While axon fasciculation plays a key role in the development of neural networks, very little is known about its dynamics and the underlying biophysical mechanisms. In a model system composed of neurons grown ex vivo from explants of embryonic mouse olfactory epithelia, we observed that axons dynamically interact with each other through their shafts, leading to zippering and unzippering behavior that regulates their fasciculation. Taking advantage of this new preparation suitable for studying such interactions, we carried out a detailed biophysical analysis of zippering, occurring either spontaneously or induced by micromanipulations and pharmacological treatments. We show that zippering arises from the competition of axon-axon adhesion and mechanical tension in the axons, and provide the first quantification of the force of axon-axon adhesion. Furthermore, we introduce a biophysical model of the zippering dynamics, and we quantitatively relate the individual zipper properties to global characteristics of the developing axon network. Our study uncovers a new role of mechanical tension in neural development: the regulation of axon fasciculation.DOI:http://dx.doi.org/10.7554/eLife.19907.001 As an animal develops, neurons within the nervous system connect with one another to form complex networks. Each neuron has a long cable-like protrusion known as an axon that establishes connections with other neurons. The axon has a structure called the growth cone at its tip, which navigates toward its target in response to signals produced by the surrounding tissues.Newly growing axons may bundle together or with previously grown axons, which helps them to move along a common path. Individual axons can later detach from the bundle to reach their specific target. It is generally thought that the growth cone controls axon bundling by latching on to the shaft of a neighboring axon and then moving along it. However, this viewpoint does not take into account possible dynamic adjustments in the adhesion of the shafts behind the growth cone.Šmít, Fouquet et al. have now grown neural explants taken from the nasal tissue of mouse embryos in the laboratory and used video microscopy to record how the axons grew. The growing axons formed progressively larger bundles without direct involvement from the growth cones. Instead, the shafts of the axons stuck together in a way that resembles fastening a zipper.Šmít, Fouquet et al. manipulated the ‘axon zippers’ and observed that zippering arises from a competition between two forces: the contact force that causes two axons to adhere to each other (which favors zippering) and the mechanical tension that arises from internal or external pulls on the axon (which favors unzippering). More research is now needed to directly observe zippering in developing animals in order to understand how it helps the nervous system to assemble.DOI:http://dx.doi.org/10.7554/eLife.19907.002 In the developing nervous system, complex neural networks are built through the growth of axons from their neuronal cell body of origin toward their target(s), according to specific pathfinding patterns . These pWhile the interaction of growth cones with other axons has been the focus of numerous studies , other aSince it is currently technically challenging to image mouse OSN axon fasciculation and defasciculation dynamics in vivo, and impossible to manipulate in vivo the individual axons in order to assess their biophysical properties, we chose to perform our study on embryonic OE cultured explants, grown on a permissive planar substrate. Using time lapse imaging, we recorded OSN axons as they grow from the explants, and characterized their dynamic interactions. Surprisingly, we observed that OSN axons interact extensively with each other through their axon shafts, leading to zippering and unzippering behaviors that trigger their fasciculation or defasciculation, respectively. In the present paper, we characterize the dynamics of these axon-axon shaft interactions, assess quantitatively the biophysical parameters of these processes, and develop a biophysical model of this dynamics. Micromanipulations of individual zippers, as well as pharmacologically induced perturbations of the fasciculated network, are used to demonstrate unzippering by forces of functionally relevant magnitude. Our analysis supports a framework in which axonal mechanical tension regulates fasciculation through the control of axon shaft zippering.OSN axons grow in cultures as unbranched axons. In our experimental conditions , the growth of these axons from OE explants was characterized by a sequence of three main stages: (1) advance of the growth cones and initiation of an axon network (first 24–48 hr), (2) maintenance of the growth cones at distance from the explants, but with little or no further growth (48–72 hr), and (3) retraction of the growth cones and collapse of the network (3–5 d). We analyzed in detail the intermediate stage, during which axon shafts interact and form bundles.During this stage, the initial axon network, composed of individual axons or bundles of few axons, progressively evolves into a less dense network with thicker bundles, indicating that individual axons and/or small bundles fasciculate together to form larger bundles . To charTo understand the processes that lead to this coarsening of the axon network, we examined its dynamics on finer time scales. On time intervals of the order of minutes, we observed elementary zippering processes, as shown in ilibrium . In the Numerous and frequent zippers were observed throughout the network, as demonstrated in To assess the axonal structure of the zippers, we performed scanning electron microscopic analyses of our cultures . We obseMore rarely, we observed zippers with entangled structure . Such stpictures  to find Besides axon shafts and their bundles, thin lateral protrusions emerging from the shafts are observed in the network . These pThe observations reported above indicate that the progressive coarsening of the axon network results from zippering events driven by adhesion between the axon shafts.In recorded time lapse sequences of network evolution in 13 explants, we typically observed that the axon network coarsened in a manner similar to First, we envisaged treatments aiming at rapidly enhancing growth cone motility, in view of increasing the pulling force exerted by the GC on axon shafts. Since the molecular cues having such effects on OSN explant cultures remain unknown, we tested in a first approach Foetal Bovine Serum (FBS), assuming that some of growth factors it contains may stimulate axon outgrowth. Interestingly, while no obvious effect on the growth cones was observed upon FBS addition to the culture, we found that the application of 5% FBS reliably induced the explant pull. This is likely due to a cell-rounding effect of FBS on cultured neurons, previously demonstrated in As an alternative means to influence axon tension, we tested blebbistatin, a well known inhibitor of neuronal Myosin II (NMII), previously shown to decrease cell cortex/membrane tension in a variety of non-neuronal cells . SomewhaWe took advantage of the stabilizing effect of blebbistatin to facilitate the analysis of the coarsening or de-coarsening effects generated by subsequent treatments. In In the example of As a complement to the observed de-coarsening induced by a pulling force, we sought to perturb the network dynamics by decreasing the tension in the axons. In previous literature, cytochalasin, an inhibitor of actin polymerization, was shown to significantly lower the tension of PC-12 neurites . Indeed To understand the conditions leading to zippering or unzippering, we analyze the force balance in a zipper of two axons . The cone zipper , the forwhere er angle  and S isondition  is no loThe balance of forces at a junction of three axon segments was previously considered in , and useTo support the explanatory framework presented above, we carried out micro-manipulation experiments designed to measure the magnitude of the inter-axon adhesion force It is known from previous literature that the typical value of mechanical tension in an isolated axon grown in culture is of the order of 1 nN  reports , 1989, aUsing the BFP technique, we determined the tension in a collection of thin network segments, presumably individual axons, even though we cannot exclude that some of them might have been fascicles of several axons. The basis of the measurement is force equilibrium between the calibrated force of the probe acting on and deforming the axon, and a restoring force, which arises from the tension in the axon shaft. The measurement is described in acements , until t∘. sinδ . The sloFor one of the axons, two distinct values of tension were measured early and late in the experiment: (432 ± 157) pN and (1665 ± 219) pN. This increase in tension was likely caused by the strong stretching of the axon that occurred during this particular experiment—see Using the slope values and their errors calculated from the seven remaining linear fits, we estimated the distribution of the tensions in the axon population, shown in Technical limitations were encountered in these experiments, including the uncontrollable bead localization along the axons and with respect to zippers, as well as early detachment of beads from the red blood cell upon pulling. The seven measurements included in As zippers that are entangled as in  may remaBased on the measured distributions of axon tensions and of equilibrium zipper angles, we then estimated the axon-axon adhesion force To determine the axon adhesion force more directly, not relying on the measurement of axon tension, we attempted to unzipper selected zippers using a calibrated pulling force. These attempts were not successful, due to insufficient strength of the bond between the bead and the red blood cell. This resulted in the detachment of the red blood cell before any significant effect on the zipper. To overcome this limitation, we bypassed the red blood cell and bead and used the pipette to drag the axon directly. This allowed us to use forces sufficiently large to induce unzippering at the price of losing the knowledge of the force magnitude. Similarly to these cases of induced unzippering/rezippering, we view the numerous individual zippering processes observed in the developing network  and 3 asdvancing  and recedvancing  zippers,Our analysis of equilibrium zipper configurations cf.  was baseWe first describe the frictional force arising from the stretching or shortening of axons (which necessarily occurs during zippering or unzippering). Within the linear viscoelasticity framework, the viscous stress in each axon is proportional to the local strain rate. Assuming a uniform elongation strain in between the axon fixed points, the strain rate is simply expressed as where In addition to axon elongation/shortening, another possible source of energy dissipation consists in changes in the axon configuration in the immediate vicinity of the vertex. When the vertex advances during zippering, new regions of the axons undergo bending/unbending , with corresponding viscoelastic losses. Possible non-equilibrium binding effects at the newly adhering membrane region may also result in dissipation. These energy losses are expected to result in a localized frictional force that acts at the vertex and is anti-parallel to the vertex velocity component along the axis of the zipper; that is, this frictional force is collinear with the adhesion force where pper see . Thus, tThe balance of forces at a moving vertex may now be readily expressed. Consider for simplicity the case of a symmetric zipper . The dynamics preserves the symmetry, that is, an initially symmetric configuration  the friction of the axons with the substrate. Our experimental observations as in  show thaOur observations of spontaneous zippering processes in the developing network showed that