorotocephala (SM and DJ, which are removed from GDs with at least 100 million years of evolutionary distance) with much higher frequency than morphologies corresponding to less related species (PF). We conclude that not only are phenotypic outcomes from physiological networks perturbation stochastic (as the same treatment leads to one of several discrete forms among individuals), but the frequencies are not equal and correlate roughly with evolutionary distance between the worm species these heads are similar. Figure 2. Canonical variable analysis of the shape of the head. ( A ) Graphic output, showing confidence-slips for means, with a 0.9 probability, of form data from wild type and experimentally derived morphologies. Ellipses are coloured to correspond to phenotype and treatment. n = 10 WT G. dorotocephala , n = 8 WT G. dorotocephala 10 days after amputation and regenerated in water, n = 9 WT D. japonica , n = 6 WT P. felina , n = 8 WT S. mediterranea , n = 7 pseudo G. dorotocephala , n = 13 pseudo D. japonica , n = 5 pseudo P. felina , and n = 6 S. mediterranea flatworms were measured; ( B ) Legend of landmark placement on a wild type G. dorotocephal headform (see Materials and methods); ( C ) Schematic demonstration of change of morphology to better resemble another species after 8-OH treatments. Procruste's distance between wild type G. dorotocephala , 8-OH treated G. dorotocephala with D. japonica headform, and wild type D. japonica shows objective change in morphology to be more similar to the non-native species. Figure 2. Canonical variable analysis of the shape of the head. ( A ) Graphic output, showing confidence-slips for means, with a 0.9 probability, of form data from wild type and experimentally derived morphologies. Ellipses are coloured to correspond to phenotype and treatment. n = 10 WT G. dorotocephala , n = 8 WT G. dorotocephala 10 days after amputation and regenerated in water, n = 9 WT D. japonica , n = 6 WT P. felina , n = 8 WT S. mediterranea , n = 7 pseudo G. dorotocephala , n = 13 pseudo D. japonica , n = 5 pseudo P. felina , and n = 6 S. mediterranea flatworms were measured; ( B ) Legend of landmark placement on a wild type G. dorotocephal headform (see Materials and methods); ( C ) Schematic demonstration of change of morphology to better resemble another species after 8-OH treatments. Procruste's distance between wild type G. dorotocephala , 8-OH treated G. dorotocephala with D. japonica headform, and wild type D. japonica shows objective change in morphology to be more similar to the non-native species. Quantification was used to determine whether the forms that looked like other species were objectively similar to these species, and to suggest a continuous morphospace within which octanol-induced form change can be visualized along a continuum (from normal to normal in another species). Canonical variable analysis supported the statistical significance of the given pre-defined morphological groupings (in this case groupings were based on experimental treatment and morphology) (Figure 2 A). Comparisons of Procruste's distance between form groups ( Figure 2 A, Table 1 ) showed that the experimentally derived morphologies were closer in shape to the wild-type morphologies they resembled than the wild-type Girardia dorotocephal main morphology. Analysis of variance (ANOVA) of both centroid size and shape between wild-type morphology and pseudomorphology groups also confirmed significant differences between groupings (F = 7.94, p < 0.0001, and F = 7.40, p < 0.0001, respectively). We conclude that amputation and treatment in 8-OH can produce regenerated worms whose morphology has changed to become significantly more similar to that of another species of planaries. Geometric morphometrics [73] was used to quantify similarities and differences between headforms of true species, as well as the experimentally derived pseudomorphologies. In short, geometric morphometric analysis involves placing a range of landmarks, which are both biologically significant and reproducible across all samples, removal of non-form variation (size, rotation, etc.). ), and the conduct of a set of statistical analyses [74]. Landmarks were selected from the common biological landmarks present over samples, and semi-mark marks were placed with prescribed relationships to these landmarks (Figure 2 B). Soil data were recorded for n > 60 worms, including GD worms whose head shape had been experimented with being disturbed by 8-OH, controlling GD worms that had regenerated in water for 10 days, and adult wild-type worms from each of the three species. Analysis of main components (no data) and analysis of canonical varieties was performed on the data set. This enabled visualization of medium-form changes between wild species morphologies, and between experimentally derived main forms. Both analyses resulted in the pseudomorphologies being separated from the wild type G. dorotocephala morphology. Procruste's distance between each of the groups was calculated, in order to produce a quantified metric for comparing shape differences. After 3 days of exposure to octanol, the worms were allowed to complete regeneration (10 days total). Noteworthy is that this process resulted in worms exhibiting main forms that were very similar to those heads suitable for other species of planarians (Figures 1 E–H). 