Abstract
A hallmark of gastrulation is the establishment of germ layers by internalization of cells initially on the exterior. In C. elegans the end of gastrulation is marked by the closure of the ventral cleft, a structure formed as cells internalize during gastrulation, and the subsequent rearrangement of adjacent neuroblasts that remain on the surface. We found that a nonsense allele of srgp-1/srGAP leads to 10-15% cleft closure failure. Deletion of the SRGP-1 C-terminal domain led to a comparable rate of cleft closure failure, whereas deletion of the N-terminal F-BAR region resulted in milder defects. Loss of the SRGP-1 C-terminus or F-BAR domain results in defects in rosette formation and defective clustering of HMP-1/α-catenin in surface cells during cleft closure. A mutant form of HMP-1 with an open M domain can suppress cleft closure defects in srgp-1 mutant backgrounds, suggesting that this mutation acts as a gain-of-function allele. Since SRGP-1 binding to HMP-1 is not favored in this case, we sought another HMP-1 interactor that might be recruited when HMP-1 is constitutively open. A good candidate is AFD-1/Afadin, which genetically interacts with cadherin-based adhesion later during embryonic elongation. AFD-1 is prominently expressed at the vertex of neuroblast rosettes in wildtype, and depletion of AFD-1/Afadin increases cleft closure defects in srgp-1 and hmp-1R551/554A backgrounds. We propose that SRGP-1 promotes nascent junction formation in rosettes; as junctions mature and sustain higher levels of tension, the M domain of HMP-1 opens, allowing maturing junctions to transition from recruitment of SRGP-1 to AFD-1. Our work identifies new roles for α-catenin interactors during a process crucial to metazoan development.
Introduction
Gastrulation is a hallmark of metazoan development that establishes the basic body plan [1]. In many organisms, internalization of founder cells that form the three primary germ layers, as well as primordial germ cells, occurs via detachment of the apical surfaces of individual cells from the embryo’s exterior [2–5]. Such internalization can involve an epithelial-mesenchymal transition (EMT), as cells dismantle their cell-cell junctional machinery and detach [6, 7]; in other cases, a true EMT does not occur [5, 8]. Neighboring cells that remain on the exterior must seal the breach left behind by internalizing cells, rearranging and making new cell-cell junctional connections as they do so. While cell internalization is essential for successful gastrulation in numerous organisms, most of the focus thus far has been on cellular events within internalizing cells; relatively less attention has been paid to neighboring cells that seal the embryonic exterior.
The early C. elegans embryo is a useful model system for understanding changes in cell-cell adhesion associated with cell internalization. Gastrulation in C. elegans involves stereotypical events on the ventral surface of the embryo that internalize endodermal, mesodermal, and germ cell precursors [5, 9]. The best studied of these events is the internalization of Ea and Ep, the endodermal precursors. Ea/p undergo myosin-mediated apical constriction [8, 10–13]. Germ cell precursors rely on a different mechanism, involving cadherin-dependent “hitchhiking” [14]. Concomitant with internalization of Ea/p, neighboring cells have been observed to produced protrusions that may aid resealing of the embryo’s surface via active crawling [15]. Together with apical constriction of internalizing cells themselves, these movements are thought to aid cell internalization and simultaneous resealing of the ventral surface [12, 15].
A ventral cleft forms on the surface of the embryo as the last sets of cells are internalized at the end of gastrulation. The ventral cleft is surrounded by neuroblasts derived from ABplp and ABprp in the posterior and ABalp and ABarp in the anterior. The ventral gastrulation cleft is subsequently closed via movements of ventral neuroblasts toward the ventral midline between 230 and 290 minutes postfertilization, causing the ventral cleft to disappear approximately one hour before the movements of ventral epidermal enclosure begin [16, 17]. Failures in ventral cleft closure lead to highly penetrant failure of ventral enclosure (for reviews of this process, see [9, 18, 19]).
Defects in the movement of neuroblasts to close the ventral cleft are observed in embryos defective in several cell signaling pathways, including those involving Eph/ephrin signaling [16, 20, 21], PTP-3/LAR (Leukocyte Common Antigen Related Receptor, a protein tyrosine phosphatase; [22]), semaphorin-2A/MAB-20/plexin signaling [23, 24], and the C. elegans Kallmann syndrome ortholog kal-1 [25, 26]. Such defects result in an enlarged or persistent ventral cleft; if the ventral cleft is not closed by the time of epidermal enclosure, enclosure movements are often disrupted.
The motile events downstream of cell signaling at the ventral cleft are poorly understood; loss of function of the SCAR/WAVE gene wve-1 leads to significant defects in ventral neuroblast organization [27], suggesting that actin-based motility may be important for ventral neuroblast movement. Filopodial protrusions have been observed during cleft closure, but their significance is unclear [28]. As ventral neuroblasts move together, surrounding cells adjacent to the cleft must rearrange as the cleft closes [12]. After the events of ventral cleft closure, the neuroblasts that seal the cleft divide and rearrange to form part of the presumptive ventral nerve cord before the embryo begins to elongate into a vermiform shape [9, 29]. Ventral neuroblasts later accumulate myosin foci and cadherin complex proteins [30].