the zippering velocity typically remained approximately constant , with 7–10 time points per experiment at which the zipper angle distribution was extracted. The typical shape of the distribution is shown in experiment 1 corresponds to the time interval in Evaluating the relation between the zipper angle distribution and the network coarsening, we found a consistent trend in the five analyzed experiments. The median zipper angle To propose an explanation of this observation, we return to the distribution of single-axon tensions obtained using the BFP technique . We assuTo evaluate how this is reflected in the distribution of zippering angles, we use To qualitatively asses the changes of Xenopus embryos. In Strong connection points can be established between our dynamical observations and the in vivo observations of Roberts and Taylor , who stuIn addition to extracting the distribution of incidence angles for fasciculated neurites, Roberts et al. determined the probability for two neurites to cross (rather than fasciculate). This crossing probability gle βinc . Using oce angle . Given tAxon fasciculation is generally thought to be controlled during development at the level of growth cones, which may adhere to other axons in their environment (hence driving fasciculation), or may separate from other axons (hence driving defasciculation). Here, we provide strong evidence for an additional mechanism playing a critical role in regulating axon fasciculation, which does not involve growth cones, but takes place at the level of axon shafts, through zippering of individual axons or small bundles of axons. In our culture of embryonic olfactory epithelium explants, this mechanism resulted in a substantial reorganization of the structure of a grown axon network, on the time scale of 10 hr. The process of axon zippering has been rarely reported or discussed in previous literature. Axon zippering in vivo was inferred in −16−16 to 4 × 10−15) Using a combination of experimental observations and biophysical modeling, we showed that axon zippering arises from the competition of two principal forces: axon-axon adhesion and mechanical tension. The adhesion force favors an increase in the length of the zippered segment. The mechanical tension tends to minimize the total length of the axons, thus favoring unzippering. The relative strength of these two forces determines the vertex angle between axons in a zipper that reached static equilibrium. We used the BFP technique to measure the mechanical tensions of the OSN axons grown ex vivo, and obtained values (interquartile range (529–833) pN) comparable to tensions reported in the previous literature for PC-12 neurites grown in culture, on average around 650 pN . CombiniWe were able to unzip selected zippers by manipulating them with micropipettes, with consequent re-zippering after the manipulation was stopped. By fitting such induced zippering/unzippering dynamics to a basic biophysical model, we obtained an independent estimate of the axon-axon adhesion force, comparable with the estimate based on static observations. Comparing the shape of the observed zipper trajectories with the trajectories predicted by the dynamical model, we inferred that the zippering dynamics is limited by energy dissipation arising at the zipper vertex, and estimated the corresponding friction coefficient nce cues , the infnce cues , as wellnce cues . The preIn the previous literature, the tendency to fasciculate was often interpreted as arising from differential adhesion, that is, the growth cone having stronger adhesion to another axon than to the substrate . The strA striking observation, in our culture system, is that the axon network, initially established as a complex network of individual axons or small bundles of axons, progressively coarsens in time, leading to the formation of large fascicles of axons. The coarsening persists over time scales of about 10 hr, which is much longer than the typical time scale for an individual zippering process (10 min). It is therefore unlikely that the coarsening is a result of protracted equilibration of zipper configurations under stationary force conditions. A possible explanation of the slow coarsening dynamics may lie in a gradual decrease of the average axon tension as the culture matures, which would lead to increasing domination of axon-axon adhesion forces over tension, hence favoring zippering. This proposal is supported by the observed sequence of stages of the culture maturation: axon elongation in the early stage, insignificant elongation in the intermediate stage and axon retraction in the final stage. According to To directly test if a decrease in average axon tension leads to coarsening, we used cytochalasin, a drug that was previously shown to significantly decrease the tension of PC-12 neurites . Indeed,As a second strategy aiming to perturb axon tension, we tested blebbistatin, an inhibitor of NMII, previously shown to decrease cell cortex/membrane tension in non-neuronal cells  and to dSince the biological agents having the ability to specifically stimulate axon growth or motility in our cultured explants are currently unknown, we tested FBS for this purpose, because of its established content of a variety of bioactive molecules and growth factors. It turned out that FBS did not have any significant boosting effect on OSN growth cones but, very interestingly, it induced the apparent contraction of the whole explant itself, leading to the generation of pulling forces on axons from the explant core. This apparent contraction of the explant is likely to be the result of changes in the shape of the individual cells constituting the explant, if such changes lead to an overall rounding of previously flattened or elongated cells. In line with this hypothesis, in , the serA quantitative estimate of the increase in axon tension due to the FBS-induced pull may be obtained from the measured stretch of the axons and the expected axon elongation stiffness. The measurements of , 1989 sheriments  is expeceriments .We have thus shown on the network level that the extent of axon fasciculation can be regulated by changes in axon tension: an overall decrease in tension leads to zippering-driven coarsening, while an overall increase in tension leads to unzippering and de-coarsening. The estimated tension increase (of order 1 nN) generated endogenously by the FBS-induced explant pull is comparable in magnitude to active changes in axon tension demonstrated in previous literature, such as the tension recovery within 30 min after axon unloading in vitro  and in vAs the axon network coarsens, the zippers become predominantly formed by axon fascicles, rather than by individual axons. We derived the expected distribution of tensions among the fascicles and combined it with the expected scaling of the fascicle-fascicle adhesion force  to predict how the distribution of equilibrium zipper angles in the network depends on the mean number of axons per fascicle. This theoretical prediction was consistent with the observed relation between the median zipper angle and network coarsening, thus supporting the framework in which zippering is controlled by the competition between tension and adhesion not only for individual axons, but also on the fascicle level.A direct test of our theoretical models, be it on the single-zipper level or on the network level, would require a measurement of tension changes in the evolving network and following pharmacological manipulations. The tension generated by the pull of the growth cone may be efficiently assessed using traction force microscopy . In thisTopologically, the structure of the axon network observed in our study is similar to the structure of foams (froths) with a low liquid fraction. A froth consists of gas bubbles that are separated by liquid film walls, with surface tension in the walls . The typT2 process is illustrated for a three-sided bubble in T1 in the literature) observed in coarsening froths is shown in T2 and T1 rearrangements, the froth coarsens  has no topological analogue in the axon network. Rather, an attempt to implement such a process leads to a complete unzippering  was converted into a ‘living froth’ when oryzalin was used to depolymerize microtubules attached to the cell walls. In the resulting tissue, the topology and geometry of the cell interfaces was consistent with a typical froth, and pronounced coarsening of the structure was observed during the plant growth. The system we investigated—the zippering axon network—presents a remarkable example of an ex vivo system exhibiting both topological and dynamical analogies to froths. In our system, the coarsening is not limited by any cell volume restrictions and can proceed rapidly, on the time scale of hours. However, structural features such as complex loop configurations and entangled zippers can limit the final extent of coarsening.Analogies between froths and biological tissues consisting of closely packed cells have been investigated in previous literature (e.g. ). Such aWe report in the present paper that OSN axons grown on a planar glass substrate covered homogeneously with polylysine and laminin display extensive zippering behavior, raising the possibility that these axons, and more generally other types of axons with adhesive interactions, may zipper in vivo.Xenopus embryos, the geometry of the sensory neurite plexus on the basal lamina of trunk skins shows features that are likely to be the result of axon zippering, as proposed by Roberts and Taylor , never myelinate, cover nor even separate individual axons from each other: they instead wrap large bundles containing up to thousands of naked axons . Within An appealing hypothesis is that the zippering of olfactory axons may participate in the sorting of olfactory axons. This sorting is indeed critical given the facts that each OSN expresses one odorant receptor (OR) gene, picked out of a large repertoire of roughly 1,000 OR genes, and that the axons of all OSNs expressing the same OR (these OSNs are distributed across a large zone of the OE) converge into a few glomeruli of the OB (reviewed in ). This pMore generally, what are the expected functional consequences of regulated axon shaft zippering in vivo? First, early on as a growth cone navigates toward its target, the ability to zipper can regulate the probability with which it would cross a fascicle or not, as we illustrated in Results with the Roberts and Taylor data . Second,In light of our analysis, there are two principal ways by which zippering and hence the extent of fasciculation may be regulated in vivo. In a developing network, the growth cone activity or shaft cytoskeleton activity  can chanThe tension of an axon shaft is influenced by the traction force generated by its growth cone, which in turn depends on the mechanical properties of its environment (reviewed in ), likelyelopment  may gradIn conclusion, our work shows that adhesion-driven zippering of axon shafts can induce the formation of axon fascicles without a direct involvement of the growth cones. However, active changes in the pulling force at the growth cone, and hence in axon tension, may be used as a mechanism to control the extent of zippering and to regulate fasciculation/defasciculation. Mechanical tension has been shown to play important roles in neural development (reviewed in ), and re2) until the day of experiment in a culture medium of DMEM/F12 (Gibco 31331) containing 1% N2 (Gibco 17502), 0.1 All animal procedures were approved by the Île de France Ethics Committee. Pregnant female Swiss mice were sacrificed by cervical elongation at embryonic day 13.5 (E13.5), embryo were extracted from the uterus, and olfactory epithelium explants were prepared from the posterio-dorsal quarter of the septum and turbinates as follows. First, these posterior and dorsal parts of septa and turbinates were cut into pieces in L15 medium  maintained on ice at 