8-OH treated fragments regenerated both head and tail correctly, but in many cases the shape of the head changed drastically. Wild type G. dorotocephala (GD) has a very pointed head shape, with two elongated auricles at the eye plane (Figure 1 A). Fragments covered by the same 8-OH treatment schedule regenerated one of: fully rounded heads such as the Planarian S. mediterranea (SM) (Figure 1 H), heads with thick neck and ‘cat-like’ auricles such as those of Planarian P. felina (PF) (Figure 1 G), heads which are triangular as Planarian D. japonica (DJ) (Figure 1 F), or heads similar to wild type G. dorotocephala (Figure 1 E). We will refer to head forms of regenerate as "pseudo" of the most morphologically similar species. Variant, other species-specific main morphologies were never observed after amputation or during regeneration in water (control state) – the normal process of main renewal has 100% fidelity to the species-specific form (Figure S1 A). The same schedule of exposure to hexanol (6-OH), a related compound to octanol that does not effectively block GJs and can thus be used as control [69], had no effect (Figure S1 B). Similarly, intact worms soaked in 8-OH for > 3 days showed no unusual morphological results. Based on these results, we conclude that discrete, species-specific main forms can be achieved by manipulating connection of physiological networks in the flatworm during head renewal. Figure 1. Characterisation of varying main morphologies produced by octanol treatment. ( A – D ) Wild type morphologies of four species of planaria flatworm. The arrows indicate auricle placement and general headform; ( E – H ) pre-tail (PT) fragments of G. dorotocephala , treated in 8-OH for three days, and then moved in water for the remainder of regeneration ( n > 243). Arrows indicate auricle placement and general headform. Scale bar 0.5 mm; ( I ) Experimental schedule for octanol treatment. PT fragments are amputated from G. dorotocephala worms. Fragments are treated in octanol (8-OH) for three days, and may regenerate in water for seven days. Figure 1. Characterisation of varying main morphologies produced by octanol treatment. ( A – D ) Wild type morphologies of four species of planaria flatworm. The arrows indicate auricle placement and general headform; ( E – H ) pre-tail (PT) fragments of G. dorotocephala , treated in 8-OH for three days, and then moved in water for the remainder of regeneration ( n > 243). Arrows indicate auricle placement and general headform. Scale bar 0.5 mm; ( I ) Experimental schedule for octanol treatment. PT fragments are amputated from G. dorotocephala worms. Fragments are treated in octanol (8-OH) for three days, and may regenerate in water for seven days. Planar flatworm species exhibit a wide range of main forms, from the very rounded to the nearly triangular, with varying forms of auricles (Figures 1 A–D). To interrogate the mechanisms responsible for regeneration and maintenance of the head shape, G. dorotocephala planarians were amputated along a plane placed posteriorly towards the pharynx but anterior to the tail, to produce a fragment before the tail (PT). Subsequently, PT fragments were treated with the gap junction (GJ) communication blocker octanol (8-OH). 8-OH is a commonly used pan-GJ blocker [20 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 ], which alters the physiological connection between populations of cells, thus disrupting the rate and pattern of transmission of bioelectric and other small molecular signals. The dosage was titrated to a level sufficiently low to allow interference with regenerative signaling without organismic toxicity. 8-OH exposure has been validated to be transient with GC-MS: drug levels are undetectable after a few hours of wormwash-out in water, and octanol does not alter genetic sequences in the mask [20 , 72 ]. 3. Discussion 3.1 Perturbation of Bioelectric Networks Produces inter-species Morphological change Our data shows that in planarian flatworm G. dorotocephala, results of morphogenes can be modified by perturbation of physiological networks. Short exposure to a well characterized disruptor of electrical synapses (gap nodes blocks) resulted in regenerated main forms quantitatively similar to those of another planar species. We observed main forms (and internal structures) similar to D. japonica, S. meditranea and P. felina. Note that other species exist with main forms similar to each (for example, 2 other species are known to exhibit P. felina-like flattened heads); since these three types are good representatives of each form class and are available as living specimens for analysis (unlike other members of the same morphological main types), we refer to our phenotypes such as pseudojaponica, pseudomediterranea, and pseudofelina respectively. Future analysis of other planaric species will reveal whether these phenotypes in fact resemble one of the other members of each form class. The head morphology change effect was instructive, and not just one of toxicity or a housekeeping defect, as the result was not only a disturbance of regenerative ability or a generalized mispattery (e.g. tumor or other unorganized growth), but rather resulted in one of several contiguous (and ecologically valid) morphologies. No intermediate morphologies were observed, highlighting the discrete nature of the complex patterning results after exposure to GJ blockers. The same 3-day treatment with the closely related alcohol, hexanol (6-OH), which does not effectively block gap coupling channels, and subsequent regeneration in water for 7 days, did not produce altered morphologies, consistent with the importance of GJ blocking activity of octanol, and no other generalized effect of long chain alcohols. Dramatically different morphologies can be derived from the same by [103,104,105]. Muler and hinnies, which differ only in the gender of respective donkey and horse parents, are quite morphologically distinct, despite nearly identical DNA sequences. Toadflax (Linaria vulgaris) has a morphological variant produced solely by variations in DNA methylation patterns, and numerous reptiles species will develop morphologically distinct genitals in a temperature-dependent manner [106]. In addition, recent data showed how changes in biochemical signalling can be selected among different species-specific morphologies [103,107]. Here we identify a new physiological entry into the process by which distinct morphologies can be derived from the same genome: physiological bioelectric networks. The compatibility of multiple morphological results with a single genomic sequence underpins the importance of learning to understand and control the instructive patterning information generated by chemical, physical, and bioelectric processes beyond hard-coupled gene-regulating networks. 