Internalization of cells during gastrulation in C. elegans involves detachment of cells from their neighbors and establishment of new connections among cells remaining at the ventral surface, so changes in cell-cell adhesion must presumably occur during this process. The C. elegans cadherin/catenin complex (CCC) has been the focus of significant attention in this regard. The core components of the CCC, HMR-1/cadherin, HMP-2/β-catenin, and HMP-1/α-catenin, are present in the early embryo before gastrulation begins [11, 14, 31]. While there is not an essential requirement for cadherin-dependent adhesion during Ea/p internalization in otherwise wild-type embryos, there is a synergistic requirement for the cadherin complex when the L1CAM homologue SAX-7 or CED-5/DOCK180 is depleted [32, 33]. Accumulation of CCC components at the interface between cells that internalize and those that remain on the surface has been proposed to aid recruitment of actomyosin contractile networks necessary for internalization [5, 11], after engagement of an actomyosin-mediated “clutch” in Ea/p [13].
Much of the focus regarding the CCC during gastrulation has been on the internalizing cells, specifically Ea/p. Requirements for the CCC in subsequent internalizations have not been specifically analyzed, nor has the role of the CCC in resealing the ventral surface after internalization been assessed. We set out to investigate roles for the core CCC component, HMP-1, in these processes. In addition, we turned our attention to SRGP-1/srGAP, the lone slit/robo GTPase activating protein in C. elegans [28, 34]. We showed previously that SRGP-1 is a modulator of cell-cell adhesion during the later events of ventral enclosure [28] and embryonic elongation [35, 36]. In addition, however, srgp-1 knockdown in hmp-1(fe4) mutants leads to Gex (Gut on the exterior) phenotypes due to a failure to complete cleft closure [28], implicating it in the earlier events of internalization and ventral sealing at the end of gastrulation.
SRGP-1 is a homolog of vertebrate Slit/Robo GTPase Activating Proteins (srGAPs), which have an N-terminal F-BAR domain that associates with curved membranes, a central RhoGAP domain, and an SH3 domain which has been shown to associate with various other factors such as WAVE, WASP, and Lamellipodin [37–39]. SRGP-1 in C. elegans does not contain an SH3 domain; nevertheless, we showed previously that the SRGP-1 C-terminus interacts with both the N-terminal half of SRGP-1 [28] and with HMP-1 [35]. Overexpression of the F-BAR domain of SRGP-1 leads to ectopic membrane tubulations. The C-terminus of SRGP-1 is required to recruit HMP-1 into these tubulations [28] and for normal HMP-1 dynamics [36], consistent with a role for the SRGP-1 C terminus is coordinating the interaction with HMP-1.
Here we investigated the role of SRGP-1 prior to epidermal morphogenesis, as the ventral surface seals the final breaches due to cell internalization at the end of gastrulation. We found that SRGP-1 is required for normal cell behavior, cell morphology, and HMP-1 recruitment during this essential process. We also found that destabilizing salt bridge mutations within the M (middle) domain of HMP-1, which cause the M domain to remain in an extended state and abrogate binding by the SRGP-1 C terminus [35], are able to suppress SRGP-1 phenotypes. This suppression may be in part due to increased recruitment of components that interact with an open conformation of the M domain, including the C. elegans afadin homologue, AFD-1.
Results
Mutations in srgp-1 lead to cleft closure defects
Our prior work established a role for SRGP-1 in the embryonic epidermis in C. elegans [28, 35], but srGAPs in vertebrates were originally identified through their roles in the developing nervous system [39–43]. In C. elegans, a majority of neuroblasts are found on the ventral side of the embryo following gastrulation [9, 17]. These neuroblasts must (1) adhere to one another to keep other tissues internalized during gastrulation (reviewed in [5, 9]), (2) divide and rearrange to form part of the ventral nerve cord [29], and (3) act as a substrate for the epidermis, which undergoes epiboly during ventral enclosure [23, 30, 44]. Using 4D DIC microscopy, we observed that an appreciable percentage (11.1%) of homozygotes for srgp-1(jc72), a nonsense allele hereafter referred to as srgp-1W122Stop (Fig. 1A), do not complete ventral cleft closure at the end of gastrulation, leading to endodermal precursors being extruded when the epidermis attempts to undergo epiboly and the contractions normally associated with embryonic elongation (Figure 1B, second row).
