During animal development, cellular morphogenesis plays a fundamental role in determining the shape and function of tissues and organs. Identifying the components that regulate and drive morphogenesis is thus a major goal of developmental biology. The four-celled tip of the Caenorhabditis elegans male tail is a simple but powerful model for studying the mechanism of morphogenesis and its spatiotemporal regulation. Here, through a genome-wide post-embryonic RNAi-feeding screen, we identified 212 components that regulate or participate in male tail tip morphogenesis. We constructed a working hypothesis for a gene regulatory network of tail tip morphogenesis. We found regulatory roles for the posterior Hox genes nob-1 and php-3, the TGF-β pathway, nuclear hormone receptors (e. g. nhr-25), the heterochronic gene blmp-1, and the GATA transcription factors egl-18 and elt-6. The majority of the pathways converge at dmd-3 and mab-3. In addition, nhr-25 and dmd-3/mab-3 regulate each others' expression, thus placing these three genes at the center of a complex regulatory network. We also show that dmd-3 and mab-3 negatively regulate other signaling pathways and affect downstream cellular processes such as vesicular trafficking (e. g. arl-1, rme-8) and rearrangement of the cytoskeleton (e. g. cdc-42, nmy-1, and nmy-2). Based on these data, we suggest that male tail tip morphogenesis is governed by a gene regulatory network with a bow-tie architecture.
Morphogenesis involves the coordinated change in the shape of cells and tissues during development, eventually giving rise to functional structures in the adult animal. Such coordinated change must occur at the correct time and in the proper position. In the case of structures that differ between the sexes, this process must also be regulated sex-specifically. While many genes and pathways are known that regulate development, the identity of genes that link regulation to the execution of morphogenesis have been more difficult to ascertain [1]. The many different cues and signals that must be integrated to control morphogenesis, combined with the complexity of the molecular machinery associated with this process, suggest that a large number of genes and gene products are involved. To elucidate the molecular mechanisms underlying morphogenesis, the first step is thus to determine what components are involved in its regulation and execution and to determine how they interact in a network. In the pursuit of such an aim, it is advantageous to use a simple model structure that still demonstrates all the properties of cellular morphogenesis. The model we use is the male tail tip of Caenorhabditis elegans. This structure is made up of four epithelial (" hypodermal" ) cells, hyp8–hyp11, which are born during embryogenesis. Embryonic morphogenesis of hyp8–hyp11 leads to the formation of a pointed, whip-like tail tip. The tail tip retains this shape throughout the lifespan of the hermaphrodite. However, during the last larval stage (L4) of males, these conical cells are dramatically remodeled to form the rounded tip of the adult [2]–[4]. Male tail tip morphogenesis begins when hyp8–11 fuse to form a syncytium; fusion is followed by detachment of the cells from the overlying cuticle. Towards the middle of the L4 stage, the syncytium changes its shape from conical to round and moves anteriorly; these morphogenetic events cease at the end of the L4 stage (Figure 1A). In hermaphrodites, the tail tip cells do not fuse and do not change shape. The tail tip model thus allows the study of a sexual dimorphism at the cellular level. Another advantage of this model is that male-specific mutations in C. elegans can be propagated through the self-fertile hermaphrodites, even if the mutations affect male fertility, mating ability, or viability. A few mutations in genes involved in the regulation of tail tip morphogenesis have been described previously [5]–[7]. Some of these mutations impede or completely block tail tip morphogenesis, resulting in the retention of the pointed larval tail tip in the adult male, a phenotype that we call" Lep" (Figure 1C). This phenotypic designation is derived from the term" leptoderan," which—in the taxonomic literature—describes the unretracted, pointed male tail tip in some nematodes related to C. elegans [8], [9]. Other mutations cause precocious onset of (and thus an extended total period for) tail tip retraction, which results in over-retracted (" Ore" ) and thus abnormally shortened adult male tails [6] (Figure 1C). Studies of these mutants have revealed a few of the important regulatory components for tail tip morphogenesis. DMD-3 was suggested to be a central regulator of tail tip morphogenesis, as it is required for tail tip retraction in males and is sufficient for inducing ectopic morphogenesis in hermaphrodite tail tips [7]. The gene encoding this DM-domain transcription factor, dmd-3, is a homolog of dmrt1 in vertebrates and doublesex in Drosophila [10], [11]. DMD-3 functions cooperatively and partially redundantly with a closely related factor, MAB-3 [7], also involved in somatic sex determination [12]. TRA-1, the most downstream global regulator in the sex-determination pathway, inhibits the expression of these genes in hermaphrodites [7], [13], [14]. The initiation of dmd-3 expression at the proper developmental stage is controlled by the" heterochronic" pathway [7]. Finally, maintenance of dmd-3 expression levels is regulated