3.2. Geometric Morphometry Quantifies Planarian Shape Change Geometric Morphometrics is a technique commonly used to quantify and compare biological forms. We used landmark-based analysis, which involves registration of landmark data, conversion of resulting forms to remove scaling and rotational noise, as well as performance of statistical analyses. Canonical variate analysis (CVA) was used to compare wild-type morphologies of four species of worm with corresponding pseudomorphologies, as well as comparing wild-type G. dorotocephal morphology with all pseudomorphologies. CVA determines the statistical significance of variation between given groups of samples [74,108]. In this case the groupings corresponded to observed phenotype and experimental treatment. We chose to use Procruste's distance to quantify “distance” between shapes, because it is a measure of the square root of the summarized square distances between the corresponding landmarks in two forms, and is often used in morphometric studies comparing different morphologies. The Procrustes distances between the pseudomorphologies and the species they resemble are less than the Procrustes distances between the pseudomorphologies and wild type G. dorotocephala. The observation was supplemented with both Procrustes centroid size and shape ANOVA (p < 0.001 and p < 0.001, respectively). This shows a significant change in morphology, and quantitatively confirms the apparent similarity between the pseudophenotypes and their wild-type counterparts. Interestingly, the centroids in the groups of wild type S. meditranea, D. japonica and G. dorotocephal morphology (the three most closely related species) all vary along a linear axis through the CV space. This reveals the morphological consequences of variation along a canonical variate. Moving from negative to positive values of canonical variate correlates with the extension of the auriculars, if they exist (with SMs located at the negative end of the spectrum, and GDs and PFs located at the positive end). Going from negative to positive values of canonical variate two correlates with movement of auricles, if they exist, from a very front position, to a more posterior one (with PFs being at the negative end of the spectrum, and GDs being at the negative end). 3.3. Morphological change is not only skin depth Important, the patterning changes caused by electrical synapse blockade affect the shape of the brain (Figure 4), not just the overall headform. Worms with pseudoheads similar to a given species possess brains similar to the same species, revealing that the functions of this regulatory pathway extend beyond setting the outer contour of the head but instead control the shape of the brain within. These data also suggest a link of brain morphology to the shape of the head. Whether the morphology of the brain drives the shape of the head, or whether both are parallel consequences of the same GJC-dependent upstream control mechanism, this finding shows the depth of reorganization of the body plan after perturbation of GJC connection. Linking or involvement of the central nervous system in the morphogenesis would not be entirely surprising, given previous work showing the necessity of long-range neural inputs for proper polarity patterning during regeneration in planaria [20] and vertebrates [109,110]. In addition, the perturbation of gap coupling communication reorganizes the distribution of planar adult stem cells (neoblasts) into spatial patterns suitable for other species (Figure 5). Along with the change in brain shape, it is clear that the morphological change to pseudomorphologies in other species is thorough, which involves not only external headform but also internal organ scaling and positioning of key cell groups. These data reveal the link between proliferative stem cells and large-scale morphology, its variability throughout phylogenine and its control through GJ-dependent signaling. It is possible that control of neoblast distribution is what drives large-scale morphology. Conversely, their spatial distribution may be one of several downstream effects of the dynamics of a GJC-mediated cellular network. 3.4. Bioelectric state of tissues and its stable change in species-specific shape change Our results include the first analysis of the effects of GJ modulation on the long-term bioelectric profile of patterning tissues in vivo. Although large-scale bioelectric gradients have been shown to functionally instruct pattern formation in a number of models [12], including determination of morphhollaxis/scaling in S. meditranea [19] and anterior-rear polarity in D. japonica [19], no studies have yet investigated patterns of V-mem distribution associated with changes in the electrical connection of tissues. We found that short exposure to a GJ blocker, known to be washed out from worm tissues within 24 hours [20], results in a greater degree of bioelectric isolation (ability to maintain many smaller regions of isopotential V mem ) as late as 10 days after exposure (Figure 6). Although these long-term changes in bioelectric patterning are not directly caused by octanol remaining in the body of the mask (as 8-OH is known to depart from planaric tissues within 24 hours [20], they are initiated by the octanol-mediated disorder of GJC, which alters bioelectric network topology, and results in a sustained perturbation of bioelectric state. In this, the effect is similar to the known properties of electrical synapses (GJs) in the nervous system, which help implement a kind of hebbian plasticity that stabilizes the ionic conductivity of cells after transient