SRGP-1 has three major functional domains: (1) an N-terminal F-BAR domain, (2) a central GAP domain, and (3) an unstructured C-terminal region that is involved in protein-protein interactions (see Figure 1A, [28, 34, 35, 45]) We explored whether one of these domains might be important for SRGP-1 function during cleft closure. Using CRISPR/Cas9 methodology, we generated the following alleles: srgp-1ΔF-BAR, missense allele srgp-1R563A, which prevents GAP activity [34, 46], and srgp-1ΔC, which deletes most of the region C-terminal to the GAP domain. Loss of the SRGP-1 F-BAR domain and C-terminal region both led to cleft closure defects following gastrulation (Figure 1B). The percentages of embryos that displayed cleft closure defects were similar between srgp-1ΔC and srgp-1W122Stop alleles; while we observed cleft closure defects in srgp-1ΔF-BAR mutant embryos, the lower frequency did not rise to the level of statistical significance compared to wildtype, in which we did not observe cleft closure defects (Figure 1C). As in our previous studies examining the epidermal functions of SRGP-1 [28], we did not observe any obvious defects in embryos lacking SRGP-1 GAP functionality. These results suggest that important aspects of SRGP-1 function during cleft closure are mediated through its C terminus, with the F-BAR domain playing a supporting role.
HMP-1 and SRGP-1 co-localize at the vertices of rosettes following the last internalization events of gastrulation
Gastrulation in C. elegans involves the internalization of progenitor cells that generate endoderm, mesoderm, and germ line tissues. As these cells move into the interior, they undergo apical constriction. As they do so, neighboring cells form transient rosettes to cover the space vacated by the departing cells [12]. We examined endogenously tagged HMP-1::mScarlet-I and SRGP-1::mNeonGreen in living embryos, beginning with ventral cleft formation through the final internalization events of gastrulation, which occur after cleft closure (Figure 2A). Two rosettes form and resolve at this stage, involving cells born on the left and right sides of the ventral cleft (Figure 2A, yellow dotted line; B, colored cells). At the vertex of the anterior rosette, where cells internalize, we observed a bright accumulation of HMP-1::mScarlet-I immediately after the internalization event (Figure 2A, white arrowhead). Subsequent to the accumulation of HMP-1 in the anterior rosette, the posterior rosette resolved and elongated along the anterior-posterior axis, forming new cell contacts as it did so (Figure 2A; yellow arrowheads at 10 min indicate direction of cell movement by 20 min). Significantly, SRGP-1 also accumulated at vertices in both the anterior and posterior rosettes (Fig. 2A, 0 min, arrowheads).
We previously demonstrated that homozygotes carrying a nonsense allele of srgp-1 display decreased HMP-1 junctional intensity [35]. We therefore sought to determine whether srgp-1 mutant backgrounds could influence the localization of HMP-1 during rosette formation, focusing on the anterior rosette. We examined localization of HMP-1::mScarlet-I within the anterior rosette before and after the final set of cell internalizations (Figure 3; blue dotted line indicates internalizing cells). In a full-length, endogenously tagged srgp-1 background, HMP-1 accumulated at the vertex formed by the disappearance of internalizing cells (Figure 3A, 0 min). In contrast, in srgp-1ΔF-BAR mutants HMP-1 at the vertex failed to coalesce into a single cluster (Figure 3B, 0 minutes). In addition, a stable rosette no longer formed, and the remaining neuroblasts instead coalesced into two rows with no central vertex (Figure 3B, yellow lines). In srgp-1ΔC mutants clusters of SRGP-1 accumulated within neuroblasts with no apparent pattern; while HMP-1 was still able to coalesce around the rosette, multiple clusters with accumulated HMP-1 were visible (Figure 3C). Taken together, these results indicate that both the SRGP-1 N- and C-terminal regions have important roles during cleft closure. The F-BAR domain appears to be important for organizing the tips of cells within rosettes into a single vertex, whereas the C-terminus may play an important role in spatially organizing and interacting with HMP-1 during this process.
Destabilizing the M domain of HMP-1 suppresses cleft closure defects in srgp-1 mutants
The M domain of HMP-1 forms a closed structure that is stabilized by multiple salt bridges (Figure 4A; [47]). Mutating Arginines 551 and 554 to alanines prevents two of these salt bridges from forming between MII and MIII; as a result, the HMP-1 M domain adopts a constitutively open conformation that prevents the recruitment of the SRGP-1 C-terminus [35]. Although the hmp-1R551/554A mutation abrogates interaction with the C-terminus of SRGP-1, there is evidence in vertebrates that an extended conformation of α-catenin may activate actin binding and/or recruitment of other binding partners, including vinculin [48–53] and afadin [54]. We therefore assessed whether a constitutively open conformation of HMP-1 could bypass the requirement for SRGP-1 at the end of gastrulation.