4°C, before being subsequently incubated for 30 min at 25°C in a solution of 1:1 of Trypsin 0.25% (Gibco 25050) and Pancreatin 4X USP (Gibco 02-0036DG) to allow the OE to separate from the lamina propria. Enzymatic reactions were stopped by adding 10% Fetal Bovine Serum , and the biological material was rinsed in ice-cold L15 containing 5% FBS. Pieces of tissue were transferred into a glass Petri dish in which the OE sheets were cut, using a micro-scalpel, into small pieces of about (100–200) μm diameter each. Explants were then carefully transferred into 50 mm diameter IBIDI video dishes  that included a 35 mm glass coverslip (for BFP experiments), or into IBIDI μ-slide eight well #1.5 polymer coverslip  (for time lapse acquisition), previously coated with poly-L-lysine  % relative humidity) using a DIC 6310 mM Hepes was added to the explant cultures 1 hr before starting time lapse acquisitions. In some experiments, the cultures were treated with FBS , blebbistatin , cytochalasin B , or trypsin . Videomicroscopy was performed on a Leica DMI 6000B  inverted microscope in a thermostated chamber , and 10 explant where FBS was added (pre-treated with blebbistatin in five cases). Major criteria for selection for quantitative analysis were good contrast, culture survival, and sufficient area and density of the network. The reported data are based on analyses performed for (i) ion Fiji  of the pion Fiji . The fieion Fiji , where aThe segmentation selections underlying the analysis shown in Scanning electron microscopy explants were cultured on a 14 mm diameter coverslip (as described above), fixed for 1h at 4°C in 2% glutaraldehyde prepared in 0.1 M sodium cacodylate buffer, rinsed in cacodylate buffer, dehydrated in a series of graded ethanol baths, and dried using a critical point dryer . They were finally mounted on a carbon stub and sputter-coated. Observations were made using a Cambridge Instruments Stereoscan 260 scanning electron microscope equipped with a digital camera.The implementation of the BFP method  was adapwhere For these experiments, 2 days in vitro (DIV) OE explants, cultured in 50 mm IBIDI dishes, were biotinylated using EZ-Link Sulfo-NHS-SS-Biotin  according to the manufacturer instructions. Dishes were then transferred into the thermostated chamber (37°C) of the Leica DMIRB inverted microscope equipped with micropipette manipulators and a CCD digital camera . Streptavidin beads were added to the culture, a micropipette (1.5–2 μm inner diameter) was filled with the culture medium and fixed onto the mechanical micropipette manipulator . The diae medium .Then, a RBC was aspirated into the micropipette  was transformed into a distribution, by convolving the dataset with a Gaussian kernel (using Matlab's kernel distribution functions).Two complementary approaches were used to estimate the value of axon-axon adhesion strength where the relation between For the general asymmetric axon zipper , one cannot in principle exclude active adjustments of axon tension accompanying the zippering or unzippering, or a coupling to active intracellular transport processes. In previous literature, a recovery of tension within 15–60 min was shown for axons that were made slack following a large rapid distension . CompareUnder these assumptions, the instantaneous zipper configuration is fully specified by the Cartesian coordinates where where A zipper is in a static equilibrium when d α2 see . In the while in the perpendicular directionGiven the parameters A nonzero driving force where the friction tensor The form of the vertex friction tensor depends on the dominant mechanism of energy dissipation. In the main text, we introduced two forms of internal energy dissipation in the axons—the viscosity of elongation/shortening, and the vertex-localized dissipation. As the corresponding frictional forces are collinear with the axon tension and with the axon-axon adhesion force, respectively, one may substitute the ‘dynamically corrected’ tension and adhesion magnitudes  and three reviewers, one of whom is a member of our Board of Reviewing Editors. The reviewers have opted to remain anonymous.Thank you for submitting your article "Axon tension regulates fasciculation/defasciculation through the control of axon shaft zippering" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Your paper has been well received, and all three reviewers thought it was of interest to the field, extending across subfields and disciplines. The theoretical and biophysical aspects of axon fasciculation have rarely been considered and so this study would be of great value. Please see the attached reviews.We will consider a revised manuscript that addresses the concerns of the reviewers. Aside from the requests for textual edits and clarification of presentation for those not in theoretical biology and biophysics, the major revisions called for the inclusion of analyses that measure tension at different time points in culture, and/or interfere with tension, to demonstrate a correlation between tension and the ratio of zippering/unzippering events. We believe that these experiments would substantially strengthen the paper.Please let us know as soon as you are able whether these requested experiments look to be feasible and if so, whether you anticipate that they can be completed in less than about two months' effort. Please be advised that your revised manuscript will be re-reviewed and acceptance is not guaranteed.Reviewer # 1This manuscript uses embryonic mouse olfactory epithelium explants to study axon fasciculation, namely to address the biophysics of axon shaft-shaft interactions in fasciculation/defasciculation dynamics. The work is well executed and well presented, and represents a paradigm shift in how the field thinks about and understands fasciculation, a phenomenon classically considered to occur at the level of growth cones rather than along the axon shaft.One of the strengths of the manuscript – its relevance to researchers across disciplines – is also its main challenge. The subject matter is relevant to developmental neurobiologists and those with a biophysical background. Some sections may be either unclear in their content or significance to those in the former audience pool. For instance, most developmental neuroscientists are likely to be unfamiliar with the details and significance of foam dynamics. This part of the Discussion as well as some of the nuances of the modeling could be clarified and better summarized in order to reach a wider audience and successfully communicate the full depth of the work. That being said, the authors have largely succeeded in presenting their findings in a way that is of interest and relevance to a wide and somewhat diverse audience.1) The estimates of force while expressed in Equation 1, should ideally be tested experimentally; this would take extra experiments that may take quite some time to execute, but would be important to perform.2) The authors touch on the possibility of the increase in zippering resulting from progressive decrease in axon-substrate adhesion, but this idea could be fleshed out a bit more. Have substrates other than polylysine/laminin been tested? Or has the concentration of laminin been varied?3) In the fifth paragraph of the Results, the authors indicate that entangled axon structures appear more rarely. Is it possible to roughly quantify the relative ratios of parallel, crossing, and entangled axon-axon interactions? It would be informative to provide a distribution of types of axon shaft interactions.4) The conclusion presented in the third paragraph of the Discussion is reasonable and accurate, i.e. that axon fasciculation observed in the experiments presented are likely OR-independent. However, the experiments and results presented here do not rule out the possibility that OR expression (or other molecules in this or other systems) could change the axon shaft-shaft dynamics if observed at a different time point. Obviously directly studying the effects of different molecular expression on axon zippering behavior is beyond the scope of this study, but it would be interesting to provide a further discussion of how molecular expression might affect zippering. This is addressed somewhat in the twelfth paragraph of the Discussion, but perhaps it is worth either combining these two parts of the Discussion or fleshing out the first section somewhat.5) Despite the thorough biophysical, computational and microscopical analyses, the question remains as to the functional significance of axon zippering along shafts observed in this culture setting.C. elegans  describes an Ig-domain molecule important for maintenance of axon tracts. One could imagine that in some diseases or in the normal lifespan that if some axons degenerated, there might be zippering (of unmyelinated axons) in tracts to preserve integrity. In addition, after injury (transection) of axons, any regenerating axons, or the remaining axons, would "zipper up" in the fashion described. The authors are urged to read up on axon regeneration, especially the recent work of Zhigang He  after optic nerve crush or transection of retinal ganglion cell axons more distally, that could suggest some points of discussion in this regard.A) Fasciculation is often studied during early development, mainly in context of a pioneer-follower axon paradigm. Are there conceivable functions relevant to circuit development that do not involve follower-axons? The authors should speculate on how dynamic axon fascicles are throughout later development and during the lifespan of a circuit. A study on B) The Discussion would benefit from addressing some other differences between the in vitro and in vivo systems, for instance, space constraints of axons growing along developing tracts, which are often within glial or parenchymal borders.C) Likewise, it would be important to compare what is known about stiffness /fluidity of brain tissue  and mature tissue where there is little, as well as glial processes, etc. compared to a tissue culture substratum/plastic or glass.Reviewer #2:This paper presents a very elegant experimental and theoretical analysis of the biophysics of axon zippering. The data is of high quality, and the modelling is well done. The model is a nice balance of biophysical detail and simplicity. A highlight is being able to compare the high-quality images with diagrams of the model  which is a great aid to intuition. The annotations on the images are also very helpful in communicating the methods efficiently.I list below a number of issues which could potentially be addressed to improve the manuscript.1) The current manuscript does not appear to offer any new insight into the molecular mechanisms underlying fasciculation/zippering .2) It is perhaps not clear enough what new insight has been gained from this work about how neural wiring develops in vivo. At the very least I suggest expanding on the three points in the eleventh paragraph of the Discussion.3) There is a lot of analysis of the topological properties of the axon network (e.g. number of vertices). This may be of mathematical interest, but the biological relevance is currently not clear. Does this network behaviour confirm or rule out particular microscopic mechanisms, or is some importance of the structure itself being claimed?4) There are a few places where results seem to be based on very low n values. For instance, in Figure 1H the results seem to be just from one small window from one explant. It would be reassuring to know that these results generalise. More generally, the number of explants contributing to each result should always be stated.5) Readability could be improved. Although very clearly written, two problems are as follows. (i) The manuscript is currently extremely long – about 15,000 words by my estimate, making it a bit of a marathon for the reader. (ii) Neither the Results or Discussion sections are currently split up into any