electrical modulation [44,111,112.13,114]. Quantitative physiological models of bioelectric dynamics will need to be developed to understand bioelectric state memory [115] and determine why different types of worms have such distinct profiles of regions of isopotential V mem . The variability and overall magnitude of the effect indicate that the result of 8-OH exposure is only partially effective in shutting down GJC (which affects a certain percentage of GJs in any animal); this is consistent with the stochasticity of the morphological phenotype (Figure 3). While 8-OH is a common method of blocking GJC (and in D. japonica, giving the same effect as RNAi as innxins genes [20]), we cannot definitively rule out that additional targets besides GJs also become perturbated either directly or secondary after 8-OH exposure. One difficulty with a molecular (gen specific) approach to GJ shutdown, is that not only would ~13 innxins need to be cloned out of the GD genome (no one has yet been characterized), but their RNAi target search would need to be tested combinatorially. The space for all possible combinations of different innxin-RNAi that would have to be tested at the same time is enormous, as GJ proteins can often compensate for each other. Despite this, our previous work tested some such combinations (in D. japonica [20]), and showed that a simultaneous impact of three Innexins of RNAi reproduces the 8-OH phenotype, reinforcing the conclusion that phenotypes are indeed the result of 8-OH's effect on gap crossings. A recent study [20] showing that transient glitches changes induce permanent changes in plane target morphology raised the question of whether this pattern memory was determined by changes in GJC that persist well after the blocking reagent leaves the tissues, or by GJC states that change biochemical or transcriptional cell states that are stable after GJC returns to normal. Although we cannot rule out further epigenetic changes that may occur, our data show that even 10 days later, the effects of short 8-OH exposure are preserved as reduced electrical connection in the somatic tissues. The ability of the GJC networks to stabilise induced changes in the coupling is consistent with GJ's known roles in memory and synapse plasticity [44.52.114.116.117.118]. Our analysis did not reveal a simple correspondence between the specific distribution of voltage gradients and morphological results of the pseudoworm regeneration, which could have several explanations. Firstly, it is possible that not tension without another molecular signal is what crosses the GJ network to determine plane headform. GJ-permeent molecules with signaling roles include serotonin [119,120] and other neurotransmitters [121], histamine [122], calcium [123], and others, which will be tested in future studies, especially as fluorescent probes for some of these physiological signals come online [124,125,126]. Another possibility is that each morphological state is achieved through several possible distributions of the voltage; in this case, the apparent variability would conceal our ability to detect a specific mapping from distribution to main pattern outcomes. However, the most likely explanation is that form outcomes must not be encoded via steady (single-image) distributions but in time-dependent activity, which occurs during information coding in the central nervous system. In this case, films of voltage change over time in vivo must be acquired and analysed—a significant challenge in the planaria model system. Our analysis of isopotential regions is just a first attempt to break the information encoded in bioelectric states. Overall, we believe it is very likely that a much more sophisticated analytical method must be developed, perhaps similar to what is used to derive the content of neural networks from brain scans [80, 127], and applied to a large selection of worm imaging data, to truly crack the bioelectric code of this system. Functional studies in planaria and Xenopus [19],25,128] have shown the relevance of bioelectric gradients for anterior patterning in general, but new computational methods will need to be taken to show what spatiotemporal pattern of bioelectric activity is required for each of the patterning results we observed. Uncovering this survey will be the focus of our efforts for future studies. 3.5. Morphological remodeling in 2 phases: Main form Change without Traumatic damage The pseudomorphologies we observed are not permanent: they remodel, after the first regeneration process is completed, to resemble G. dorotocephala again. In this they differ from the permanent double-headed phenotype induced in D. japonica, representing a stable change in worm target morphology after physiological perturbation [20.129]. Thus, in G. dorotocephala, repair takes place in two phases: main regeneration after amputation, which is completed within the normal time frame (< 10 days) and results in the forms of different species, and a subsequent much longer remodeling back to the genome-specific G. dorotocephala headform. This latter process occurs in the absence of injury, within a complete head, over several weeks, illustrating the ability of cellular networks to detect deviations from the correct shape, even in the absence of trauma or ulcers, and that effect remodeling. Interestingly, the same biphasic process occurs in vertebrates: when a salamander tail blast is transplanted on a flank, a tail is first formed, but later it is redesigned into a limb in a much slower process (130.131). Morphology appears to be a homeostatic parameter, a parameter that is assessed and adjusted consistently to maintain any ‘goal' morphology. Morphology must first be decided within days of amputation, since it is possible to observe the main form already day six after amputation. In 8-OH treated worms, this mechanism produces faulty, pseudomorphologies. However, morphology must then be