by Wnt signaling and a feedback loop involving both MAB-3 and DMD-3 [7]. Only one effector of a cellular process is known to be downstream of DMD-3 and MAB-3, namely the fusogen-encoding gene eff-1, which is important for the many fusions that occur between epithelial cells in C. elegans [15], [16]. DMD-3 and MAB-3 induce expression of eff-1 through an unknown post-transcriptional mechanism [7]. To find additional genes with roles in tail tip morphogenesis, we carried out a genome-wide, post-embryonic RNAi-feeding screen. This screen identified 212 candidates. Starting with these candidates, we used network model-building [17], transgenic reporter lines and expression epistasis analysis to construct a first draft of the gene regulatory network for tail tip morphogenesis. We found that dmd-3 expression is regulated by a nuclear hormone receptor (NHR-25), a new heterochronic gene, Hox anteroposterior patterning factors, and GATA transcription factors. NHR-25 is in turn negatively regulated by dmd-3 and mab-3. In addition, dmd-3 and mab-3 negatively regulate other signaling modules, including the TGF-β pathway. We also found that DMD-3 and MAB-3 regulate the localization or expression of genes involved in vesicular trafficking/endocytosis, cell polarity and cytoskeletal organization. Our data thus strongly support the hypothesis that DMD-3, MAB-3 and NHR-25 are central regulators of tail tip morphogenesis. The genetic architecture for tail tip morphogenesis which emerges from this analysis closely resembles the" bow-tie" architecture, a possibly universal characteristic of robust, evolvable systems [18]. A bow-tie network has many inputs and outputs that are connected through a conserved core. Versatile weak linkages form the interface between the core and the input and output. In such a network, there is not a simple flow of information from input to output through the core; instead, there is extensive global and local feedback regulation found at every level [19]. A bow-tie architecture has been found to underlie a variety of biological networks: metabolic networks [20], [21], the Toll-like receptor signaling network [22], the epidermal growth factor receptor signaling network [23], the osmolarity glycerol signal-transduction pathway in yeast [24], stress response networks [25] and the immune system [26], [27]. In fact, it has been proposed that the bow-tie architecture of regulatory networks is ubiquitous because this structure ensures not only robustness but also evolvability [18], [19]. We found evidence for the existence of each aspect of a bow-tie architecture in the gene network governing tail tip morphogenesis. To our knowledge, this is the first time that this kind of network architecture has been explicitly identified in the context of development and morphogenesis. However, we believe that other developing systems are also governed by bow-tie genetic networks, supporting the proposition that this network architecture is universal.
To identify tail tip morphogenesis genes, we cultured animals on dsRNA-expressing bacteria from L1 to adult and looked for evidence of defective morphogenesis. Using the Ahringer RNAi-feeding library [28], we screened 16,131 genes, approximately 83% of the genome. Genes that gave a positive Lep or Ore phenotype were screened again; only repeatable positives were kept as candidates. We identified 212 genes, of which 190 produced Lep phenotypes, 14 produced Ore phenotypes, and 8 produced both Lep and Ore phenotypes in a single experiment (Figure 1D). Positives in each category were analyzed for GO-attribute enrichment using FuncAssociate [29]. Relative to the genome, Lep positives showed enrichment of GO-attributes associated with components or processes involved in morphogenesis, such as anchoring junctions and cell migration. However, Ore and Ore/Lep positives were enriched for genes involved in cell division/cytokinesis, and nuclear/chromosome organization (Figure 1D). The 212 candidates are listed and categorized by developmental pathway or annotated cellular process (if known) in Table S1. Raw RNAi data are publicly available along with representative images via the" Male Tail Tip Database" (MTTdb, at http: //wormtails. bio. nyu. edu). We identified components of conserved and widely studied developmental regulatory pathways (e. g. Hox anteroposterior patterning factors, TGF-β signaling module, heterochronic pathway, and GATA transcription factors). Importantly, the screen also identified components of fundamental cellular structures and processes that are likely to be important for the execution of morphogenesis. These include vesicular trafficking/endocytosis, cellular polarity, cytoskeleton, cell junctions, and nuclear export/import. Genes with the most severe and/or most penetrant RNAi phenotypes were selected for further study (Table 1). Hox genes play central roles in shaping animal body plans by distinguishing different fields of cells along the anteroposterior axis [30]. Hox proteins regulate not only the expression of other transcription factors, but also of genes involved in specific processes such as morphogenesis [31]. Our screen has shown that posterior Hox patterning is crucial for tail tip morphogenesis. RNAi knockdowns of the Abd-B homologs php-3 and nob-1 postembryonically cause Lep phenotypes (Figure S1A, Tables S1 and S2). Likewise, a php-3 null allele, ok919, results in a 100%-penetrant Lep phenotype of moderate severity (Figure S1B, Table S2). The severity of the php-3 (ok919) phenotype is increased with nob-1 RNAi treatment (Table S2). A nob-1: : gfp translational fusion construct (containing a genomic fragment with the nob-1 gene and 9 Kb of the 5′-regulatory region) is expressed in the tail tip cells hyp8–11 throughout larval development in both sexes (Figure 2A and data not shown). A php-3: : gfp translational fusion driven only by the short 500-bp intergenic region between nob-1 and php-3 shows variable expression in the nuclei of hyp8–11 during tail tip morphogenesis (data not shown). This latter transgene only partially rescues the tail tip phenotypes of php-3 mutants (Table S2), suggesting that regulatory elements upstream of both php-3 and nob-1 are required for appropriate expression of php-3. Using a tail tip-specific promoter (from the gene lin-44 [32]), we observed that expression of the PHP-3: : GFP fusion protein product in hyp8–11 is sufficient to rescue the php-3 (ok919) Lep phenotype (Figure S2A and Table S2). Mosaic animals that only express PHP-3: : GFP in a subset of tail tip cells do not show rescue (Figure S2B). These data suggest that Hox-mediated patterning by PHP-3 and NOB-1 is carried out cell-autonomously and is required in all tail tip cells (hyp8–11) for proper morphogenesis. GATA transcription factors play important regulatory roles during the differentiation of multiple cell types in normal development [33], [34] and during tumorigenesis [35]. RNAi knockdown of the gene for the GATA factor egl-18 resulted in Lep phenotypes (Figure S1C, Tables S1 and S2). An egl-18 null allele, ok290, causes Lep phenotypes of varying severity with 45% penetrance (Figure S1D, Table S2). Another GATA transcription factor that was missed in our RNAi screen, elt-6, shares an operon with egl-18 [36]. We repeated the RNAi treatment against elt-6 and observed low-penetrance, low-severity tail tip defects (Table S2). RNAi treatment for elt-6 in the egl-18 (ok290) mutant strain, however, dramatically increased the penetrance and severity of the tail tip defects, such that tail tip morphogenesis failed altogether in some individuals (Table S2). Interestingly, an egl-18: : gfp translational fusion is expressed in the nuclei of the main body epidermal syncytium hyp7, but appears to be excluded from the tail tip cells hyp8–11 (Figure 2B). This observation suggests that EGL-18 and ELT-6 function in cells adjacent to the tail tip to regulate tail tip morphogenesis cell-nonautonomously. Furthermore, transforming wild-type animals with the egl-18/elt-6 operon regulated by the lin-44 promoter disrupted morphogenesis (data not shown), suggesting that morphogenesis requires exclusion of these GATA factors from hyp8–11. In other epidermal cells, it has been observed that EGL-18 and ELT-6 repress cell fusion [37]. Thus, their exclusion from the tail tip cells may be required to allow tail tip cell fusion and subsequent morphogenesis. TGF-β signaling is a major conserved cell-signaling module which regulates multiple processes during the development of all animals and also during the progression of cancer [38]. The screen identified two components of this pathway. RNAi treatment against sma-3, which encodes a Smad protein, and daf-4, encoding the TGF-β receptor, resulted in Lep phenotypes (Figure S1E, Tables S1 and S2). A sma-3 null allele, e491, resulted in a 57%-penetrant low-severity Lep phenotype (Figure S1F, Table S2). Of the other known components of the TGF-β pathway, only sma-2 showed any RNAi phenotype: RNAi against sma-2 significantly enhanced the penetrance of the Lep phenotype of sma-3 (e491) (Table S2). A transgenic strain expressing a sma-3: : mCherry translational reporter shows expression at low levels in the cytoplasm of the tail tip of both sexes at the beginning of the L4 stage. In males, the SMA-3: : mCherry fusion protein enters the nuclei of hyp8–11 prior to tail tip morphogenesis and remains in both the cytoplasm and nuclei during morphogenesis (Figure 2C). In hermaphrodites, SMA-3: : mCherry remains cytoplasmic throughout L4 and never enters the nuclei (data not shown). The dynamic localization pattern of SMA-3: : mCherry suggests that TGF-β-mediated gene expression occurs concurrently with tail tip morphogenesis. Consistent with this hypothesis, DAF-4: : YFP fusion protein localizes to the plasma membranes of hyp8–11 during tail tip morphogenesis (Figure 2D). Taken together, the RNAi results, loss-of-function mutant phenotypes and expression patterns of sma-3 and daf-4 suggest that TGF-β signaling is required during tail tip morphogenesis. Previous work has shown that Wnt signaling plays a crucial role in the regulation of tail tip morphogenesis [5]. Consistent with those findings, the RNAi screen identified additional genes that are in or interact with the Wnt pathway. RNAi treatments against sys-1 (β-catenin) and lit-1 (Nemo-like kinase) each resulted in Lep phenotypes (Table S1, Figure S3C, S3D). A transgenic strain expressing a SYS-1: : GFP fusion protein shows faint cytoplasmic expression in hyp8 and hyp11 but not in hyp9 or hyp10 (Figure S3C). A strain expressing a LIT-1: : GFP fusion protein shows nuclear expression in hyp9 and hyp10 but not in hyp8 or hyp11 prior to morphogenesis (Figure S3D). We identified multiple nuclear hormone receptor genes in our screen: nhr-9, nhr-23, nhr-25 and nhr-165. NHR-25 is the C. elegans homolog of FTZ-F1 [39] which is a highly conserved protein