clearly delineated subsections.6) New results are introduced in the Discussion section : I feel these should be in the Results.7) The reader is assumed to be familiar with the biophysics of foams. Some introductory background information in this regard would be helpful, and more discussion of what the reader should take away from this comparison.Reviewer #3:In this manuscript, Smit et al. present measurements of axon tension in a 2D culture system, observe axonal network morphologies, zippering and unzippering events of axons, and develop a mathematical model to describe the process. The biophysics of axon fasciculation has so far rarely been considered, and a thorough understanding of the process would be important to better understand the development of the nervous system.While the presented idea is very nice, and the model looks good to me, the experiments (on which the model is based) seem to be rather preliminary and not conclusive. I have the following specific concerns with the manuscript in its current form:1) The relation of branching angles  and axon tension has been investigated in the past. Particularly the 1979 paper from Dennis Bray and Shefi at al., Neurocomputing, 2004, which already present mathematical models to connect tension and axon morphology / branching angles, need to be cited.2) The measurement of axon tension (only in static neurons) using the BFP technique is not ideal. Biotinylation of axons might already change their mechanical properties, calibration of the force probe (an RBC) seems to be a bit crude and not very accurate (how large is the estimated error of the measurements?), numbers of experiments are very small, and a dynamic system could not be investigated with it. To validate the model, and to directly test the relation between tension and zippering angle, one would have to measure the development of tension during zippering and unzippering events, which really should be done. This would also show if un/zippering of axons in the network is primarily because of a change in tension or adhesion. Other methods  might be better suited for such measurements.3) The force balance in Equation 1 does not seem to take the force required to deform / bend the axons into account. I'm not sure if this is an oversimplification.4) An important experiment, which is currently missing but which would be required to test Equation 1, is the direct measurement of the adhesion force. This should be done. Again, potentially suitable techniques include magnetic tweezers, calibrated microneedles, or AFM.5) Ideally, the system should also be experimentally perturbed to test the model. For example, tension could be manipulated by interfering with contractility , and adhesion using knockdown of cell adhesion molecules.6) It appears that the zippering process is considered a purely passive event. Active cellular processes, such as transport etc., will, however, also be involved. This should at least be discussed.7) The interpretation of the development of zippering angles shown in Figure 12C is not supported by the data shown. For example, the statement "the mean angle decreased slightly from 62° to 56°" suggests a steady decrease over time, which, however, is not the case. The distributions at 60 minutes and 180 minutes look very similar to me. The statement "The predicted decrease in mean zipper angle is thus 7.5° , in good agreement with the experimentally observed change of 6° " doesn't work for that reason – it is only 1° difference after 2 hours (if comparing 60 mins and 180 mins), which might very well be noise. I doubt that a statistical analysis (which should be done) would reveal a low P value. Also, the boxplots actually suggest that the distribution is more asymmetric at 180 mins than at 60 mins – so I'm not convinced by the usefulness of the skewness parameter s. N is very low here as well for a proper statistical analysis. Together, I don't see the trend the authors mention, and I strongly recommend them to reconsider the interpretation of these experiments.eLife. Your revised article has been favorably evaluated by David Van Essen (Senior Editor), a Reviewing Editor and two reviewers.Thank you for resubmitting your work entitled "Axon tension regulates fasciculation/defasciculation through the control of axon shaft zippering" for further consideration at The two reviewers found that you have addressed the bulk of their comments and that your revised manuscript is improved, but there are some remaining issues that need to be addressed before acceptance, as outlined below.One of the reviewers had a remaining concern – to test your model experimentally and directly measure using the BFP technique the change in tension following the application of blebbistatin. The hope was that you could have related the changes you observed in network morphology to changes in tension.When the Senior Editor asked on February 25 whether this was possible, you wrote in a letter on March 3 that several technical and logistical factors made it extremely difficult to make the requested tension measurements. You explained why the experiment would not be possible at present, and provided significant new information that further strengthened your case.In our discussion online, reviewer 3 further commented that in published experiments, blebbistatin decreases tension and agrees that there is currently no study showing that this is also true for axons. He pointed out that if axonal tension was decreased after treatment, then the network would not behave as the model predicts, and therefore, this reviewer continued to feel that it would be important to show how blebbistatin impacts axonal tension. However, he now acknowledges, based on your response letter, that this experiment is not feasible in your lab.We are therefore pleased to inform you that we will proceed with your manuscript without including the force measurements.We request revisions that would incorporate the data and arguments in your rebuttal letter:1) Comment on the blebbistatin experiments, as above.2) Discuss the possibility (and caveats) of using thermal fluctuation spectroscopy).3) Highlight your (novel) use of embryonic olfactory epithelium explants, highlighting that this preparation presents "the right balance of axon tension and axon-axon adhesion" that provides insight into axon zippering and progressive fasciculation.4) Add from your response letter the figure on FBS-induced increase of axon tension, as Figure 5—figure supplement 1.5) Add the data from the figure on cytochalasin-induced reduction of axon tension, pointing out, as per your prediction, that the cytochalasin-induced tension reduction was expected to be comparable for all directions within the network, in contrast to the FBS-induced pull in one dominant direction.6) Strengthen the "take-home" message that your results demonstrate that the extent of axon zippering can be controlled by axon tension forces, and that future work could aim for force measurements to directly verify the increase in tension in the FBS experiments and the decrease in tension in the cytochalasin experiments.7) Throughout the manuscript, stress that axon zippering is controlled by tension changes of functionally relevant magnitude.We all agree that the paper shows a fundamentally new mechanism of axon fasciculation via axon zippering, and that it will be an important contribution to the field.Reviewer #2:I am happy with how the authors have addressed my comments, and have nothing further to add.Reviewer #3:The authors have addressed most points and have overall improved the manuscript.However, they fail to provide experimental evidence confirming their model. Particularly the experiments aiming to perturb network tension are inconclusive: As the authors state in their rebuttal, the question here was to find a means to experimentally perturb axon tension. However, I do not see any measurement of axon tension in the perturbed systems , so how do we know if and how the treatments changed axon tension? What if, for example, axon tension does drop after blebbistatin treatment and yet the network zippering does not change as predicted? This would contradict the model. Thus, these experiments would only be meaningful is axon tension was measured.I am also a bit puzzled by the BSA experiments. It is not clear to me what BSA is doing, or how a cell (body) rounding should lead to the contraction of the whole network? How do the authors know that FBS induces strong pulling of axons? Tension was not measured. Instead, FBS could, for example, simply interfere with adhesion, which could easily cause the observed retraction and / or de-coarsening without changing tension at all. I don't think that this approach is very helpful. Combining it with blebbistatin treatment, whose effect on the network is also not understood, does not help.As a minor point, I do not understand the motivation of the authors to use drugs known to interfere with growth cone motility. Is growth cone motility coupled to axon tension? In none of the figures presented growth cones are shown, so they are probably not relevant for the zippering / unzippering of the established network?What is shown in the new Figure 5 are a few images of parts of a network. Without proper quantification, it is very difficult to say what the effect of the treatments is. For example, the authors state that blebbistatin has a stabilizing effect on the network; however, Figure 6G-I indicate that the network is behaving the same way in treated and untreated cultures before cytochalasin treatment?Finally, I still think that it would be important to directly measure the adhesion force to test Equation 1 . […] Reviewer #1:This manuscript uses embryonic mouse olfactory epithelium explants to study axon fasciculation, namely to address the biophysics of axon shaft-shaft interactions in fasciculation/defasciculation dynamics. The work is well executed and well presented, and represents a paradigm shift in how the field thinks about and understands fasciculation, a phenomenon classically considered to occur at the level of growth cones rather than along the axon shaft.One of the strengths of the manuscript – its relevance to researchers across disciplines – is also its main challenge. The subject matter is relevant to developmental neurobiologists and those with a biophysical background. Some sections may be either unclear in their content or significance to those in the former audience pool. For instance, most developmental neuroscientists are likely to be unfamiliar with the details and significance of foam dynamics. This part of the Discussion as well as some of the nuances of the modeling could be clarified and better summarized in order to reach a wider audience and successfully communicate the full depth of the work. That being said, the authors have largely succeeded in presenting their findings in a way that is of interest and relevance to a wide and somewhat diverse audience.We thank reviewer 1 for this very positive evaluation of our paper.We recognize that, in the first version of the manuscript, some of the biophysical sections were not easy to fully understand for most developmental neurobiologists. In revising the text of the manuscript, we paid close attention to this point.We consider that our new presentation of the relation to foam dynamics  is now fully accessible to a wide audience including neuroscientists, thanks to a didactic text and clear figure illustrations . Likewise, we clarified some important aspects of the modeling in order to allow non-specialists to more readily understand our approach.1) The estimates of force while expressed in Equation 1, should ideally be tested experimentally; this would take extra experiments that may take quite some time to execute, but would be important to perform.Indeed, while starting work on this project, we had planned to directly measure the adhesion force (denoted S in Equation 1) by