reconsidered again after this first decision. When the current morphology is not consistent with target morphology, reconstruction begins sometime between day 10 and day 17. The activity of these post-regeneration remodeling processes has been completed is fascinating, and means that morphology is consistently re-evaluated and edited, even after large-scale reorganization events have been completed. The nature of the processes that drive cell behavior towards a specific, stable final state (when reconstruction ends) is almost completely obscure. At this time, it is not possible in any model system to derive specific form information (to know exactly what anatomical pattern will be a sufficient end-to-end state to cause remodeling to cease) from genomic data. 3.6. Calculation modelling of head formation The various main morphologies we observed, which are distinct in external anatomy, distribution of stem cells, and the shape of the brain, must be the result of differential cell migration and cell-cell communication. To link cell-level behaviors to the discrete large-scale morphologies and explore the consequences of reduced GJ communication (induced by octanol exposure) on emergent cell signaling and morphogenes, we analyzed an agent-based model. In this model (Figure S2), some cells migrate actively, and change the shape of the resulting tissues. Their movements are controlled by a chemical gradient formed by other static cells in the head, which activation patterns are established by GJC, which is known to occur in the worm [20.70] and in many other organisms [51.132.133]. Our model shows the morphogenetic consequences of disruptions in this communication, which parallels the interaction between signaling of flat neoblasts (stem cells) and surrounding somatic tissues. In this model, we propose that significant instructive (GJ-dependent) signaling takes place from the somatic tissues to the neoblasts or their offspring, to guide the new tissue generation and shape during regeneration. The computational simulations showed how a small number of cell configurations could result in main forms quite closely matching the observed morphologies (Figure 9 and Table 2). Interestingly, when specific static cells (black) lose the ability to send their signal due to GJC blocking of octanol, the movement of the moving cells (red) changes which gives rise to one of the 4 outcomes. The model thus exhibits the two main characteristics of the experimental data set: several possible outcomes (due to different patterns of GJC-blocked static cells, possibly from physiological noise in pharmacological drug-target interaction), and discrete specific outcomes (as a result of deterministic signaling of static "black" cells and consequent behavior of moving "red" cells). It should be noted that our model is also easily applied to an organ that grows from an initial cell position to its final position: if we allow the division of cells B, cell migration continues in the same way as shown: they will leave behind deactivated (differentiated) cells B that do not move and do not divide. The future work will test the applicability of this model class to other regeneration systems. Figure 9. A conceptual model of shape change driven by physiological network dynamics. Planaria regeneration parallels classical neural network behavior; both can be described in terms of free energy landscapes with multiple attracting states. (A) Behavior of a classic Hopfield neural network is trained to reproduce three types of patterns, in this case forms of the letters “F”, “H” or “G”, which are the three stable states of the network’s free energy landscape. The state of the Hopfield network nodes directly relates to the brightness of pixels on a display, generating output. Perturbation of the network from a stable state (red arrow) by removing (damaging) part of the pattern is tantamount to moving a ball to an unstable point on the free energy landscape, and leading to regeneration of the nearest learned attraction state (blue arrow). In this, such networks are well known ability to carry out memory analogous to regenerating organisms restoring a specific target morphology in the event of damage; (B) The parallel concept of planaria regeneration to main forms of one of three different species, which are attracting states of the free energy landscape. The result morphology is driven by the dynamic outputs of physiological cellular networks. Amputation (red arrow) is similar to moving the system to an unstable point on the free energy landscape. Normal regeneration would return the system to its main attraction, but changing cell network dynamics via gap crossing blockade enables regenerative transitions (blue arrow) to alternative local minima, corresponding to morphospace regions normally occupied by P. felina and S. meditranea worms. Over time, remodeling (green arrow) transfers these morphologies to the global minimum of wild-type condition (G. dorotocephala ). Figure 9. A conceptual model of shape change driven by physiological network dynamics. Planaria regeneration parallels classical neural network behavior; both can be described in terms of free energy landscapes with multiple attracting states. (A) Behavior of a classic Hopfield neural network is trained to reproduce three types of patterns, in this case forms of the letters “F”, “H” or “G”, which are the three stable states of the network’s free energy landscape. The state of the Hopfield network nodes directly relates to the brightness of pixels on a display, generating output. Perturbation of the network from a stable state (red arrow) by removing (damaging) part of the pattern is tantamount to moving a ball to an unstable point on the free energy landscape, and leading to regeneration of the nearest learned attraction state (blue arrow). In this, such networks are well known ability to carry out memory analogous to regenerating organisms