with diverse functions regulating embryonic patterning [40], [41], and ecdysone-mediated molting in Drosophila [42]. Both nhr-25 RNAi and a hypomorphic allele, ku217, showed Lep phenotypes (Figure S1G and S1H, Tables S1 and S2). Furthermore, nhr-25 RNAi treatments on the ku217 strain showed a complete lack of male tail morphogenesis in 33% of the animals (N = 24, Table S2), a phenotype reminiscent of the mab-3 (e1240); dmd-3 (ok1327) double mutant [7]. We were not able to produce transgenic lines expressing NHR-25 fusion proteins due to lethality, as previously reported [43]. Instead, we made a transgenic strain expressing a transcriptional reporter containing the 5′- and 3′-regulatory regions for nhr-25. This reporter shows a dynamic expression pattern in which expression in hyp8–11 is intense prior to and at the beginning of morphogenesis, rapidly shuts off in late L4, and is never on in adult animals (N = 34 adults) (Figure 2E). RNAi against another nuclear hormone receptor gene, nhr-165, showed a low-penetrant, mild, yet reproducible Lep phenotype upon RNAi treatment (Table S1, and Figure S3A). An nhr-165: : gfp translational reporter shows nuclear expression in the lateral hypodermis (hyp7, but not hyp8–11) near the time of larval molts. This expression is highest and seen in the largest number of cells in the tail prior to the L3–L4 molt (Figure S3A). The heterochronic pathway ensures that tail tip morphogenesis is initiated precisely at the beginning of the L4 stage [6]. We identified genes in this pathway: the zinc-finger transcription factor gene blmp-1, and known suppressors of the miRNA let-7 [44]. BLIMP-1 is a transcriptional repressor that regulates ecdysone-mediated molts in Drosophila [45] and differentiation of multiple cell types in humans and mice, such as lymphocytes [46] and primordial germ cells [47], [48]. Intriguingly, BLIMP-1 is a target of let-7-mediated degradation in Reed-Sternberg cells, a Hodgkin-lymphoma cell line, suggesting a possibly conserved interaction with the heterochronic pathway, of which let-7 is a member [49], [50]. RNAi treatments directed against blmp-1 produced Ore phenotypes (Table S1). A deletion allele, tm548, produces an Ore phenotype with 100% penetrance (Table S2) due to precocious initiation of tail tip morphogenesis during the L3 stage (N = 26, data not shown). A transgenic line expressing a BLMP-1: : GFP fusion protein shows both nuclear and cytoplasmic expression in hyp8–11 throughout development, although cytoplasmic expression is most intense during tail tip retraction (Figure 2F). Of the 41 suppressors of let-7 lethality identified by Ding et al. [44], six were positives in our screen. RNAi knockdown of two (pri-2, npp-6) resulted in the Ore phenotype, of two others (spg-7, smo-1) in the Lep phenotype and of two further genes (cdt-1, xpo-2) in both phenotypes in a single experiment. Four of these genes—pri-2, npp-6, cdt-1, and xpo-2—are predicted by N-Browse [17] to interact in a subnetwork that includes other genes for which RNAi knockdown also produced Ore phenotypes (Figure 3). It is thus possible that these additional genes also influence the timing of tail tip morphogenesis. It is still unclear why both Ore and Lep phenotypes are observed in a single experiment. One possible explanation is that precision in timing of tail tip morphogenesis is lost when certain gene-products are removed. In this case, morphogenesis might begin too early in one animal and too late in another, leading to Ore tails and Lep tails, respectively. Post-transcriptional regulation appears to play an important role during the coordination of tail tip morphogenesis, as our screen has identified multiple genes that encode RNA splicing factors, kinases, phosphatases and ubiquitinating enzymes. One such gene is ubc-12, which is a part of the NED-8 conjugating system and has been shown to be important in epidermal differentiation during embryogenesis in C. elegans [51]. RNAi-knockdown of ubc-12 resulted in larval lethality in the RNAi hyper-sensitive rrf-3 (-) background (Table S4). However, we identified ubc-12 in a pilot screen which was carried out in the wild-type background; ubc-12 RNAi treatments produced a highly penetrant and severe tail tip defect (Figure S3B). UBC-12: : GFP expresses intensely in the cytoplasm of hyp10 just prior to and during tail tip retraction (Figure S3B). We identified many genes known to play central roles during the execution of morphogenesis. Such genes encode proteins involved in vesicular trafficking, endocytosis, cell-cell communication, cytoskeletal rearrangement, establishment of cellular polarity and cell-cell transport. Adjacent to the tail tip cells lie the dendritic projections of the PHC neurons. Two genes that produced Lep phenotypes upon RNAi treatment are expressed in these neurons but not in the tail tip cells: the calcipressin-encoding gene rcn-1 [54] and ptl-1, a gene encoding a tau-like microtubule-associated protein [55], [56]. A null allele of ptl-1, ok621, produced Lep phenotypes with 23% penetrance (Table S2). PTL-1: : GFP and RCN-1: : GFP fusion proteins are expressed in the PHC neurons prior to and during hypodermal morphogenesis. In adults, PTL-1: : GFP is expressed in most neurons of the tail (data not shown). RCN-1: : GFP is expressed in most tail neurons and in the support cells of the phasmid neurons (socket cells, Figure 4H). This pattern is consistent with the previously described adult