unzippering axon shafts using a calibrated force. In conjunction with the determined tension T (using BFP or another method), this would have permitted the direct test of Equation 1 on the level of an individual zipper. Unfortunately, this procedure was not possible to carry out using the BFP technique at our disposal, as described in the manuscript. Instead, we were able to unzipper axon shafts using an uncalibrated force manipulation ; the results of these induced unzippering experiments are consistent with Equation 1, but do not provide a direct quantitative test of it.Given the limited time we had to carry out the revisions, and the expected longer time frame that would be needed to implement an alternative calibrated manipulation setup for our system, we gave priority to the pharmacological manipulations of axon tension.2) The authors touch on the possibility of the increase in zippering resulting from progressive decrease in axon-substrate adhesion, but this idea could be fleshed out a bit more. Have substrates other than polylysine/laminin been tested? Or has the concentration of laminin been varied?We indeed envisaged modifying axon-substrate adhesion because it would have allowed us to test directly the consequences on zippering. However, given the focus of our studies – namely the coarsening of a previously established network, we were concerned that changing the substrate of our culture would be likely to lead to changes in the initial development of the axon network. Hence, it would have been very difficult to establish a causal link between a new substrate with different adhesive properties, and possible changes in the dynamics of coarsening through zippering. Moreover, very little is known about the molecular mechanisms involved in OSN axon/substrate adhesion in culture. Thus, the extent to which such substrate change (or changes in laminin concentration) would change the axon/substrate adhesion would have been difficult to assess. It would require a large body of experiments that included many different substrate conditions to find conditions in which adhesion would have seem to be changed while the initial establishment of the network would have been unchanged.To bypass these difficulties, we used trypsin to induce a modification of adhesion once the network was developed on our polylysin/laminin substrate. This treatment rapidly degrades extracellular proteins, among them cell-cell and cell-substrate adhesion proteins. Unfortunately, the trypsin treatments induced a rapid collapse of the network (retraction onto the explant due to detachment of axons from the substrate), before any visible change in axon-axon adhesion. This experiment thus nicely confirmed that the entire network was under tension, but did not allow us to study changes in zippering.For all these reasons, as we undertook new experiments in the perspective of revising this paper, we decided to focus our efforts into experiments aiming at modifying axon tension.3) In the fifth paragraph of the Results, the authors indicate that entangled axon structures appear more rarely. Is it possible to roughly quantify the relative ratios of parallel, crossing, and entangled axon-axon interactions? It would be informative to provide a distribution of types of axon shaft interactions.We thank reviewer 1 for this suggestion. We have now quantified the relative abundance of simple and entangled zippers, as well as axon crossings, based on electron micrographs of the network configuration. We now provide these data in the manuscript, and show the underlying analysis in a supplementary figure .4) The conclusion presented in the third paragraph of the Discussion is reasonable and accurate, i.e. that axon fasciculation observed in the experiments presented are likely OR-independent. However, the experiments and results presented here do not rule out the possibility that OR expression (or other molecules in this or other systems) could change the axon shaft-shaft dynamics if observed at a different time point. Obviously directly studying the effects of different molecular expression on axon zippering behavior is beyond the scope of this study, but it would be interesting to provide a further discussion of how molecular expression might affect zippering. This is addressed somewhat in the twelfth paragraph of the Discussion, but perhaps it is worth either combining these two parts of the Discussion or fleshing out the first section somewhat.The Discussion has now been extensively reorganized, and it includes a full subsection dedicated to the regulation and functional significance of axon zippering in vivo, in which this issue is addressed.5) Despite the thorough biophysical, computational and microscopical analyses, the question remains as to the functional significance of axon zippering along shafts observed in this culture setting.We recognize that these aspects were not discussed enough in the first version of the paper. As reviewer 1 will see, this very important point is now extensively discussed in the “Axon zippering in vivo: its regulation and functional significance” subsection of the Discussion.A) Fasciculation is often studied during early development, mainly in context of a pioneer-follower axon paradigm. Are there conceivable functions relevant to circuit development that do not involve follower-axons? The authors should speculate on how dynamic axon fascicles are throughout later development and during the lifespan of a circuit. A study on C. elegans  describes an Ig-domain molecule important for maintenance of axon tracts. One could imagine that in some diseases or in the normal lifespan that if some axons degenerated, there might be zippering (of unmyelinated axons) in tracts to preserve integrity. In addition, after injury (transection) of axons, any regenerating axons, or the remaining axons, would "zipper up" in the fashion described. The authors are urged to read up on axon regeneration, especially the recent work of Zhigang He  after optic nerve crush or transection of retinal ganglion cell axons more distally, that could suggest some points of discussion in this regard.We are very grateful to reviewer 1 for suggesting these specific points and papers. The work from Aurelio indeed provides a wonderful example illustrating how axon shafts may zipper in vivo, in this case with detrimental consequences, and further shows how inhibition of axon/axon adhesion negatively regulates zippering. This paper is now discussed in this section. We also briefly speculate on the possible consequences of zippering in pathological conditions, and upon regeneration. However, it was unclear to us how to directly link our framework to the paper by Bei et al.B) The Discussion would benefit from addressing some other differences between the in vitro and in vivo systems, for instance, space constraints of axons growing along developing tracts, which are often within glial or parenchymal borders.This was indeed a great suggestion, and we have now addressed these differences between the in vitro and in vivo system, including space constraints, and with a particular attention on the glia/axon interactions in peripheral and central systems .C) Likewise, it would be important to compare what is known about stiffness /fluidity of brain tissue  and mature tissue where there is little, as well as glial processes, etc. compared to a tissue culture substratum/plastic or glass.Indeed, the changes in tissue stiffness may have important consequences for axon zippering. We now discuss this aspect in a newly added paragraph in the eighth paragraph of the subsection “Axon zippering in vivo: its regulation and functional significance”.Reviewer #2:This paper presents a very elegant experimental and theoretical analysis of the biophysics of axon zippering. The data is of high quality, and the modelling is well done. The model is a nice balance of biophysical detail and simplicity. A highlight is being able to compare the high-quality images with diagrams of the model  which is a great aid to intuition. The annotations on the images are also very helpful in communicating the methods efficiently.I list below a number of issues which could potentially be addressed to improve the manuscript.1) The current manuscript does not appear to offer any new insight into the molecular mechanisms underlying fasciculation/zippering .We agree that we did not provide any new insights into the molecular mechanisms underlying fasciculation/zippering. This was out of the scope of this paper which rather aims at documenting, analysing, and modeling the dynamical zippering process and its role in axon shaft fasciculation, both in vitro and in vivo. All these aspects are new and they provide a paradigm shift in the field of axon fasciculation, as stated by reviewer 1.2) It is perhaps not clear enough what new insight has been gained from this work about how neural wiring develops in vivo. At the very least I suggest expanding on the three points in the eleventh paragraph of the Discussion.As suggested by reviewer 2 and 1, we have reorganized extensively our Discussion, and have included a subsection devoted to the regulation and functional significance of zippering in vivo. In this subsection, we indeed expanded the paragraph mentioned by reviewer 2.3) There is a lot of analysis of the topological properties of the axon network (e.g. number of vertices). This may be of mathematical interest, but the biological relevance is currently not clear. Does this network behaviour confirm or rule out particular microscopic mechanisms, or is some importance of the structure itself being claimed?The quantification of network characteristics was instrumental for evaluating the overall outcome of the individual zippering and unzippering processes. Using these characteristics, we were, for example, able to clearly demonstrate that cytochalasin (which lowers tension) promotes fasciculation . Individual zippers are highly dynamic and often alternate between zippering and unzippering periods, depending on the network rearrangements occurring in their vicinity. The network structure provides a robust reading of the overall tendency to fasciculate or defasciculate .At this point we do not claim a particular biological significance of the network structure, beyond the formation of bigger or smaller fascicles. Such potential significance would need to be evaluated in the context of a particular in vivo system.4) There are a few places where results seem to be based on very low n values. For instance, in Figure 1H the results seem to be just from one small window from one explant. It would be reassuring to know that these results generalise. More generally, the number of explants contributing to each result should always be stated.In the revised manuscript, we now clearly state the number of explants contributing to each result. For example, see subsection “Progressive fasciculation in cultures of olfactory epithelium explants is due to axon shaft zippering”, second paragraph; subsection “Manipulation of axon tension alters the relative abundance of zippering and unzippering”, first paragraph; subsection “Measurement of axon tension allows to estimate the axon-axon adhesion energy”, third and seventh paragraphs; subsection “Induced or spontaneous dynamics of individual zippers”, last paragraph; subsection “Progressive fasciculation is reflected in the network distribution of zipper angles”, second paragraph; subsection “Analysis of the videomicroscopy recordings”, first and last paragraphs. Moreover, the total number of analyzed experiments has been very significantly expanded for the revised submission.5) Readability could be improved. Although very clearly written, two problems are as follows. (i) The manuscript is currently extremely long – about 15,000 words by my estimate, making it a bit of a marathon for the reader. (ii) Neither the Results or Discussion sections are currently split up