Jeff Hardin

Reciprocal asymmetry of SYS-1/β-catenin and POP-1/TCF controls asymmetric divisions in Caenorhabditis elegans

β-Catenins are conserved regulators of metazoan development that function with TCF DNA-binding proteins to activate transcription. In Caenorhabditis elegans, SYS-1/β-catenin and POP-1/TCF regulate several asymmetric divisions, including that of the somatic gonadal precursor cell (SGP). In the distal but not the proximal SGP daughter, SYS-1/β-catenin and POP-1/TCF transcriptionally activate ceh-22 to specify the distal fate. Here, we investigate the distribution of SYS-1/β-catenin and its regulation. Using a rescuing transgene, VNS::SYS-1, which fuses VENUS fluorescent protein to SYS-1, we find more VNS::SYS-1 in distal than proximal SGP daughters, a phenomenon we call “SYS-1 asymmetry.” In addition, SYS-1 asymmetry is seen in many other tissues, consistent with the idea that SYS-1 regulates asymmetric divisions broadly during C. elegans development. In particular, SYS-1 is more abundant in E than MS, and SYS-1 is critical for the endodermal fate. In all cases, SYS-1 is reciprocal to POP-1 asymmetry: cells with higher SYS-1 have lower POP-1, and vice versa. SYS-1 asymmetry is controlled posttranslationally and relies on frizzled and dishevelled homologs, which also control POP-1 asymmetry. Therefore, upstream regulators modulate the SYS-1 to POP-1 ratio by increasing SYS-1 and decreasing POP-1 within the same cell. By contrast, SYS-1 asymmetry does not rely on WRM-1, which appears specialized for POP-1 asymmetry. We suggest a two-pronged pathway for control of SYS-1:POP-1, which can robustly accomplish differential gene expression in daughters of an asymmetric cell division.

The secret life of α-catenin: Moonlighting in morphogenesis

Cadherin-based intercellular adhesions are important determinants of proper tissue architecture. These adhesions must be both stable and dynamic to maintain tissue integrity as cells undergo morphogenetic movements during development. The role of α-catenin in this process has been vigorously debated due to conflicting in vitro and in vivo evidence regarding its molecular mechanism of action. Recent data supports the classical view that α-catenin facilitates actin attachments at adherens junctions, but also suggests that α-catenin may act as a force transducer, and may have additional roles in the cytoplasm. These multiple functions for α-catenin converge on the regulation of adhesion and may help to explain its stable yet dynamic nature.

Requirements for F-BAR proteins TOCA-1 and TOCA-2 in actin dynamics and membrane trafficking during Caenorhabditis elegans oocyte growth and embryonic epidermal morphogenesis.

The TOCA family of F-BAR–containing proteins bind to and remodel lipid bilayers via their conserved F-BAR domains, and regulate actin dynamics via their N-Wasp binding SH3 domains. Thus, these proteins are predicted to play a pivotal role in coordinating membrane traffic with actin dynamics during cell migration and tissue morphogenesis. By combining genetic analysis in Caenorhabditis elegans with cellular biochemical experiments in mammalian cells, we showed that: i) loss of CeTOCA proteins reduced the efficiency of Clathrin-mediated endocytosis (CME) in oocytes. Genetic interference with CeTOCAs interacting proteins WSP-1 and WVE-1, and other components of the WVE-1 complex, produced a similar effect. Oocyte endocytosis defects correlated well with reduced egg production in these mutants. ii) CeTOCA proteins localize to cell–cell junctions and are required for proper embryonic morphogenesis, to position hypodermal cells and to organize junctional actin and the junction-associated protein AJM-1. iii) Double mutant analysis indicated that the toca genes act in the same pathway as the nematode homologue of N-WASP/WASP, wsp-1. Furthermore, mammalian TOCA-1 and C. elegans CeTOCAs physically associated with N-WASP and WSP-1 directly, or WAVE2 indirectly via ABI-1. Thus, we propose that TOCA proteins control tissues morphogenesis by coordinating Clathrin-dependent membrane trafficking with WAVE and N-WASP–dependent actin-dynamics.