Mutants homozygous for hmp-1R551/554A and either the srgp-1W122Stop or the srpg-1ΔC allele displayed fewer cleft closure defects compared to srgp-1W122Stop or srpg-1ΔC homozygotes with wild-type hmp-1; in contrast, there was no change in frequency of cleft closure defects in srgp-1ΔF-BAR homozygotes when the salt bridge mutations were introduced (Figure 4B). These results suggest that an open conformation of the HMP-1 M domain can bypass some functions normally performed by the SRGP-1 C-terminus, but that the SRGP-1 F-BAR domain is still required, presumably independently of binding of the SRGP-1 C terminus to the HMP-1 M domain.
To test if the salt bridge mutations in HMP-1 truly behave as gain-of-function mutations, we introduced these mutations into the hmp-1(fe4) background. The hmp-1(fe4) allele, hereafter referred to as hmp-1S823F, replaces a serine with a phenylalanine (S823F) within the actin binding domain of HMP-1 and behaves as a hypomorph (Figure 4A; [55, 56]). hmp-1S823F homozygotes display morphogenetic failure and developmental arrest at a variety of stages, including during cleft closure. Using CRISPR/Cas9 we introduced the R551/554A mutations into the hmp-1S823F background. Hermaphrodites homozygous for hmp-1R551/554A; S823F on average laid more embryos and had minimal body morphology defects compared to hmp-1S823F homozygotes (Figure 4C,F). hmp-1R551/554A; S823F homozygous embryos also showed reduced lethality compared to embryos homozygous for hmp-1S823F (Figure 4D,E). However, when we imaged hmp-1R551/554A; S823F embryos, we found that they were more sensitive to mechanical pressure in 10% agar mounts and required mounting on 5% agar pads (Figure S1), which we used for subsequent experiments utilizing this allele. We also observed that hmp-1S823F homozygotes exhibited increased embryonic lethality at colder temperatures (Figure 4D). We examined whether the hmp-1R551/554A mutation suppressed hmp-1S823F phenotypes at 15°C. The introduction of the salt bridge mutations partially suppressed embryonic lethality at 15°C, and, although it did not rise to the level of statistical significance, hmp-1R551/554A;S823F homozygotes reared at 15°C had an increase in brood size compared to hmp-1S823F homozygotes (Figure 4C,D). Taken together, these results indicate that the hmp-1R551/554A mutation acts as a gain-of-function allele that can partially offset reduction of actin binding activity conferred by the C-terminal S823F mutation.
We previously utilized hmp-1S823F hypomorphic (hmp-1(fe4)) homozygotes as a sensitized background to identify modulators of cadherin-dependent adhesion [57] and identified srgp-1 as a strong enhancer of embryonic lethality in the hmp-1S823F background. RNAi knockdown of srgp-1 resulted in nearly total embryonic lethality, while srgp-1 knockdown by feeding RNAi in wild-type embryos had minimal effects. At least some of the synergistic lethality was caused by Gex phenotypes during ventral enclosure [28]. We therefore examined the synergistic effect of srgp-1 RNAi knockdown in hmp-1R551/554A; S823F embryos. The salt bridge mutation was able to reduce the embryonic lethality of srgp-1(RNAi);hmp-1S823F embryos significantly (Figure 4E). These results confirm that an open conformation of the HMP-1 M domain is able to bypass some requirements for SRGP-1, in addition to its ability to compensate for reduced actin binding activity mediated by the HMP-1 C terminus.
Loss of afd-1/afadin function leads to increased frequency of cleft closure defects
Conformational changes within the α-catenin M domain can affect its ability to recruit components that modulate cell adhesion. One such modulator in vertebrates is vinculin; when the αE-catenin M domain is extended, either via direct mechanical distension [58–60] or by introducing salt bridge mutations [51], its binding affinity for vinculin is increased. However, DEB-1, the vinculin homolog in C. elegans does not interact with HMP-1 [47], and its expression is confined to muscle cells during development [61–63], ruling it out as a candidate HMP-1 interactor that could be positively affected by the R551/554A salt bridge mutations in the context of cleft closure. Another candidate modulator is AFD-1/afadin. Vertebrate afadin can bind to αE-catenin [64, 65]. While there is currently no published evidence for direct binding of the Drosophila afadin, Canoe, to α-catenin (M. Peifer and U. Tepass, pers. commun.), Canoe localizes to cell-cell junctions and modulates morphogenesis in a variety of contexts in Drosophila [66–71]. Moreover, we showed previously that AFD-1 can be co-immunoprecipitated with HMP-1 [72] and that loss of afd-1 function synergizes with the hmp-1S823F mutation during later morphogenesis [57]. We therefore examined whether afd-1 loss of function showed genetic interactions with srgp-1 and with hmp-1 salt bridge mutations.