restoring a specific target morphology in the event of damage; (B) The parallel concept of planaria regeneration to main forms of one of three different species, which are attracting states of the free energy landscape. The result morphology is driven by the dynamic outputs of physiological cellular networks. Amputation (red arrow) is similar to moving the system to an unstable point on the free energy landscape. Normal regeneration would return the system to its main attraction, but changing cell network dynamics via gap crossing blockade enables regenerative transitions (blue arrow) to alternative local minima, corresponding to morphospace regions normally occupied by P. felina and S. meditranea worms. Over time, remodeling (green arrow) transfers these morphologies to the global minimum of wild-type condition (G. dorotocephala ). Our agent-based model includes physical parameters of cells [95,134], as well as traditional chemical gradient diffusion [135,136]. In the model, octanol interference is modeled by cell:cell communication by deactivation of signaling from a specific cell type; a candidate for this unique cell population are the rare but widely distributed cells expressing the gap crossing protein Innexin 5 [71]. Future experiments will test the specific predictions of this model and assign molecular-genetic identities of the multiple different cell types postulated in the model. Another important line of investigation is to mechanically link large-scale dynamics of physiological signaling to the specific behavior of cells modeled in our simulations. 3.7. One hypothesis: Physiological Network Attractors Underlie Regions of Anatomical Morphospace The outcome of regeneration in this model is a set of contiguous, discrete main forms—not a confusing amalgam of different patterns that would be expected if cells made purely local decisions and were only dis-coordinated by octanol treatment. Currently, the area largely lacks conceptual models to think of this type of input to development and renewal; it is not clear how to best represent or model discrete, yet stochastically chosen, emerging large-scale patterns. Yet this is an increasingly important goal as advances in regenerative medicine and synthetic biotechnology require the ability to predict and control global outcomes by adjusting cell level parameters. The area of developmental bioelectricity is still searching for an appropriate formalism, but an obvious candidate is that of the neural network paradigm used by computational neuroscientists to understand emergent dynamics of decision-making in brains composed of electrically coupled cells. We have previously argued that since somatic cells have many of the same components as nerves (ion channels, neurotransmitters, GJs act as electrical synapses, gene expression is regulated by electrical activity), we can usefully use some of the concepts of neuroscience to understand similar phenomena in slower development networks, where production is not muscle-driven behavior but pattern formation [1.13,137]. Scientists in computational neuroscience have modeled the states of power-connected networks in an energy area that reveals the landscape of trajectories implemented by the networks [138,139,140,142,143]. Such (neural) networks can be stabilized by attractors in this virtual space, and their activities can be explained in a couple of ways by the network's efforts to minimize a global "energy" function [144.145.146.148.148]. Thus, we propose that one way to conceptualize the connection between bioelectric connectivity among worm cells (GJC) and discrete global behaviors achieved through regeneration is to regard worm cells as a neural-like network, processing developmental information slower but obeying the same statistical dynamic rules that are known to occur in the brain (Figure 9A). It is unlikely that GJs are well known to be important for information processing in the brain [54,149,150]. We assume that distinct form results may be attractions in a state space that describes distinct modes in the bioelectric network of planar body cells connected by gap crossings (electric synapses); essentially, these attractions correspond to regions of a morphospace classically understood by evolution theory [151]. In the experiments, octanol interferes with the normal dynamics of this network, destabilizing and knocking its stochastic dynamics to one of several nearby attractions, normally belonging to different species of flatworm (Figure 9B). The development of a quantitative, computational model of cell networks that have such modes (with electrical states preventing cell proliferation, apoptosis and migration behaviors resulting in various main forms) poses the next deep challenge in this area. Such dynamic networks form the basis of virtually every complex process known in the biological world, from metabolism to group behavior [152.153.154]. These richly interconnected nonlinear networks, composed of many simple units, have the ability to produce complex patterns in open and thermodynamically unstable systems, without any single governing instance [155.156]. Within a given system, these networks converge into one or more discrete, stable states [157]. Neutral networks are just one example of complex dynamic networks; they are implemented via electrical signaling and gap crossings showing discrete attractions corresponding to global conditions [141,158] and stable re-employment of large-scale permits (memory) after perturbation [54,147]. Given that neural signaling developed by specializing in much more ancient somatic physiological communication mechanisms, it is reasonable to postulate fundamentally similar network dynamics for physiological networks under pattern formation [137,159. In this view (Figure 9), the discrete main patterns are the result of bioelectric network dynamics with the lowest free energy; cutting off the grid raises the free energy