expression pattern of RCN-1 [54]. With a list of genes required for tail tip morphogenesis, we next sought to characterize the interactions between these genes. We constructed a working hypothesis for these interactions using N-Browse, a publicly available database which integrates the information from numerous genome-wide studies to build gene networks [17]. We manually entered our candidate genes into N-Browse, excluding those that did not have predicted functions or known interactors. N-Browse produced a genetic network that included not only our candidate tail tip morphogenesis genes, but additional genes predicted to be nearest-neighbor interactors. To the resulting N-Browse network, we manually added gene interactions (edges) based on published work not represented in N-Browse [6], [7], [44], [51], [57]–[61] (Figure 5). This analysis predicts the involvement of genes not identified in our screen. We tested two of these predictions by repeating RNAi knockdown with different methods and/or by scoring larger numbers of males. We could thus validate roles in morphogenesis for elt-6 and vav-1. elt-6 has genetic interactions with egl-18 (positive in our screen) in other contexts. It showed a low-penetrance Lep phenotype when the RNAi experiment was repeated and more males were scored. In addition, elt-6 (RNAi) enhances the Lep phenotype of egl-18 (ok290) mutants (Table S2). Also, the network model predicts that vav-1 has interactions with php-3, cdc-42 and inx-12 (all positives in our screen, Figure 5). Although vav-1 treatment by RNAi via feeding did not result in detectable phenotypes, administering RNAi against vav-1 by soaking did cause tail tip defects (data not shown). We next asked whether the network model—developed from information in other systems—has biological relevance for tail tip morphogenesis. To this end, we tested a selection of the predicted interactions by genetic and expression epistasis analyses. The results are detailed below.
Here, we used systemic RNAi to identify components that are involved in male-specific morphogenesis of the tail tip of C. elegans. RNAi via feeding in C. elegans provides a simple yet powerful means for identifying the regulatory and structural components and pathways of developmental processes [28], [62]. The methodology we employed (Materials and Methods and Figure 1B) allowed us to quickly score for subtle defects in morphogenesis at high magnification. Many of the genes we identified have roles in embryonic processes and are lethal when knocked down (e. g. nob-1, cdc-42). This justifies our approach to treat worms with RNAi postembryonically and it underscores the power of RNAi as a tool for identifying postembryonic roles of genes that have essential embryonic functions. To minimize the number of false negatives, we performed the screen on the RNAi hypersensitive strain, rrf-3 [63]. For a number of reasons, however, we believe that there are still other tail tip genes to be identified. First, our screen did not cover the entire genome (approx. 83%). Second, because of complete larval lethality, about 2% of the genes in our screen were not scored, including the known tail tip regulator lin-41 [6] (Table S4). Third, of the previously known tail tip genes, only lin-44, which encodes for the Wnt ligand, was found in the screen. Other known tail tip genes, tlp-1 and dmd-3, which have representative clones in the library, were missed. Finally, two genes (elt-6 and vav-1) not found in the screen, but predicted by the N-Browse network analysis, turned out to have RNAi-induced tail tip phenotypes when treated in a different genetic background (i. e. elt-6 RNAi in the egl-18 (ok290) background) or by soaking instead of feeding (vav-1). The number of false positives is likely to be very small since only genes which were positive in the primary and secondary screen were considered. The 212 candidate genes identified in this process were significantly enriched with morphogenesis-related GO attributes relative to the genome at large, consistent with what would be predicted if our screen were successful. Some candidates were studied further to elucidate their roles in regulating or effecting tail tip morphogenesis. Developmental decisions, such as the initiation of morphogenesis, require the input of multiple signaling pathways and result in a coordinated response by many different components of the cell. The response must be robust against perturbations from the internal and external environment. Robustness and precision of biological processes are thought to be facilitated by a bow-tie (or hourglass) architecture of the gene regulatory network [18], [19]. Characteristics of bow-tie networks include the following. (1) Many inputs and outputs are connected to a conserved core. (2) Versatile weak linkages form the interface between input and core and between core and output. (3) Systems control is facilitated by positive and negative feedback at every level. (4) Modularity and partial redundancy or degeneracy are two other properties of the bow-tie network architecture that contribute to the robustness of biological systems [19], [64]. We used the data about gene interactions described here in combination with published information to delineate a first draft of the gene regulatory network underlying tail tip morphogenesis in C. elegans males. Although the reconstruction of this network has only just begun, we already find many features that are consistent