We first determined whether afd-1 RNAi led to cleft closure defects and lethality in wild-type embryos or in embryos homozygous for the hmp-1S823F allele (Figure 5A-C). Knockdown of srgp-1 or afd-1 led to comparable levels of lethality and cleft closure defects in otherwise wild-type embryos on agar mounts. Moreover, RNAi against either srpg-1 and afd-1 caused a significant increase in cleft closure defects in hmp-1S823F embryos on plates or agar mounts (Figures 5A-C). Since both srgp-1 and afd-1 knockdown increased the frequency of cleft closure defects in hmp-1S823F, we examined how afd-1 knockdown genetically interacted with srgp-1 loss of function and with the hmp-1R551/554A mutation. In wild-type, hmp-1R551/554A, and srgp-1W122Stop backgrounds, afd-1 knockdown resulted in an increase in cleft closure defects (Figure 5C). In srgp-1W122Stop; hmp-1R551/554A double mutants, loss of afd-1 resulted in a higher frequency of cleft closure defects than in hmp-1R551/554A alone, but less than in srgp-1W122Stop mutants alone, suggesting that there may be additional factors beyond AFD-1 that may stabilize HMP-1 when it adopts an open conformation.
Computational work was previously used to engineer the actin binding domain of human αE-catenin to bind actin with higher affinity [50]. Using protein alignment, we identified the homologous amino acids in HMP-1 and generated hmp-1QNLM676-679GSGS, which is predicted to bind actin with higher affinity (Figure S2A,B). Embryos homozygous for hmp-1QNLM676-679GSGS have a low level of embryonic lethality, which causes developmental arrest at various embryonic stages, including during cleft closure. These phenotypes were suppressed by loss of function of srgp-1 and afd-1 (Figure S2C,D). These results suggest that HMP-1 stability and linkage to the actin network must be maintained within a dynamic range during processes that contribute to cleft closure.
AFD-1 localizes to the vertex of the anterior rosette at the end of gastrulation
Since genetic perturbation of afd-1 had consequences for cleft closure, we next assessed the localization of AFD-1 during cleft closure. We visualized mKate2::AFD-1 and HMP-1::GFP from cleft closure through rosette formation. While expression of mKate2::AFD-1 in the ventral neuroblasts was weak, we observed strong accumulation of AFD-1 at the vertex of the anterior rosette immediately following the final internalization events of gastrulation, which quickly dispersed as the rosette resolved (Figure 6A). We next examined HMP-1 accumulation and localization at the vertex of the anterior rosette in various mutant backgrounds (Figure 6B). Total accumulation of HMP-1::GFP increased in the hmp-1R551/554A mutant background, but RNAi against afd-1 reduced total HMP-1 accumulation at the vertex, as well as the spatial extent of HMP-1 accumulation at the vertex. We did not find a significant change in AFD-1 accumulation in srgp-1W122Stop mutants; while there was an increase in AFD-1 accumulation in hmp-1R551/4A homozygotes, it did not quite rise to statistical significance (Figure S3). These results suggest that while SRGP-1 may play an important role in orienting cells during rosette formation and organizing HMP-1 around the vertex, AFD-1 is essential for normal HMP-1 accumulation at the vertex following the final internalization events of gastrulation. Unfortunately, we could not perform the converse experiment to address whether AFD-1 recruitment requires HMP-1 at this stage of development, because depletion of maternal and zygotic HMP-1 leads to catastrophic morphogenetic failure, including lack of cleft closure [31, 73].
Discussion
Rosette formation during C. elegans gastrulation requires cadherin-based adhesion
Ventral cleft closure is the culmination of gastrulation in the C. elegans embryo. It is essential for proper organization and cohesion of neuroblasts following gastrulation, which in turn is crucial for the embryo to survive the mechanical forces that operate during later morphogenesis [5, 9, 12]. Here we have characterized the cell rearrangements that accompany sealing of the ventral surface of the embryo, as cells on the surface change position to accommodate loss of cells that internalize near the end of gastrulation. Specifically, we have demonstrated that HMP-1/α-catenin and two of its functional modulators, SRGP-1/srGAP and AFD-1/afadin, facilitate the adhesion of cells during this critical stage in embryogenesis in the C. elegans embryo. Based on prior work, rosettes that form as a result of earlier cell internalization events in C. elegans appear similar [12], so insights gleaned from studying these later events will likely be useful in understanding other internalization events in the earlier embryo.
Cell internalization is a common event during gastrulation in metazoan embryos, as cells destined for the embryo’s interior detach their apical surfaces from the embryo’s exterior [2–5]. Given the apical-to-basal axis of such movements during C. elegans gastrulation, internalization also bears similarities to other basal extrusion events, often triggered by apoptosis or cell crowding in a variety of epithelia (reviewed in [74–76]). In all these cases, however, relatively little attention has been paid to how the cells that remain on the surface seal breaches on the embryonic exterior left behind by internalizing cells. At least in some cases, such tissue sealing involves multicellular rosette formation. The geometry of these rosettes bears similarities to those associated with other morphogenetic processes, such as convergent extension [77]. An intriguing parallel to the rosettes we observed are those observed in the chick epiblast [3, 78, 79]. Although the functional significance of the rosette structures in the primitive streak is unclear, these may reflect similar events at sites where cells depart from the surface of the embryo during gastrulation.