by derogating the system from its optimal morphology (damage), and initiates the regenerative process—a journey back down to a low-energy attraction. Interrupting the physiological bioelectric network with 8-OH results in system dynamics being moved from this attractor to another attractor, and subsequent regeneration of a changed morphology. Apparently, these pseudoforms are only local minima, because after about 3–4 weeks, the pseudomasks return to the global minimum found in the standard G. dorotocephala morphology. In this class of models, the output of the physiological network consists of signals that route cells to proliferate, apoptosis, or migrate, so that head morphogenes is achieved. The details of this process at cell level begin to be understood, both in the CNS (control of gene expression by brain activity [160]) and developmental bioelectricity [12,23,32]. Variations in membrane potential between cell groups can act as electrophorizing forces, drawing small signaling molecules, small RNAs, or charged transcription factors to specific regions of the planer body, yielding many possible long-term effects [47,161,162,163,164]. Additional mechanisms include voltage sensitive phosphatases [165] and voltage controlled action of chromatin modifying enzymes [34,43,166]. It is obvious that this is only one way of thinking about this data, and others are possible. The advantage of this paradigm is the rich set of mathematical results available from studies of computational neuroscience that can limit future model building and help make testable predictions. 3.8. Stochastic phenotypes and Evolutionary implications One of the most important aspects of these phenotypes is their stochastic nature: the same treatment is performed on a cohort of clonic (isogenic) animals resulting in one of several patterns within any exposed population. The frequencies with which species-specific patterns are observed correspond to the evolutionary distances estimated for these species of planaria (Figure 5). There are far more pseudo G. dorotocephala, D. japonica, and S. meditranea morphologies, which correspond to a minor evolutionary distance from G. dorotocephala, while P. felina pseudomorphologies are still relatively rare. One possibility is that a core network (which consists of gene regulatory elements and physiological gap coupling communication (GJC)—mediated signals) is working among many planarians, with species-specific differences adjusting its key dynamics. Therefore, the evolutionary distance between the planar types can be expected to reflect how easy it is to transfer this network to its various potential outcomes states (as happens during GJC inhibition). It should be noted that wild type P. felina has several eyes along the doso-ventral body boundary, a trait that our pseudo-PF does not exhibit. Incomplete mimicry of the most distant species, P. felina, is consistent with the relationship between evolutionary distance and ease with which species-specific morphology can be recapitulated entirely during perturbed regeneration. It should be noted that Phagocata gracilis and Phagocata vella planarians also have main morphologies that are quite similar to P. felina, and therefore also pseudo-PF. These two species possess only two eyes, and both species are similarly evolutionaryly removed from G. dorotocephala. However, no living wild-type worms of these two species could be procured for this work, and therefore our analysis focused on P. felina. Overall, the ability to generate distinct morphologies from the same by likely represents an extremely flexible mechanism that could be exploited through evolution via many known environmental and genetically encoded modulators of gap coupling states. An apparent absence of ‘intermediate’ morphologies, or main forms similar to a mixture of two or more species of planaries, may be the result of limitations limiting the morphological space available for the regenerating planari. Within this framework, the limitations of phenotypic space become driving factors in the morpogenesis. These limitations are likely an amalgam of developmental, environmental, and evolutionary factors, which serve to limit the stable positions in patterning networks, and thus the morphogenetic possibilities available for a regenerating flatworm. Efforts currently underway in our lab aim to produce mechanistic bioelectric models of individual cells and cell networks to quantitatively explain the discrete stable positions of such networks, the stochastic nature of each ‘run’ of such a network after amputation, and the molecular links to downstream cell behaviors [94]. Biologists at Tufts University have managed to get a species of flatworm to grow heads and brains characteristic of another species of flatworm without changing the genome sequence. The work reveals physiological circuits as a new kind of epigenetics -- information found outside genomic sequence -- which determines large-scale anatomy. The finding that the shape of the head is not hardwired by the genome but can be overpowered by manipulating electrical synapses in the body suggests that differences in species could be partially determined by the activity of bioelectric networks. The discovery could help improve understanding of foetal damage and renewal by revealing a new path to control complex pattern formation. It has long been known that neural networks utilize bioelectric synapses to store and rewrite information in the brain. The results are detailed in the cover of the November 2015 edition of the International Journal of Molecular Sciences, which is published online Nov 24. "It is generally intended that the sequence and structure of chromatin -- materials that make up chromosomes -- determines the shape of an organism, but these results show that the function of physiological networks can override the species-specific standard anatomy," says the magazine's senior and