Intermediate filaments are required for C. elegans epidermal elongation

Cytoplasmic intermediate filaments (cIFs) are thought to provide mechanical strength to vertebrate cells; however, their function in invertebrates has been largely unexplored. The Caenorhabditis elegans genome encodes multiple cIFs. The C. elegans ifb-1 locus encodes two cIF isoforms, IFB-1A and IFB-1B, that differ in their head domains. We show that both IFB-1 isoforms are expressed in epidermal cells, within which they are localized to muscle-epidermal attachment structures. Reduction in IFB-1A function by mutation or RNA interference (RNAi) causes epidermal fragility, abnormal epidermal morphogenesis, and muscle detachment, consistent with IFB-1A providing mechanical strength to epidermal attachment structures. Reduction in IFB-1B function causes morphogenetic defects and defective outgrowth of the excretory cell. Reduction in function of both IFB-1 isoforms results in embryonic arrest due to muscle detachment and failure in epidermal cell elongation at the 2-fold stage. Two other cIFs, IFA-2 and IFA-3, are expressed in epidermal cells. We show that loss of function in IFA-3 results in defects in morphogenesis indistinguishable from those of embryos lacking ifb-1. In contrast, IFA-2 is not required for embryonic morphogenesis. Our data indicate that IFB-1 and IFA-3 are likely the major cIF isoforms in embryonic epidermal attachment structures.     

The cytoskeleton and epidermal morphogenesis in C. elegans

During Caenorhabditis elegans development, the process of epidermal elongation converts the bean-shaped embryo into the long thin shape of the larval worm. Epidermal elongation results from changes in the shape of epidermal cells, which in turn result from changes in the epidermal cytoskeleton, the extracellular matrix, and in cell-matrix adhesion junctions. Here, we review the roles of cytoskeletal filament systems in epidermal cell shape change during elongation. Genetic and cell biological analyses have established that all three major cytoskeletal filament systems (actin microfilaments, microtubules, and intermediate filaments (IFs)) play distinct and essential roles in epidermal cell shape change. Recent work has also highlighted the importance of communication between these systems for their integrated function in epidermal elongation. Epidermal cells undergo reciprocal interactions with underlying muscle cells, which regulate the position and function of IF-containing cell-matrix adhesion structures within the epidermis. Elongation thus exemplifies the reciprocal tissue interactions of organogenesis.     

The F-BAR domain of SRGP-1 facilitates cell-cell adhesion during C. elegans morphogenesis

Robust cell-cell adhesion is critical for tissue integrity and morphogenesis, yet little is known about the molecular mechanisms controlling cell-cell junction architecture and strength. We discovered that SRGP-1 is a novel component of cell-cell junctions in Caenorhabditis elegans, localizing via its F-BAR (Bin1, Amphiphysin, and RVS167) domain and a flanking 200-amino acid sequence. SRGP-1 activity promotes an increase in membrane dynamics at nascent cell-cell contacts and the rapid formation of new junctions; in addition, srgp-1 loss of function is lethal in embryos with compromised cadherin-catenin complexes. Conversely, excess SRGP-1 activity leads to outward bending and projections of junctions. The C-terminal half of SRGP-1 interacts with the N-terminal F-BAR domain and negatively regulates its activity. Significantly, in vivo structure-function analysis establishes a role for the F-BAR domain in promoting rapid and robust cell adhesion during embryonic closure events, independent of the Rho guanosine triphosphatase-activating protein domain. These studies establish a new role for this conserved protein family in modulating cell-cell adhesion.     

Mechanisms of cell positioning during C. elegans gastrulation

Cell rearrangements are crucial during development. In this study, we use C. elegans gastrulation as a simple model to investigate the mechanisms of cell positioning. During C. elegans gastrulation, two endodermal precursor cells move from the ventral surface to the center of the embryo, leaving a gap between these ingressing cells and the eggshell. Six neighboring cells converge under the endodermal precursors, filling this gap. Using an in vitro system, we observed that these movements occurred consistently in the absence of the eggshell and the vitelline envelope. We found that movement of the neighbors towards each other is not dependent on chemotactic signaling between these cells. We further found that C. elegans gastrulation requires intact microfilaments, but not microtubules. The primary mechanism of microfilament-based motility does not appear to be through protrusive structures, such as lamellipodia or filopodia. Instead, our results suggest an alternative mechanism. We found that myosin activity is required for gastrulation, that the apical sides of the ingressing cells contract, and that the ingressing cells determine the direction of movement of their neighboring cells. Based on these results, we propose that ingression is driven by an actomyosin-based contraction of the apical side of the ingressing cells, which pulls neighboring cells underneath. We conclude that apical constriction can function to position blastomeres in early embryos, even before anchoring junctions form between cells.     