into any clearly delineated subsections.We are grateful to reviewer 2 for this excellent suggestion. We have now introduced subsections in both the Results and Discussion sections, with titles summarizing the main message of each subsection, which indeed improves significantly the readability of the manuscript.We have also reduced the length of some subsections, notably on the BFP experiments (particularly in Methods). We have also removed one figure  and most of the associated text from the manuscript; it is replaced by a simplified analysis presented as part of the current Figure 14. As a result, the corresponding subsection is substantially shortened.6) New results are introduced in the Discussion section : I feel these should be in the Results.We agree with the reviewer. The new organization of the paper accommodates this change in a natural way. These results now form the last subsection of Results, allowing us to finish the Results with an analysis of zippering in vivo, and to clearly distinguish it from the discussion of the functional role of zippering in vivo in the corresponding subsection of Discussion. .7) The reader is assumed to be familiar with the biophysics of foams. Some introductory background information in this regard would be helpful, and more discussion of what the reader should take away from this comparison.We acknowledge that an introduction of foams for non-physicist readers was missing, and that the relevance of the discussion of foams in the context of this work was not sufficiently highlighted. As reviewer 2 will see, we have now remedied this by preparing a subsection of the Discussion  which introduces the foam structure and dynamics, discusses the topological and dynamical analogies to our system , and makes a comparison to other biological literature in which analogies to foams were invoked.Reviewer #3:In this manuscript, Smit et al. present measurements of axon tension in a 2D culture system, observe axonal network morphologies, zippering and unzippering events of axons, and develop a mathematical model to describe the process. The biophysics of axon fasciculation has so far rarely been considered, and a thorough understanding of the process would be important to better understand the development of the nervous system.While the presented idea is very nice, and the model looks good to me, the experiments (on which the model is based) seem to be rather preliminary and not conclusive. I have the following specific concerns with the manuscript in its current form:1) The relation of branching angles  and axon tension has been investigated in the past. Particularly the 1979 paper from Dennis Bray and Shefi at al., Neurocomputing, 2004, which already present mathematical models to connect tension and axon morphology / branching angles, need to be cited.Indeed the condition of force balance at a branching point, formulated by Bray and followers, is related to our Equation 1. We now acknowledge this when Equation 1 is introduced, and explicitly discuss the differences as compared to the case of an axon zipper. We also refer to additional literature in which this force balance at a branching point was experimentally tested.2) The measurement of axon tension (only in static neurons) using the BFP technique is not ideal. Biotinylation of axons might already change their mechanical properties, calibration of the force probe (an RBC) seems to be a bit crude and not very accurate (how large is the estimated error of the measurements?), numbers of experiments are very small, and a dynamic system could not be investigated with it. To validate the model, and to directly test the relation between tension and zippering angle, one would have to measure the development of tension during zippering and unzippering events, which really should be done. This would also show if un/zippering of axons in the network is primarily because of a change in tension or adhesion. Other methods  might be better suited for such measurements.While the BFP technique did not allow us to perform dynamic manipulations of zippers, we consider the measurements of axon tension we performed with BFP to be reliable. The error of calibration of the BFP probe is about 15%, as estimated by us and as stated in previous BFP literature. This error is lower than the variability of tensions recorded in our set of measurements from 7 axons. All the errors contributing to BFP measurements are quantified in the Methods . We wish to point out that the BFP technique has been successfully used in numerous previous biophysical investigations. Please also see the methodological paper of  p. 2080–2088), in which the BFP technique was analyzed in detail and verified using data obtained with magnetic tweezers.We did attempt to use optical tweezers at an early stage of our experiments, but encountered the same difficulty as with BFP – the force was insufficient to significantly move the axons. Moreover, this method was likewise dependent on biotinylation, and was significantly less flexible than the micropipette-based BFP.Due to material and time constraints, we have been unable to implement an alternative method of probing the tension, although we agree with the reviewer that it would be of benefit.3) The force balance in Equation 1 does not seem to take the force required to deform / bend the axons into account. I'm not sure if this is an oversimplification.Indeed, the bending force was neglected in the model. As we argued in the original manuscript, this simplification is justified for zippers consisting of single axons or thin fascicles. In the revised manuscript, we expand on this point in the second paragraph of the subsection “Dynamical model of axon zippering”. We make a theoretical estimate of the bending force, showing that it is roughly one order of magnitude lower than the forces we include in the model, for a zipper made of single axons. We then use a scaling argument to show that the bending force will play an important role only for fascicles containing more than about 10 axons.4) An important experiment, which is currently missing but which would be required to test Equation 1, is the direct measurement of the adhesion force. This should be done. Again, potentially suitable techniques include magnetic tweezers, calibrated microneedles, or AFM.Please refer to the response to point 1 of reviewer 1.5) Ideally, the system should also be experimentally perturbed to test the model. For example, tension could be manipulated by interfering with contractility , and adhesion using knockdown of cell adhesion molecules.We thank reviewer 3 for his suggestion to experimentally perturb the system to test the model. Regarding adhesion, OSNs express many adhesion molecules in vivo, but the expression of these molecules is not yet characterized in vitro. Therefore, targeting specific adhesion molecules would have required first characterizing their expression in our system (an approach requiring a significant amount of time) before developing knock-down strategies, which furthermore would not be obvious. Using RNA interference through transfection would be the fastest way to knock-down a candidate adhesion molecule, but the knock-down would concern only a subset of neurons in such explant cultures. Viral vectors would be likely to transduce higher proportions of OSNs in the explants, but producing viruses requires time consuming constructs preparation and viral production. Taking into account the fact that we could not target specific adhesion molecules, we attempted to decrease global adhesion through proteolysis of extracellular proteins using trypsin treatment. Unfortunately, such trypsin treatments induced a rapid collapse of the network, as described in the answer to point 2 of reviewer 1. We thus decided to concentrate our efforts onto manipulations allowing to perturb axon tension.identify a biological molecule having a specific effect, but to find a means to experimentally perturb axon tension. It turned out that FBS did not have any significant effect on OSN growth cones but, very interestingly, it induced a rapid contraction of the whole explant itself, leading to the pulling of axons by the explant core, leading to the de-coarsening of the network . As we discuss in the paper , such a serum-induced “rounding” effect, already observed by Jalink and Moolenaar (1992) on cultured differentiating neural cells, may be due to LPA , but we did not attempt to characterize further this phenomenon in the limited time we had for these revisions.We are grateful to reviewer 3 for suggesting the use of blebbistatin to interfere with axon contractility. It turns out that blebbistatin's main effect, in our system, was to stabilize the network dynamics, inhibiting the coarsening of the network while the individual zippers remained mobile. However, blebbistatin did not have any significant effect on axon outgrowth in our system, in contrast to its previously reported effects on dorsal root ganglion axons, for example, which were either positive or negative, depending of the studies and experimental conditions (see for details the second paragraph of the subsection “Structure and dynamics of the axon network in light of the zippering framework” in Discussion). We have also tested several other treatments aiming at interfering with axon tension, including a biologically-derived solution, the foetal bovine serum (FBS). The rationale for testing FBS was as follows. We first sought specific biological molecules to boost axon growth, motility or contractility in our system, but it was challenging because guidance cues or growth factors having such effects in vitro on axons growing from olfactory epithelia explants are not yet known. FBS, known to contain a number growth factors, appeared to be an interesting alternative candidate, considering that some of its active molecules may have a boosting effect on growth cone motility, which may increase their pulling force onto shafts. Even though we were aware of the fact that we would have no clue about the identity of the active molecule in the event that we did find an interesting effect on zippering, we considered that this would not be an issue in this particular context. The question here was not to before treating it with FBS, allowing us to observe repeatedly a reliable de-coarsening induced by the explant contraction- dependent pulling of axons .Often, however, FBS-induced strong pulling of axons resulted in a rapid collapse of the network, probably due to the pull-generated disturbance of the axon attachment onto the substrate. Having observed that blebbistatin was able to stabilize the network (see above), we used it as a stabilizer As a complement to the observed decoarsening induced by a pulling force, we sought to decrease the tension of axons in view of testing whether it would enhance coarsening, as expected from our framework. Because Dennerll et al. (1988) previously showed that cytochalasin, an inhibitor actin polymerization, significantly decreases the tension of PC-12 cell neurites, we selected this drug for this purpose. We observed that cytochalasin B changed the network dynamics in a way which is consistent with a drop of average axon tension, leading to coarsening . As the networks generally have a tendency to coarsen in our experimental paradigm, we sought to better isolate the effect of cytochalasin by applying it to networks that were pre-stabilized by blebbistatin. As shown in Figure 6D-I, cytochalasin B induces strong network coarsening within 30 minutes of application.Overall, we have shown in the course of these new experiments that FBS-dependent pulling of axons induced unzippering and de-coarsening, while treating the network with a drug known to decrease neurite tension increased coarsening . These results are in full agreement with our core framework, in which axon shaft fasciculation is controlled by the competition between tension and adhesion.6) It