corresponding author Michael Levin, Ph.D., who holds the Vannevar Bush chair in biology and leads the Center for Regenerative and Developmental Biology in the School of Arts and Sciences at Tufts. "By modulating the connectivity of cells via electrical synapses, we were able to derive head morphology and brain patterning belonging to a completely different species from an animal with a normal genome." Knowing how form is determined and how to influence it is important because biologists could use that knowledge for example to remedy birth defects or make new biological structures grow after an injury," Levin adds. "These findings raise major questions about how genes and bioelectric networks interact to build complex body structures," he says. Working with Girardia dorotocephala -- free-living flatworms, which have remarkable regenerative capacity -- researchers were able to induce the development of various species-specific main forms by interrupting gap crossings, which are protein channels that enable cells to communicate with each other by sending electrical signals back and forth. The changes were more than skin depth; they included not only the overall shape of the head but also the shape of the brain and the distribution of the worm's adult stem cells. The ease with which a particular form could be coaxed from a G. dorotocephala mask was proportional to the proximity of the target mask on the evolutionary timeline. The closer the two species were related, the easier it was to bring about the change. This observation strengthens the connection to evolutionary history, suggesting that modulation of physiological circuits may be another tool developed to change animal body plans. However, unlike the previous work of the Levin Lab, in which another species of planaria could be permanently changed to a two-headed morphology, this change of form was only temporary. Weeks after planaria completed regeneration to the main forms of the second species, the worms again began to reshape and re-acquire their original main morphology. Further research is needed to determine how this happens. The authors also presented a calculation model that explains how changes in cell-to-cell communication can give rise to the different form types. The first author of the paper was Tuft's undergraduate Maya Emmons-Bell, a senior majoring in biology. "We have shown that the electrical connections between the cells provide important information for species-specific patterning of the head during regeneration in flat flat worms," she says. "This type of information will be crucial for advances in regenerative medicine, as well as a better understanding of evolutionary biology. As an undergraduate, it has been a fantastic experience to conduct and author important research with leading biologists. The interdisciplinary research involved U.S. and Canada-based biologists and European mathematicians. What is a summary of the above article?
– Kan maskar odla en annan arts huvud och hjärna? Då kanske vi kan göra bättre ifrån oss på att regenerera våra egna organ och vävnader. Det är syftet med en ny studie av forskare som fick plattmaskar att odla huvuden av andra plattmaskar utan någon förändring av maskarnas DNA, rapporterar LiveScience. Forskarna ändrade precis hur proteiner kommunicerade mellan plattmaskarnas celler. "Det är vanligt att man tror att kromatinets sekvens och struktur – det material som utgör kromosomer – bestämmer formen på en organism", säger forskaren Michael Levin i ett pressmeddelande. "Men dessa resultat visar att funktionen av fysiologiska nätverk kan åsidosätta den artspecifika standardanatomin." Forskarna började med att halshugga sötvattensplattmaskar som kallas Girardia dorotocephala, som är i stånd att returnera avhuggna vävnader; de har stamceller som kan förvandlas till någon annan typ av cell (vi har liknande celler, men bara som tidiga embyros). Forskare använde sedan en sorts alkohol för att förändra elektriska impulser som celler sänder genom proteinkanaler i maskarna, rapporterar IFL Science. Otroligt nog växte några av maskarna huvuden som var rundade, trekantiga eller hade spetsiga öron – och alla från andra arter. Maskarna växte så småningom tillbaka sina egna huvuden, med sitt DNA helt intakt. "Denna typ av information kommer att vara avgörande för framsteg inom regenerativ medicin, samt en bättre förståelse för evolutionsbiologi", säger Maya Emmons-Bell, studiens första författare.
- Can worms grow another species' head and brain? Then perhaps we can do better to regenerate our own organs and tissues. This is the purpose of a new study by scientists who made flatworms grow heads of other flatworms without any change in the worms' DNA, reports LiveScience. The researchers just changed the way proteins communicated between the cells of the flatworms. "It is common to believe that the sequence and structure of chromatin – the material that makes up chromosomes – determines the shape of an organism," says researcher Michael Levin in a press release. "But these results show that the function of physiological networks can override the species-specific standard anatomy." The researchers began by decapitating freshwater flatworms called Girardia dorotocephala, which are able to return severed tissues; they have stem cells that can turn into any other type of cell (we have similar cells, but only as early embyros). Scientists then used a kind of alcohol to alter electrical impulses that cells transmit through protein channels in the worms, reports IFL Science. Amazingly, some of the worms grew heads that were rounded, triangular, or had pointed ears — and all from other species. The worms eventually grew back their own heads, with their DNA completely intact. "This type of information will be crucial for advances in regenerative medicine, as well as a better understanding of evolutionary biology," says Maya Emmons-Bell, the first author of the study.