with bow-tie architecture. A network of interactions is called modular if it can be subdivided into relatively autonomous components (modules) that are built of highly connected parts but are more loosely connected to other modules [65]. Modularity is a major contributor to the robustness and evolvability of a system, since perturbations and mutations can occur within a module with minimal effects on the whole system [19]. It has been proposed that modularity facilitates evolutionary change by allowing new connections to be made between modules without disrupting the core function of the modules [66]. Modularity has been observed in many networks [67], [68]. The modules of metabolic networks have bow-tie structure, just like the networks themselves [21]; that is, bow-tie architecture can be nested. In the gene regulatory network of male tail tip morphogenesis, we find evidence for many conserved regulatory and effector modules. Regulatory modules include Hox patterning, the sex-determination and heterochronic pathways and TGF-β signaling. We also identified tail tip roles for additional Wnt pathway components, i. e. the SYS-1 beta-catenin, the MIG-1 Frizzled-like receptor, and the LIT-1 Nemo-like MapK. Effector modules consist of conserved components controlling vesicular trafficking and endocytosis, establishment of cellular polarity, cytoskeletal rearrangement, and cellular fusion. Degeneracy describes the coexistence of structurally or mechanistically different components that can perform similar roles or are interchangeable under certain conditions [64]. Degeneracy confers robustness because, in a system composed of partially redundant elements, one element can compensate for the failure of another. One mechanism that generates degeneracy is gene duplication. In the male tail tip morphogenesis system, we find several examples for degeneracy due to gene paralogy. We think that MAB-3 and DMD-3 function partially redundantly because the phenotype of the mab-3 (e1240); dmd-3 (ok1327) double mutant is more severe than that of dmd-3 (ok1327) or mab-3 (e1240) alone [7]. Similarly, two other pairs of paralogs function semi-redundantly: the Hox genes php-3 and nob-1 and the GATA transcription factors egl-18 and elt-6, which form an operon. In both cases, removal of both genes results in a much more severe disruption of morphogenesis than removal of only one gene. Both modularity and degeneracy contribute in a major way to the robustness of a system [19]. Indeed, the majority of RNAi knockdown phenotypes suggest that male tail tip morphogenesis is very robust against genetic perturbations. In most cases, the effects of RNAi (as well as some of the mutations tested) were subtle and the penetrance was low, suggesting that there is extensive buffering of the system against partial depletion of individual transcripts (or reduced functionality of genes). The architecture of the genetic network regulating male tail tip morphogenesis in C. elegans is congruent with the bow-tie model, since we find evidence for all the characteristics of bow-tie networks. We find modularity, degeneracy, a conserved core, weak linkage and positive and negative feedback loops connecting spatial, sexual and temporal inputs to the cellular responses required for morphogenesis. To our knowledge, this is the first time that a genetic network regulating a morphogenetic process has been specifically investigated for bow-tie architecture. However, it is likely that other morphogenetic events—e. g. , the development of the eye in flies and possibly mammals and the formation of the pharynx in C. elegans—are also controlled by bow-tie regulatory networks. In both examples, components have been identified which are likely to be part of the conserved core. Drosophila eye development is in part controlled by the products of eight eye specification genes. Deletion of either one of these genes leads to a drastic reduction or loss of the adult eye, whereas ectopic expression of all but one results in retinal development outside of normal eye tissue [88], [89]. The fly eye specification genes are conserved with orthologs in mammals. Expression of one of them, dachshund, is regulated by at least 36 upstream factors, including the TGF-β signaling pathway, the transcription factor Zerknüllt and several other patterning genes (e. g. krüppel, snail and dorsal) [88], suggesting the existence of an extensive input fan in this system. The FoxA transcription factor PHA-4 is a central regulator of pharynx development in C. elegans. pha-4 is the only zygotic gene that deletes the entire pharynx when mutated [90]. FoxA transcription factors are conserved from Cnidaria to mammals and are always associated with the digestive tract [90]. PHA-4 has hundreds of targets, many of which are directly regulated at the transcriptional level [91], [92]. In this system, feedback loops have been identified as well [90]. Thus, there is evidence for a conserved core, an output fan and system control as elements of a bow-tie network architecture for pharynx morphogenesis. Finding bow-tie networks in multiple developmental systems supports the notion that this architecture is a universal feature of evolved gene regulatory networks and is favored by selection due to its robustness. The male tail tip thus provides a simple model for investigating not only morphogenetic mechanisms, but also the properties of a universally important genetic regulatory architecture.