Rosettes in other contexts, such as during convergent extension in the Drosophila germband, involve modulation of adhesion complexes as cells change their connections to one another [80, 81]. In contrast, little is known about adhesive changes among neighboring non-intercalating cells that seal gaps left behind by ingressing cells. In the case of sea urchin primary mesenchyme cells, which exhibit many aspects of standard epithelial-mesenchymal transition [82], cells lose cadherin-catenin complex components at the time of ingression [82, 83]. The situation may be different in gastrulating cells in C. elegans and Drosophila neuroblasts; in the former, at least in the case of internalization of endodermal founder cells Ea and Ep, CCC components are transiently upregulated during apical constriction [11], while in the latter, post-translational loss of CCC components can be uncoupled from ingression events [84]. These differences indicate that while many processes may be conserved during internalization events, there may be a variety of mechanisms involved.
Our results shed light on this relatively understudied process by demonstrating that rosette formation at the end of gastrulation in C. elegans requires a robust cell-cell adhesion machinery. Reduction in the ability of HMP-1/α-catenin to bind F-actin in hmp-1S823F mutants leads to an increase in ventral cleft closure failure, as does loss of the HMP-1 binding partner, SRGP-1. The cells surrounding the position of the vacated cell at the end of ventral cleft closure form a rosette, which ultimately resolves as cells make new connections to one another at the site of sealing.
Rosette formation is fostered by activating HMP-1/α-catenin
We and others have shown that the α-catenin M domain engages in interactions that regulate the C-terminal F-actin binding region of α-catenins [50, 85]. In this context it is striking that the hmp-1R551/554A mutation suppresses phenotypes associated with the S823F mutation, which we have shown previously measurably decreases the F-actin binding activity of HMP-1 [86]. Previous intragenic suppressors all clustered in the C terminus of HMP-1, not in the M domain [86]. Our present results provide further evidence that the conformation of the M domain is relevant to the ability of HMP-1 to interact, either directly or indirectly, with the actin cytoskeleton.
Rosette formation depends on proper HMP-1/a-catenin localization mediated by both the C-terminus and F-BAR domains of SRGP-1/srGAP
Salt bridges in the M domain of mammalian αE-catenin stabilize the M domain in a “closed” conformation, reducing the likelihood of association of vinculin [51, 59]. In C. elegans, however, we have shown previously that DEB-1/vinculin is confined to myoblasts in the early embryo, and that it does not bind HMP-1 [47, 86], suggesting that HMP-1 interacts with other effectors in non-muscle cells. In addition to its utility in identifying intramolecular interactions that regulate HMP-1 activity, the hmp-1S823F mutation has been useful as a sensitized background for identifying such functional interactors. Both SRGP-1/srGAP and AFD-1/afadin were identified in a genome-wide RNAi screen for such interactors [57]. Our previous analysis indicated that the C terminus of SRGP-1 can physically bind the HMP-1 M domain, but, unlike the case with vertebrate aE-catenin and vinculin, not when the HMP-1 M domain is fully extended. We also showed that both the N-terminal F-BAR and C-terminal domains of SRGP-1 are functionally important during elongation [36].
Our analysis here also revealed roles for the N- and C-terminal regions of SRGP-1 during ventral cleft closure. HMP-1 becomes highly concentrated at the tips of cells at rosette vertices in embryos expressing endogenously tagged, full-length SRGP-1. The greater severity of gross morphological defects in srgp-1 nonsense and C-terminal deletion mutants further suggests a more stringent requirement for the C terminus, which is lacking in both mutants, in stabilizing HMP-1. Since the SRGP-1 C terminus is intact in srgp-1ΔF-BAR mutants, it is possible that, whereas SRGP-1ΔF-BAR can no longer interact with the membrane directly to stabilize the CCC, when the N-terminus is absent SRGP-1 can still interact with HMP-1 in some functional capacity. In this case HMP-1 presumably exclusively relies on its association with the HMP-1/HMP-2/HMR-1 heterotrimeric complex to associate with the plasma membrane, which is less efficient in recruiting HMP-1 at sites of high membrane curvature, such as cell tips at rosette vertices. The likelihood of this possibility is strengthened by our observation that srgp-1ΔF-BAR homozygous embryos exhibit less lethality than srgp-1W122Stop and srgp-1ΔC homozygotes. In our previous work we suggested that there may be a second region of SRGP-1, which lies N-terminal to the C-terminal region, that can interact with some junctional component— possibly including HMP-1 [28, 36]; our present work is consistent with this possibility.