appears that the zippering process is considered a purely passive event. Active cellular processes, such as transport etc., will, however, also be involved. This should at least be discussed.Indeed, this is an important point to acknowledge. We now discuss it in the third paragraph of the subsection “Dynamical model of axon zippering”.7) The interpretation of the development of zippering angles shown in Figure 12C is not supported by the data shown. For example, the statement "the mean angle decreased slightly from 62° to 56°" suggests a steady decrease over time, which, however, is not the case. The distributions at 60 minutes and 180 minutes look very similar to me. The statement "The predicted decrease in mean zipper angle is thus 7.5° , in good agreement with the experimentally observed change of 6° " doesn't work for that reason – it is only 1° difference after 2 hours (if comparing 60 mins and 180 mins), which might very well be noise. I doubt that a statistical analysis (which should be done) would reveal a low P value. Also, the boxplots actually suggest that the distribution is more asymmetric at 180 mins than at 60 mins – so I'm not convinced by the usefulness of the skewness parameter s. N is very low here as well for a proper statistical analysis. Together, I don't see the trend the authors mention, and I strongly recommend them to reconsider the interpretation of these experiments.shape of the distribution. We have updated and simplified the corresponding text, and removed the original Figure 12 . We thank the reviewer for bringing us to improve the robustness of this analysis.In the revised manuscript, we have substantially improved this part of the paper. We have now analyzed a total of 5 experiments of the type originally presented in Figure 12. Rather than relying on a few time points from one experiment as in the original Figure 12, we now perform a correlation analysis of the relation between the median zipper angle and the network length, based on a much larger number of data points. Overall, we find a trend that is consistent with the originally stated behavior of the mean zipper angle. For the skewness, however, the originally stated behavior is not confirmed (the correlation is not significant). We now refrain from claiming any overall trend for changes in the […] We request revisions that would incorporate the data and arguments in your rebuttal letter:1) Comment on the blebbistatin experiments, as above.In line with the comment of reviewer 3, we now introduce blebbistatin in Results as a drug previously shown to reduce tension in non-neuronal cells . We then comment on the blebbistatin experiments more extensively in Discussion, subsection “Structure and dynamics of the axon network in light of the zippering framework”, third paragraph. There we cite experiments from the literature on non-neuronal cells, in which a blebbistatin-induced decrease in tension was shown, but also discuss experiments from the literature on neuronal growth cones, in which some variable effects of blebbistatin were seen.2) Discuss the possibility (and caveats) of using thermal fluctuation spectroscopy).We have added a new paragraph in Discussion  in which we acknowledge that we did not monitor axon tension changes during the network development and zippering, and explain the potential use of thermal fluctuation spectroscopy for such a task, including the strengths and caveats of this method. Traction force microscopy is also briefly discussed.3) Highlight your (novel) use of embryonic olfactory epithelium explants, highlighting that this preparation presents "the right balance of axon tension and axon-axon adhesion" that provides insight into axon zippering and progressive fasciculation.We now highlight the novel use of embryonic olfactory epithelium explants. This is done both in the Abstract and in the first paragraph of Discussion, stressing the right balance of axon tension and axon-axon adhesion forces.4) Add from your response letter the figure on FBS-induced increase of axon tension, as Figure 5—figure supplement 1.This figure has been added as Figure 5—figure supplement 1, together with the corresponding source data file. The results shown in the figure are now described in the corresponding section of the Results . The corresponding quantitative estimate of the increase in tension is given in Discussion .5) Add the data from the figure on cytochalasin-induced reduction of axon tension, pointing out, as per your prediction, that the cytochalasin-induced tension reduction was expected to be comparable for all directions within the network, in contrast to the FBS-induced pull in one dominant direction.We now describe these observations in the second paragraph of the subsection “Structure and dynamics of the axon network in light of the zippering framework”.6) Strengthen the "take-home" message that your results demonstrate that the extent of axon zippering can be controlled by axon tension forces, and that future work could aim for force measurements to directly verify the increase in tension in the FBS experiments and the decrease in tension in the cytochalasin experiments.This take-home message is now stressed more in several places, including the end of Introduction. The force measurements to verify the pharmacologically induced changes are discussed in connection with point 2 (see above).7) Throughout the manuscript, stress that axon zippering is controlled by tension changes of functionally relevant magnitude.This is now stressed in a new sentence in the last paragraph of the Introduction and also in two places in Discussion: in the paragraph following the discussion of the FBS experiment , and in the paragraph discussing changes in traction force on substrates of differing stiffnesses .


These pathways also ranked at top in sensitivity analyses. Two positive controls in KEGG, the hedgehog pathway and the BCC pathway, showed significant association with BCC risk in both main and sensitivity analyses. Our results indicate that SNPs that are undetectable by conventional GWASs are significantly associated with BCC when tested as pathways. Biological studies of these gene groups suggest their potential roles in the etiology of BCC.Genome-wide association studies (GWASs) have primarily focused on the association between individual genetic markers and risk of disease. We applied a novel approach that integrates skin expression-related single-nucleotide polymorphisms (eSNPs) and pathway analysis for GWAS of basal cell carcinoma (BCC) to identify potential novel biological pathways. We evaluated the associations between 70,932 skin eSNPs and risk of BCC among 2,323 cases and 7,275 controls of European ancestry, and then assigned them to the pathways defined by KEGG, GO, and BioCarta databases. Three KEGG pathways , two GO pathways , and four BioCarta pathways  showed significant association with BCC risk with  Basal cell carcinoma (BCC), a major type of non-melanoma skin cancer, is the most common malignancy among populations of European ancestry –3. ThougBoth environmental and genetic factors contribute to the genesis of BCC. Though exposure to ultraviolet (UV) radiation is generally accepted as the most important environmental risk factor for BCC, other known risk factors include family history of skin cancer and pigmentary characteristics, such as fair complexion, red or blond hair, and light eye color –9. Most Although GWASs have revolutionized our ability to identify disease susceptibility loci or markers associated with them, they usually yield only the most significant SNPs, and the percentage of genetic variation explained by GWAS signals has generally been modest , 14. Oneet al. first proposed pathway-based analysis of GWAS data in 2007 [in trans by genetic variants that are physically distant [et al. suggested integration of expression-related SNPs (eSNPs) into conventional pathway analysis [Borrowing ideas from gene set enrichment analysis (GSEA) in the gene expression microarray field , Wang et in 2007 . They us distant . Having  distant , Zhong eanalysis . Two maianalysis ; secondlanalysis ; howeveret al. applied this novel pathway analysis to the GWAS of basal cell carcinoma for the first time [In 2012, Zhang rst time . Though rst time , which irst time , ideallyrst time . In the −5 , 5×10−5 (sensitivity 1), and 10−4 (sensitivity 2) respectively. Among them, 69,988, 86,325, and 96,603 are available in our BCC GWAS, respectively. Because all these eSNPs have MAF >1% and imputation R-square >0.4 in our BCC GWAS, they were used for further analysis.From the MuTHER data, we identified 70,932, 87,481, and 97,903 eSNPs in skin tissue using the threshold of 10p value < 0.05, which was 1.54-fold higher than the number expected by chance . Three out of the 11 pathways had a FDR <0.2: the colorectal cancer pathway , the regulation of actin cytoskeleton pathway , and the basal cell carcinoma pathway . In sensitivity 1 analysis, the numbers of genes that can be represented by eSNPs increased to 2,649 when we used the threshold of 5×10−5 for eSNP identification. A total of 151 KEGG pathways that contain between 3 and 200 genes were examined for their associations with BCC. Twelve reached a nominal p<0.05, which was 1.59-fold higher than the number expected by chance. Five out of the 12 pathways had a FDR <0.2. Besides the three that have already been found in the main analysis, the other two pathways are the adherens junction pathway  and the pancreatic cancer pathway . In sensitivity 2 analysis, 3,158 genes were included, and 164 KEGG pathways were tested. Fifteen reached a nominal p<0.05, which was 1.83-fold higher than the number expected by chance. Only one out of the 15 pathways had a FDR < 0.2 -- the colorectal cancer pathway . In total, five KEGG pathways have shown significant associations with risk of BCC in either main analysis or sensitivity analysis. Results of main and sensitivity analyses for the five significant pathways are listed in Table In our main analysis, 2,049 genes with surrogate eSNPs were assigned to the pathways defined in the KEGG database. Using the cut-off of containing 3 to 200 genes, 143 pathways were tested for their associations with BCC risk using our GWAS data. Eleven pathways reached a nominal BCCP = 0.025), SOS1-rs12473092 (BCCP = 0.029), ARHGEF7-rs7984371 (BCCP = 0.039), ITGA2-rs3212544 (BCCP = 0.040), VCL – rs12360087 (BCCP = 0.002), BMP2-rs6054443 (BCCP = 0.0006), BIRC7-rs1075557 (BCCP = 0.014), PIK3R1-rs9291926 (BCCP = 0.016), and RAC1-rs2689420 (BCCP = 0.013).For certain pre-defined pathways identified through the pathway databases, only some of the genes could be represented by eSNPs. Therefore, more attention should be given to the genes and eSNPs that were included in the gene set enrichment analysis rather than to the entire pathway. For significant pathways, we summarized information on such genes and their corresponding eSNPs in Table Moreover, we chose two established BCC-related pathways in the KEGG database as positive controls – the basal cell carcinoma pathway and the hedgehog signaling pathway. The gene set enrichment p-value for these two pathways reached nominal significance in both the main and sensitivity analyses, though the FDRs of the hedgehog signaling pathway are above 0.2 Table .Conventional GWASs have primarily focused on the associations between individual genetic markers and risk of diseases. In the current study, we applied a novel approach that integrates skin eSNPs and pathway analysis for GWAS of BCC. Three KEGG pathways , two GO pathways , and four BioCarta pathways  cell-mediated cytotoxicity, and links between Pyk2 and Map Kinases) showed significant associations with BCC risk. Our results demonstrate that SNPs and genes of moderate effect that are undetectable by conventional GWASs are significantly associated with risk of BCC as groups. These gene sets might be implicated in the etiology of BCC.Some