Genetic manipulations and culturing of C. elegans were performed as previously described [98]. We use the following nomenclature for transgenes (similar to that used by Ziel et al. [99]). Transcriptional reporters are designated by the name of the gene associated with the promoter, followed by a “>” and the reporter gene to which it is fused (e. g. , dmd-3>yfp). Translational reporters are designated by the gene, followed by “: : ” and the reporter to which it is fused (e. g. , sma-3: : mCherry). Unless otherwise stated, the endogenous promoter is used to drive expression of translational reporters. If a different promoter is used, we use both designations (e. g. , lin-44>php-3: : gfp represents the php-3 gene fused to the gfp gene, with expression driven by the promoter of the lin-44 gene). Unless otherwise stated, the unc-54 3′UTR is used for all constructs. The protein product of a construct is designated with capital letters (e. g. , PHP-3: : GFP). No construct employed cDNA; all introns were included. Strains with transgenes generated for this paper are listed in Table S3. Other strains used for this study are listed below. CB4088 = him-5 (e1490) V. This is the otherwise wild-type, male-producing strain used as the background genotype in this study (from Caenorhabditis Genetics Center, CGC). BW2020 = ctIs57[nob-1: : gfp + rol-6] (a gift from Zhongying Zhao, University of Washington, Washington). Hermaphrodites of this strain were crossed with CB4088 males to obtain a him-59 (e1490) V; ctIs57 strain. DF125 = php-3 (ok919) III; him-5 (e1490) V. Made by crossing CB4088 males with RB998 hermaphrodites. RB998 = php-3 (ok919) III (from CGC). DF159 = rme-8 (b1023) I; him-5 (e1490) V. Made by crossing CB4088 males with DH1206 hermaphrodites. DH1206 = rme-8 (b1023) I (from CGC). DF160 = blmp-1 (tm548) I; him-5 (e1490) V; fsIs3[dmd-3>yfp + unc-122>gfp]. DF161 = blmp-1 (tm548) I; him-5 (e1490) V. Made by crossing CB4088 males with hermaphrodites carrying the blmp-1 (tm548) allele (from Shohei Mitani, National BioResource Project, Tokyo Women' s Medical University School of Medicine, Tokyo, Japan). DF163 = sma-3 (e491) III; him-5 (e1490) V. Made by crossing CB4088 males with CB491 hermaphrodites. CB491 = sma-3 (e491) III (from CGC). DF164 = egl-18 (ok290) IV; him-5 (e1490) V. Made by crossing CB4088 males with JR2370 hermaphrodites. JR2370 = egl-18 (ok290) IV (from CGC). DF167 = him-5 (e1490) V; nhr-25 (ku217) X. Made by crossing CB4088 males with MH1955 hermaphrodites. MH1955 = nhr-25 (ku217) X (from CGC). DF171 = him-5 (e1490) V; bIs34[rme-8: : gfp + rol-6]. Made by crossing CB4088 males with DH1336 hermaphrodites. DH1336 = bls34[rme-8: : gfp + rol-6] (from CGC). DF177 = him-5 (e1490) V; nhr-25 (ku217) X; fsIs3[dmd-3>yfp + unc-122>gfp]. DF178 = mab-3 (e1240) II; dmd-3 (ok1327) him-5 (e1490) V; bIs34[rme-8: : gfp + rol-6]. DF196 = him-5 (e1490) V; xnIs8[pJN343: nmy-2: : mCherry + unc-119 (+) ]. This strain was made by crossing CB4088 males to hermaphrodites carrying the transgene xnls8 which were a generous gift from Jeremy Nance (NYU Skirball Institute, New York, New York). DF197 = mab-3 (e1240) II; dmd-3 (ok1327) him-5 (e1490) V; xnIs8[pJN343: nmy-2: : mCherry + unc-119 (+) ]. DF199 = ptl-1 (ok621) III; him-5 (e1490) V. Made by crossing CB4088 males to RB808 hermaphrodites. RB808 = ptl-1 (ok621) III (from CGC). JJ1473 = unc-119 (ed3) III; zuIs45[nmy-2: : gfp + unc-119 (+) ] (from CGC). KC447 = rrf-3 (pk1426) II; him-5 (e1490) V. A generous gift from King L. Chow (Hong Kong University of Science and Technology, Hong Kong, China). KC529 = eri-1 (mg366) IV; him-5 (e1490) V. (from K. L. Chow). UR157 = fsIs2[dmd-3>yfp + unc-122>gfp]I? ; him-5 (e1490) V. A generous gift from Douglas Portman (Rochester University, New York). UR279 = mab-3 (e1240) II; dmd-3 (ok1327) him-5 (e1490) V (from D. Portman). WM79 = rol-6 (n1270) II; neEx[lit-1: : GFP + rol-6 (su1006) ] (from CGC). Hermaphrodites of this strain were crossed with CB4088 males to obtain a rol-6 (n1270) II; him-5 (e1490) V; neEx[lit-1: : GFP + rol-6 (su1006) ] strain. The genome-wide RNAi-feeding screen was carried out in the RNAi-hypersensitive background rrf-3; him-5 (strain KC447). RNAi effects on embryogenesis were bypassed by feeding siRNA-expressing bacteria to synchronized L1 larvae. Following a recommendation by K. Chow (pers. comm.), L1 larvae were plated onto a thin film of agar (1. 