A distinct role for the SRGP-1 N terminus is also suggested by our results. When the SRGP-1 F-BAR domain is deleted the tips of cells in the rosette are blunted and HMP-1 forms multiple aggregates in cells in the rosette, leading to less robust rosettes. In our previous work we observed reduced membrane curvature at the leading edge during ventral epidermal enclosure, suggesting that SRGP-1 promotes highly curved membranes [28]. Our present results are consistent with a similar role at nascent rosette vertices, which require that the plasma membranes of cell tips in the rosette adopt a high degree of curvature. SRGP-1 may either stabilize such highly curved regions of the plasma membrane or be recruited to such sites, leading to clustering of HMP-1 at such sites to stabilize nascent adhesions. The loss of normal HMP-1 accumulation at cell tips in hmp-1ΔF-BAR mutants is consistent with this possibility and suggests that not only is membrane curvature adversely affected, but that HMP-1 recruitment to sites of high membrane curvature is reduced, with adverse effects on rosettes.
The hmp-1R551/554A mutation, which maintains the HMP-1 M domain in an open conformation [36], can suppress cleft closure defects caused by loss of the SRGP-1 C-terminus, but not those resulting from loss of the SRGP-1 F-BAR domain (see Figure 4B). There are several potential explanations for this result. One possibility is that the C terminus of SRGP-1 regulates HMP-1 function beyond localization. For example, the C terminus of SRGP-1, once bound, could facilitate further opening and activation of the HMP-1 M domain, leading to recruitment of other binding partners. Constitutive opening of the HMP-1 M domain in hmp-1R551/554A mutants could obviate this requirement. Alternatively, if the major role of the SRGP-1 C terminus is to fine-tune the localization or stability of HMP-1, the enhanced activity of a fully open HMP-1 could offset the quantitative loss of HMP-1 at nascent junctions in rosettes.
Rosette formation is fostered by recruitment of AFD-1/afadin
AFD-1 accumulation at the vertex of the anterior rosette is striking compared to the low levels of AFD-1 accumulation elsewhere at this stage of development, including the posterior rosette, within which we did not see a similar accumulation of AFD-1. This indicates that junctions at the anterior rosette vertex are unique among cell-cell adhesions between ventral cells in the embryo at this stage, and that AFD-1/afadin is crucial for stabilizing them. Vertebrate afadin and Drosophila Canoe are recruited to junctions under increased tension or to sites with increased cellular (and hence actomyosin) dynamics [87–89]. In C. elegans, during later development when the epidermis is under substantial mechanical tension, AFD-1 appears at epidermal cell-cell junctions [57], consistent with tension-induced recruitment at that stage. That AFD-1 is recruited to the tips of cells in the anterior rosette vertex at the end of gastrulation suggests that the tips of these cells likewise experience increased tension. As the internalization event that forms the anterior rosette concludes, multiple cells must converge to create new contact points, which are susceptible to mechanical failure. A similar accumulation of AFD-1 is not observed in the posterior rosette, however. Notably, the cells of the posterior rosette undergo rapid extension shortly after rosette formation, whereas the anterior rosette persists. Work in Drosophila has demonstrated a necessity for Canoe localization to maintain tricellular junctions experiencing high tension; however, prolonged and continued accumulation of Canoe at junctions prevents vertex resolution during cell rearrangement [90]. If AFD-1 works in a similar fashion in C. elegans, this could imply that AFD-1 is required to stabilize the anterior rosette under higher mechanical loads.
We also found that, as is the case for srgp-1, loss of afd-1 function leads to ventral cleft closure defects that can be suppressed via the hmp-1R551/554A mutation. Moreover, simultaneous depletion of SRGP-1 and AFD-1 leads to synergistic ventral cleft closure defects. One reasonable model that accounts for this data is that, while SRGP-1 fosters the initial recruitment of HMP-1 to nascent contact sites within rosettes, AFD-1 subsequently stabilizes more mature adhesions, allowing them to withstand tension prior to rosette resolution. In this case, forcing HMP-1 into an open conformation may be able to bypass functional requirements for SRGP-1 by increasing the stability of adhesions through additional AFD-1 recruitment. It remains unclear whether AFD-1 can directly interact with HMP-1 in the way that their vertebrate counterparts do [54, 65], or if AFD-1 is recruited to cell-cell adhesion sites through other effectors that in turn depend on an open conformation of HMP-1. Since defects in afd-1(RNAi); srpg-1W122Stop double loss-of-function embryos are still suppressed by hmp-1R551/554A, there may be additional mechanisms that are stimulated by an open conformation of the HMP-1 M domain.
In conclusion, this work has clarified how cadherin-dependent adhesion between non-internalizing neighbors of internalizing cells, supported by SRGP-1/srGAP and AFD-1/afadin, stabilizes nascent cell-cell adhesions following the internalization events of gastrulation. Future work focused on identifying other factors that play a role in anterior rosette formation and that dissects the mechanisms through which SRGP-1, AFD-1, and HMP-1 work together in this process should continue to clarify the cellular events of tissue sealing following internalization.