Loss of the RhoGAP SRGP-1 promotes the clearance of dead and injured cells in Caenorhabditis elegans

Multicellular animals rapidly clear dying cells from their bodies. Many of the pathways that mediate this cell removal are conserved through evolution. Here, we identify srgp-1 as a negative regulator of cell clearance in both Caenorhabditis elegans and mammalian cells. Loss of srgp-1 function results in improved engulfment of apoptotic cells, whereas srgp-1 overexpression inhibits apoptotic cell corpse removal. We show that SRGP-1 functions in engulfing cells and functions as a GTPase activating protein (GAP) for CED-10 (Rac1). Interestingly, loss of srgp-1 function promotes not only the clearance of already dead cells, but also the removal of cells that have been brought to the verge of death through sublethal apoptotic, necrotic or cytotoxic insults. In contrast, impaired engulfment allows damaged cells to escape clearance, which results in increased long-term survival. We propose that C. elegans uses the engulfment machinery as part of a primitive, but evolutionarily conserved, survey mechanism that identifies and removes unfit cells within a tissue.

The C. elegans Zonula Occludens Ortholog Cooperates with the Cadherin Complex to Recruit Actin during Morphogenesis

The dramatic cell-shape changes necessary to form a multicellular organism require cell-cell junctions to be both pliable and strong. The zonula occludens (ZO) subfamily of membrane-associated guanylate kinases (MAGUKs) are scaffolding molecules thought to regulate cell-cell adhesion [1-3], but there is little known about their roles in vivo. To elucidate the functional role of ZO proteins in a living embryo, we have characterized the sole C. elegans ZO family member, ZOO-1. ZOO-1 localizes with the cadherin-catenin complex during development, and its junctional recruitment requires the transmembrane proteins HMR-1/E-cadherin and VAB-9/claudin, but surprisingly, not HMP-1/alpha-catenin or HMP-2/beta-catenin. zoo-1 knockdown results in lethality during elongation, resulting in the rupture of epidermal cell-cell junctions under stress and failure of epidermal sheet sealing at the ventral midline. Consistent with a role in recruiting actin to the junction in parallel to the cadherin-catenin complex, zoo-1 loss of function reduces the dynamic recruitment of actin to junctions and enhances the severity of actin filament defects in hypomorphic alleles of hmp-1 and hmp-2. These results show that ZOO-1 cooperates with the cadherin-catenin complex to dynamically regulate strong junctional anchorage to the actin cytoskeleton during morphogenesis.

SAX-7/L1CAM and HMR-1/cadherin function redundantly in blastomere compaction and non-muscle myosin accumulation during Caenorhabditis elegans gastrulation

Gastrulation is the first major morphogenetic movement in development and requires dynamic regulation of cell adhesion and the cytoskeleton. Caenorhabditis elegans gastrulation begins with the migration of the two endodermal precursors, Ea and Ep, from the surface of the embryo into the interior. Ea/Ep migration provides a relatively simple system to examine the intersection of cell adhesion, cell signaling, and cell movement. Ea/Ep ingression depends on correct cell fate specification and polarization, apical myosin accumulation, and Wnt activated actomyosin contraction that drives apical constriction and ingression (Lee et al., 2006; Nance et al., 2005). Here, we show that Ea/Ep ingression also requires the function of either HMR-1/cadherin or SAX-7/L1CAM. Both cadherin complex components and L1CAM are localized at all sites of cell-cell contact during gastrulation. Either system is sufficient for Ea/Ep ingression, but loss of both together leads to a failure of apical constriction and ingression. Similar results are seen with isolated blastomeres. Ea/Ep are properly specified and appear to display correct apical-basal polarity in sax-7(eq1);hmr-1(RNAi) embryos. Significantly, in sax-7(eq1);hmr-1(RNAi) embryos, Ea and Ep fail to accumulate myosin (NMY-2Colon, two colonsGFP) at their apical surfaces, but in either sax-7(eq1) or hmr-1(RNAi) embryos, apical myosin accumulation is comparable to wild type. Thus, the cadherin and L1CAM adhesion systems are redundantly required for localized myosin accumulation and hence for actomyosin contractility during gastrulation. We also show that sax-7 and hmr-1 function are redundantly required for Wnt-dependent spindle polarization during division of the ABar blastomere, indicating that these cell surface proteins redundantly regulate multiple developmental events in early embryos.