5 ml per 60 mm plate) containing 2 mM IPTG and 100 µg/ml ampicillin. Worms were cultured to adulthood (3 days) at 20°C at which time a square of agar with worms was removed and mounted directly onto a glass slide and covered with a coverslip (Figure 1A). All scoring was carried out at 400x with a Zeiss Axioskop equipped with Nomarski differential interference contrast. Images were recorded with a C4742-95 “Orca” Hamamatsu digital camera and Openlab software, ver. 3. 0. 9 (Improvision). The secondary screen was carried out in the same way but in a different RNAi hypersensitive background, eri-1 (strain KC529). RNAi clones consistently conferring a Lep or Ore tail tip phenotype were sequenced to confirm the targeted genes. All scoring data and images are available via our male tail tip database, MTTdb, at http: //wormtails. bio. nyu. edu. Translational fusions and transcriptional reporters were constructed by overlap-extension PCR as previously described [100]. The 5′ upstream sequence and coding sequences for php-3 (-500 bp to the stop codon), egl-18 (-3691 to stop), rcn-1 (-4797 to stop), ptl-1 (-2105 to stop), arl-1 (-550 to stop), abcx-1 (-437 to stop), cdc-42 (-1814 to stop), sys-1 (-3411 to stop), inx-12 (-3975 to stop), blmp-1 (-4916 To stop), nhr-165 (-1559 to stop), and ubc-12 (-363 to stop), were PCR-amplified from genomic DNA and fused to gfp and the unc-54 3′-UTR amplified from pPD95. 75 (Addgene). The 5′-upstream sequence and coding region of sma-3 (from -1169 bp), amplified from genomic DNA, was fused to mCherry [101] and to cfp, which were PCR-amplified from pGC326 (a gift from E. J. Hubbard) and pPD136. 61 (Addgene), respectively. To make the DAF-4 reporters, the upstream sequence and coding region (from -5091) was fused to yfp that was amplified from pPD136. 64 (Addgene). A transcriptional reporter of daf-4 fused the upstream sequence (-4865 to -1) to the NLS and gfp amplified from pPD122. 13 (Addgene). The transcriptional reporter for nhr-25 fused the upstream sequence (-9100 to -1) to the NLS and gfp from pPD122. 13 (Addgene) followed by the 3′-UTR for nhr-25 (stop to +760 bp). Transcriptional reporters for inx-12 (-3975 to -1) and inx-13 (-1557 to -1) were fused to yfp (pPD136. 64 (Addgene) ) and cfp (pPD136. 61 (Addgene) ). Transgenes were microinjected at concentrations ranging from 5–20 ng/µl along with 100 ng/µl pRF4 (rol-6 (su1006) ) as injection marker. Multiple lines were analyzed for each construct using epifluorescence (Axioskop with mercury lamp, 400 or 1000x). Representative images of fluorescence expression patterns are available via the MTTdb database at http: //wormtails. bio. nyu. edu. Strain names and primer sequences are provided in Table S3. Genes identified in our screen were manually entered into N-Browse2 (http: //Aquila. bio. nyu. edu/NBrowse2/nbrowsetest. jnlp) [17]. Only a subset of these genes showed annotated interactions, and only those with an interaction one or two edges away from another candidate gene were added to our network (Figure 5). Information from other studies [6], [7], [44], [51], [57]–[60] which show genetic or direct interactions with known or newly identified tail tip genes were also included (Figure 5).
Morphogenesis is a process in which cells change their shape and position to give rise to mature structures. Elucidation of the molecular basis of morphogenesis and its regulation would be a major step towards understanding organ formation and functionality. We focus on a powerful model for morphogenesis, the four-celled tail tip of the C. elegans male, which undergoes morphogenesis during the last larval stage. To comprehensively determine the components that regulate and execute male tail tip morphogenesis, we performed a genome-wide RNAi screen. We identified 212 genes that encode proteins with roles in fundamental processes like endocytosis, vesicular trafficking, cell–cell communication, and cytoskeletal organization. We determined the interactions among several of these genes to reconstruct a first draft of the genetic network underlying tail tip morphogenesis. The structure of this network is consistent with the" bow-tie architecture" that has been proposed to be universal and confers evolvability and robustness to biological systems. Bow-tie networks have a conserved core which is linked to numerous input and output components. Many components of the network underlying tail tip morphogenesis in C. elegans are conserved all the way to humans. Thus, understanding tail tip morphogenesis will inform us about morphogenesis in other organisms.
developmental biology
genetics
biology
morphogenesis
gene networks
genetics and genomics
A Bow-Tie Genetic Architecture for Morphogenesis Suggested by a Genome-Wide RNAi Screen in Caenorhabditis elegans