Contributions
JMS designed, implemented, and analyzed experiments, designed and generated the novel srgp-1 and hmp-1 CRISPR alleles, and wrote and edited drafts of the manuscript. MMS generated the afd-1 knock-in allele. BG commented on the manuscript. JH oversaw experimental design, implementation, and analysis and edited the manuscript.
Materials and Methods
Strains and genetics
C. elegans were maintained using standard methods. Bristol N2 was used as wildtype. A complete list of strains and genotypes used in this manuscript can be found in Supplementary Table 1.
DIC imaging
Four dimensional DIC movies were collected on either a Nikon Optiphot-2 microscope connected to a QiCAM camera (QImaging) or an Olympus BX50 microscope connected to a Scion CFW-1512M camera (Scion Corp.) using Micro-Manager software (v. 1.42) [91, 92]. ImageJ plugins (https://worms.zoology.wisc.edu/research/4d/4d.html) were used to compress and view DIC movies. All embryos were mounted on 10% agar pads in M9 solution unless otherwise specified.
Confocal imaging
Embryos were dissected from adult hermaphrodites and mounted onto 10% agar pads in M9 solution and imaged. For fluorescence imaging, a Dragonfly 500 spinning disc confocal microscope (Andor Corp.), mounted on a Leica DMi8 microscope, equipped with an iXon-EMCCD camera and controlled by Fusion software (Andor Corp.) was used to collect images using 0.21 μm slices with a 63×/1.3 NA glycerol Leica objective at 20°C.
CRISPR/Cas9 genome editing
All novel knock-in and deletion alleles with jc## designation were generated via plasmid-based CRISPR/Cas9 [93] using repair templates cloned by SapTrap cloning [94]. Small substitution mutations were made via marker-free genome editing [95]. Guides, homology arm primers, and single-stranded repair templates for all CRISPR/Cas9 editing can be found in Supplementary Table 2.
Injection RNAi
Injection RNAi was performed by synthesizing double-stranded RNA (dsRNA) using a T7 Megascript kit (Invitrogen). The templates for srgp-1 and control RNAi were obtained from a feeding library [96]. pIC386 was used as a template for production of afd-1 dsRNA. dsRNA was injected at a concentration of 2μg/μL in nuclease free water. L4 worms were injected and aged overnight before embryos were dissected from mature adults for imaging.
Quantification and analysis
Percentage cleft closure defects were measured from embryos mounted for DIC imaging. Embryonic lethality was quantified by dividing the number of unhatched embryos laid on a plate by the total number of embryos on the plate from a single hermaphrodite. Total accumulation (integrated signal) and aggregation size for HMP-1::GFP were measured by drawing a circle around GFP signal at the vertex immediately following the internalization event.
Statistical analysis
Data from control and experimental groups were compared using one-way ANOVA with Tukey post hoc testing to assess significance between individual groups. All statistical analyses were carried out in Prism (GraphPad Corp.).
Acknowledgements
Some strains were provided by the C. elegans Genetics Center, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440). JS and JH were supported by NIH grants R01GM058038, R01GM127687, and R35GM145312. BG was supported by NIH grant R35GM134838. MMS was supported by NIH grant F32GM119348
References
- 1.↵
- 2.↵
- 3.↵
- 4.
- 5.↵
- 6.↵
- 7.↵
- 8.↵
- 9.↵
- 10.↵
- 11.↵
- 12.↵
- 13.↵
- 14.↵
- 15.↵
- 16.↵
- 17.↵
- 18.↵
- 19.↵
- 20.↵
- 21.↵
- 22.↵
- 23.↵
- 24.↵
- 25.↵
- 26.↵
- 27.↵
- 28.↵
- 29.↵
- 30.↵
- 31.↵
- 32.↵
- 33.↵
- 34.↵
- 35.↵
- 36.↵
- 37.↵
- 38.
- 39.↵
- 40.
- 41.
- 42.
- 43.↵
- 44.↵
- 45.↵
- 46.↵
- 47.↵
- 48.↵
- 49.
- 50.↵
- 51.↵
- 52.
- 53.↵
- 54.↵
- 55.↵
- 56.↵
- 57.↵
- 58.↵
- 59.↵
- 60.↵
- 61.↵
- 62.
- 63.↵
- 64.↵
- 65.↵
- 66.↵
- 67.
- 68.
- 69.
- 70.
- 71.↵
- 72.↵
- 73.↵
- 74.↵
- 75.
- 76.↵
- 77.↵
- 78.↵
- 79.↵
- 80.↵
- 81.↵
- 82.↵
- 83.↵
- 84.↵
- 85.↵
- 86.↵
- 87.↵
- 88.
- 89.↵
- 90.↵
- 91.↵
- 92.↵
- 93.↵
- 94.↵
- 95.↵
- 96.↵