well-known cancer-related pathways have been mapped in both the colorectal cancer pathway and the BCC pathway in KEGG, including the p53 signaling pathway, the Wnt signaling pathway, the PI3K-Akt signaling pathway, the TGF-β signaling pathway, and other pathways related to cell cycle and survival. Studies have shown that a personal history of non-melanoma skin cancer was significantly associated with a higher risk of other primary cancers , 29. CerIn the current study, we made a major improvement by using high-quality eSNPs data on disease-relevant tissue. Although detailed gene-expression studies have profiled transcripts and genotyped SNPs across the human genome in several population-based cohorts, gene expression data in skin tissue from a fairly large cohort was not accessible until the publication of the MuTHER project. In that study, the GWAS data and expression data had undergone stringent quality controls before testing the association of expression levels with probabilities of imputed genotypes. Also, skin eSNP identified in the MuTHER study had been replicated in independent cohorts . Other s−5 had been included in the main analysis. For example, the colorectal cancer pathway in the KEGG database is composed of 114 genes, whereas only 7 genes (6%) were involved in the gene set enrichment analysis. Specifically, we found that a subgroup of seven genes – BIRC5, CYCS, FZD3, FZD8, MAPK9, SMAD3, and SOS1 – that belong to the KEGG colorectal cancer pathway showed significant association with risk of BCC. Similar conclusions could be drawn for other significant pathways, with the subgroups presented in Table The main limitation of our study is that the proportion of genes that could be represented by eSNPs within a predefined pathway is too small, because only 69,988 SNPs that were significantly associated with expression of 2,049 genes at significance level of 10p<0.05) in both main and sensitivity analyses.A further limitation is that no replication was conducted for the identified gene groups, because we used all our BCC GWAS at the discovery stage to maximize statistical power. However, the significant gene groups in the main analysis also ranked top among all pathways being tested in sensitivity analyses. Besides, the positive controls – the Hedgehog signaling pathway and the BCC pathway – were significantly associated with risk of BCC , the Nurses' Health Study II (NHS2), and the Health Professionals Follow-up Study (HPFS) were included in the current BCC GWAS: the postmenopausal invasive breast cancer case-control study nested within the NHS (BC_NHS), the type 2 diabetes case-control studies nested within the NHS and the HPFS (T2D_NHS & T2D_HPFS), the coronary heart disease case-control studies nested within the NHS and the HPFS (CHD_NHS & CHD_HPFS), and the kidney stone case-control studies nested within the NHS, the NHS2, and the HPFS . See BCC cases who had other common cancers before diagnosis of BCC were excluded. Eligible controls were free of BCC and other common cancers. According to the National Cancer Institute and the American Cancer Society, common cancers include melanoma, SCC, breast cancer, endometrial cancer, ovarian cancer, colorectal cancer, bladder cancer, lung cancer, pancreatic cancer, kidney  carcinoma, leukemia, non-Hodgkin lymphoma, thyroid cancer, and oral cancer. Participants with identical genetic information but different cohort IDs were removed; participants whose data appeared in more than one of the eight case-control studies were included only once. In total, the BCC GWAS comprised 2,323 BCC cases and 7,275 controls of European ancestry in the United States.Samples from BC_NHS were genotyped using Illumina HumanHap550 array as part of the National Cancer Institute's Cancer Genetic Markers of Susceptibility (CGEMS) Project . We usedDisease follow-up procedures are identical for NHS, NHS2, and HPFS. Self-reported BCC case-control status is updated every two years without further pathological confirmation, however its validity has been assessed in previous studies , 39, 40.http://www.muther.ac.uk/Data.html). In their study, eSNPs were called with a false discovery rate (FDR) of 1%, which corresponds to P-value < 3.8 × 10−5 [−5, 5×10−5, and 10−4) around this p-value for eSNPs selection in the current study. We used the significance level of 10−5 in main analysis and the other two in sensitivity analysis.A detailed description has been published previously . Briefly8 × 10−5 . We specWe used a multivariate logistic regression model, adjusted for age, and the first three principal components of genetic variation, to evaluate the associations between eSNPs and BCC risk in each of the eight nested case-control studies. The principal components were calculated for all individuals on the basis of ca. 10,000 unlinked markers using the EIGENSTRAT software . The witet al. [We integrated the eSNP information into pathway-based GWAS analysis using the method of Zhong et al. . For a get al. . We set http://www.genome.jp/kegg/pathway.html/) database [http://geneontology.org/) and BioCarta (http://www.biocarta.com/) databases were also included as secondary pathway collections. All pathways that contain at least 3 but at most 200 genes represented by eSNPs were tested.We used human biological pathways as defined in the Kyoto Encyclopedia of Genes and Genomes  of all tested SNP-gene expression pairs are published on the MuTHER website. The threshold to identify SNPs that are significantly associated with at least one gene's expression in skin tissue is arbitrary. As the threshold becomes less stringent, the number of genes that can be represented by eSNPs increases and the surrogate eSNP for a particular gene may change. Therefore, we changed our threshold for eSNP selection to 5×10


Salmonella enterica serovar Napoli are uncommon in Europe. However, these infections represented 5.9% of salmonellosis cases in Italy during 2014–2015. The source of infection is unknown. We analyzed surveillance data and compared strain genetic similarities and found that contaminated vegetables and surface water are probable sources of human infection.Human infections with  Salmonella enterica serovar Napoli has attracted interest since 1982, when chocolate contaminated with this serovar and imported from Italy caused 2 foodborne outbreaks  has received 8 reports of Salmonella Napoli infection, which involved 6 countries and vegetables from Italy as the source of infection.Salmonella Napoli were reported before and after the first outbreak was reported in 1984  for Salmonella spp. isolates from 2011–2015; patient identity was anonymous. Enter-Net is coordinated by the Istituto Superiore di Sanità  and contains information on strains from human and environmental samples. We excluded samples from nonhuman and nonenvironmental sources (n = 43), unspecified sample types (n = 21), and undefined Salmonella serovars  from analysis. We obtained data (aggregated for 2011–2012 and 2013–2014) for Salmonella spp. isolated from food, animals, and feed by using the passive and voluntary veterinary surveillance system Enter-Vet .We extracted data on February 16, 2017, from the passive, voluntary, laboratory-based, surveillance system Enter-Net  serovars other than Napoli  by using simple 2-way frequency tables. Univariate analysis (p<0.05 by χ2 test) was based on comparing categories for each variable and excluding missing values. We developed a multiple logistic regression model  to evaluate potential confounding among variables. We also used data for patients hospitalized with Salmonella Napoli infections or bacteremia as outcomes adjusted for other factors. The top 5 other NTS represented 73% of human Salmonella serovars isolated during 2011–2015. Results were consistent when we compared Salmonella Napoli with all reported NTS.We evaluated potential epidemiologic risks associated with human and environmental Salmonella spp. isolates by using the White–Kauffmann–Le Minor scheme . We performed dendrogram and cluster analyses by using BioNumerics 7.5 . We scored similarity between chromosomal fingerprints by using the Dice coefficient and used the unweighted pair-group method with arithmetic means, 1.00% tolerance limit, 1.00% optimization, and coefficient of similarity >80% to define clonal relationships between strains.We serotyped Salmonella serovars identified for 21,486 human records. The proportion of Salmonella Napoli cases increased from 4.3% in 2011 to 5.8% in 2015 (p<0.001). Salmonella Napoli infection was more common in men, infants, children, and the elderly in northern and southern Italy during July–September than other reported serovars  than patients who traveled abroad. Although patients infected with Salmonella Napoli were hospitalized at rates similar to those infected with other serovars (The monophasic variant of Typhimurium (37.3%), Typhimurium (21.6%), Enteritidis (9.3%), Napoli (4.7%), Derby (3.1%), and Infantis (1.9%) were the most frequent of 331 serovars . For Salserovars , they haSalmonella spp. were reported . These isolates included 187 Salmonella Napoli .Among nonhuman isolates, 15,579 Salmonella Napoli ranked fifth (3.7%) among environmental Salmonella spp. and showed a significant increase from 1.6% in 2011 to 2.9% in 2015 (p<0.005). A total of 85% of Napoli isolates were from surface water. Multivariate analysis  from wild boars in northern Italy. The low proportion (0.4%) from food remained stable during 2011–2015. Of 21 Napoli samples, 7 were from shellfish. One alfalfa sample contaminated with Napoli was reported in southern Italy in 2014.Among Salmonella Napoli strains isolated from different sources. We detected 121 unique profiles; 32 identical profiles contained 2–13 isolates. Despite high genetic variability among strains, we identified 4 main clusters that had genetic homologies >80% and matched the 3 areas in Italy: cluster A in northern Italy, cluster B in central Italy, and clusters C and D in southern Italy (p<0.0001) (We performed pulsed-field gel electrophoresis for 182 <0.0001) . In soutSalmonella Napoli infection reemerged in Italy during 2011–2015. In Italy, Salmonella Napoli patients accounted for 5.9% of salmonellosis cases reported during 2014–2015. France had highest percentage of infections (1.2%) during this period (http://atlas.ecdc.europa.eu/public/index.aspx). Consistent with a study conducted in Italy , reports of the Rapid Alert System for Food and Feed on vegetables from Italy contaminated with Salmonella Napoli, and that the fact that ≈30% of irrigative surface water in Italy is used for vegetables (https://www.istat.it/it/files/2014/11/Utilizzo_risorsa_idrica.pdf), we believe that ready-to-eat vegetables irrigated with contaminated surface water are the main food vehicle for Salmonella Napoli.Outbreaks of Salmonella Napoli from wild boars, which suggests a role for dissemination through the environment (Salmonella Napoli belonging mainly to cluster A in northern Italy and cluster B in central Italy suggest direct infection. Different clusters for human and environmental strains from southern Italy might indicate wider spread in the environment.Marked seasonality of cases matching environmental reports might be caused by increased irrigation and consumption of ready-to-eat vegetables during summer and fall, and more frequent human contact with contaminated surface water during summer and fall. We also reported frequent isolation of Salmonella Napoli increases public health concern in Italy. Uncontrolled spread of Salmonella Napoli through the environment and associated foodborne and waterborne outbreaks, the increasing proportion of young patients, and high risks for development of bacteremia make infections with Salmonella Napoli a serious public health concern in Italy.Heterogeneous sampling of nonhuman isolates through Italy was a limitation of this study. Travel history within Italy, rather than abroad, of patients infected with