The assembly and maintenance of epithelial junctions in C. elegans

The epithelial tissues of the C. elegans embryo provide a minimalist system for examining phylogenetically conserved proteins that function in epithelial polarity and cell-cell adhesion in a multicellular organism. In this review, we provide an overview of three major molecular complexes at the apical surface of epithelial cells in the C. elegans embryo: the cadherin-catenin complex, the more basal DLG-1/AJM-1 complex, and the apical membrane domain, which shares similarities with the subapical complex in Drosophila and the PAR/aPKC complex in vertebrates. We discuss how the assembly of these complexes contributes to epithelial polarity and adhesion, proteins that act as effectors and/or regulators of each subdomain, and how these complexes functionally interact during embryonic morphogenesis. Although much remains to be clarified, significant progress has been made in recent years to clarify the role of these protein complexes in epithelial morphogenesis, and suggests that C. elegans will continue to be a fruitful system in which to elucidate functional roles for these proteins in a living embryo.

4 The Cellular Basis of Sea Urchin Gastrulation

Publisher Summary This chapter discusses the cellular mechanisms of morphogenesis in the sea urchin gastrula. The sea urchin embryo has been historically important system for investigating the cellular basis of gastrulation. Sea urchin embryos can be obtained in large numbers; they develop synchronously, they are optically transparent, and their organization is relatively simple. The simplicity of the organization of the sea urchin embryo makes it an appealing model system for undertaking a cellular analysis of gastrulation. Mechanical interactions are significant during gastrulation. Forces capable of shaping the embryo may be produced by single cells or groups of cells. Understanding the response of other cells to such forces is a part of the analysis of gastrulation. Behaviors exhibited by a cell, may serve multiple functions during gastrulation. A clear example of such multiplicity is the role played by the secondary mesenchyme cells. Their mechanical influence seems to be important for elongating the archenteron, but they are also required for correct attachment and positioning of the tip of the archenteron.

Sarcomeric actin organization is synergistically promoted by tropomodulin, ADF/cofilin, AIP1 and profilin in C. elegans

Sarcomeric organization of thin and thick filaments in striated muscle is important for the efficient generation of contractile forces. Sarcomeric actin filaments are uniform in their lengths and regularly arranged in a striated pattern. Tropomodulin caps the pointed end of actin filaments and is a crucial regulator of sarcomere assembly. Here, we report unexpected synergistic functions of tropomodulin with enhancers of actin filament dynamics in Caenorhabditis elegans striated muscle. Pointed-end capping by tropomodulin inhibited actin filament depolymerization by ADF/cofilin in vitro. However, in vivo, the depletion of tropomodulin strongly enhanced the disorganization of sarcomeric actin filaments in ADF/cofilin mutants, rather than antagonistically suppressing the phenotype. Similar phenotypic enhancements by tropomodulin depletion were also observed in mutant backgrounds for AIP1 and profilin. These in vivo effects cannot be simply explained by antagonistic effects of tropomodulin and ADF/cofilin in vitro. Thus, we propose a model in which tropomodulin and enhancers of actin dynamics synergistically regulate elongation and shortening of actin filaments at the pointed end.

The N- or C-terminal domains of DSH-2 can activate the C. elegans Wnt/β-catenin asymmetry pathway

Dishevelleds are modular proteins that lie at the crossroads of divergent Wnt signaling pathways. The DIX domain of dishevelleds modulates a beta-catenin destruction complex, and thereby mediates cell fate decisions through differential activation of Tcf transcription factors. The DEP domain of dishevelleds mediates planar polarity of cells within a sheet through regulation of actin modulators. In Caenorhabditis elegans asymmetric cell fate decisions are regulated by asymmetric localization of signaling components in a pathway termed the Wnt/beta-catenin asymmetry pathway. Which domain(s) of Disheveled regulate this pathway is unknown. We show that C. elegans embryos from dsh-2(or302) mutant mothers fail to successfully undergo morphogenesis, but transgenes containing either the DIX or the DEP domain of DSH-2 are sufficient to rescue the mutant phenotype. Embryos lacking zygotic function of SYS-1/beta-catenin, WRM-1/beta-catenin, or POP-1/Tcf show defects similar to dsh-2 mutants, including a loss of asymmetry in some cell fate decisions. Removal of two dishevelleds (dsh-2 and mig-5) leads to a global loss of POP-1 asymmetry, which can be rescued by addition of transgenes containing either the DIX or DEP domain of DSH-2. These results indicate that either the DIX or DEP domain of DSH-2 is capable of activating the Wnt/beta-catenin asymmetry pathway and regulating anterior-posterior fate decisions required for proper morphogenesis.