C. elegans PAR-3 and PAR-6 are required for apicobasal asymmetries associated with cell adhesion and gastrulation

PAR proteins distribute asymmetrically across the anterior-posterior axis of the 1-cell-stage C. elegans embryo, and function to establish subsequent anterior-posterior asymmetries. By the end of the 4-cell stage, anteriorly localized PAR proteins, such as PAR-3 and PAR-6, redistribute to the outer, apical surfaces of cells, whereas posteriorly localized PAR proteins, such as PAR-1 and PAR-2, redistribute to the inner, basolateral surfaces. Because PAR proteins are provided maternally, distinguishing apicobasal from earlier anterior-posterior functions requires a method that selectively prevents PAR activity after the 1-cell stage. In the present study we generated hybrid PAR proteins that are targeted for degradation after the 1-cell stage. Embryos containing the hybrid PAR proteins had normal anterior-posterior polarity, but showed defects in apicobasal asymmetries associated with gastrulation. Ectopic separations appeared between lateral surfaces of cells that are normally tightly adherent, cells that ingress during gastrulation failed to accumulate nonmuscle myosin at their apical surfaces and ingression was slowed. Thus, PAR proteins function in both apicobasal and anterior-posterior asymmetry during the first few cell cycles of embryogenesis.     

mab-20 encodes Semaphorin-2a and is required to prevent ectopic cell contacts during epidermal morphogenesis in Caenorhabditis elegans

The Semaphorins are a family of secreted and transmembrane proteins known to elicit growth cone repulsion and collapse. We made and characterized a putative null mutant of the C. elegans gene semaphorin-2a (Ce-sema-2a). This mutant failed to complement mutants of mab-20 (Baird, S. E., Fitch, D. H., Kassem, I. A. A. and Emmons, S. W. (1991) Development 113, 515-526). In addition to low-frequency axon guidance errors, mab-20 mutants have unexpected defects in epidermal morphogenesis. Errant epidermal cell migrations affect epidermal enclosure of the embryo, body shape and sensory rays of the male tail. These phenotypic traits are explained by the formation of inappropriate contacts between cells of similar type and suggest that Ce-Sema-2a may normally prevent formation or stabilization of ectopic adhesive contacts between these cells.     

The cell junction protein VAB-9 regulates adhesion and epidermal morphology in C. elegans

Epithelial cell junctions are essential for cell polarity, adhesion and morphogenesis. We have analysed VAB-9, a cell junction protein in Caenorhabditis elegans. VAB-9 is a predicted four-pass integral membrane protein that has greatest similarity to BCMP1 (brain cell membrane protein 1, a member of the PMP22/EMP/Claudin family of cell junction proteins) and localizes to the adherens junction domain of C. elegans apical junctions. Here, we show that VAB-9 requires HMR-1/cadherin for localization to the cell membrane, and both HMP-1/alpha-catenin and HMP-2/beta-catenin for maintaining its distribution at the cell junction. In vab-9 mutants, morphological defects correlate with disorganization of F-actin at the adherens junction; however, localization of the cadherin-catenin complex and epithelial polarity is normal. These results suggest that VAB-9 regulates interactions between the cytoskeleton and the adherens junction downstream of or parallel to alpha-catenin and/or beta-catenin. Mutations in vab-9 enhance adhesion defects through functional loss of the cell junction genes apical junction molecule 1 (ajm-1) and discs large 1 (dlg-1), suggesting that VAB-9 is involved in cell adhesion. Thus, VAB-9 represents the first characterized tetraspan adherens junction protein in C. elegans and defines a new family of such proteins in higher eukaryotes.     

Autophagy and apoptosis are redundantly required for C. elegans embryogenesis

Apoptosis, the main form of regulated (or programmed) cell death, allows the organism to tightly control cell numbers and tissue size, and to protect itself from potentially damaging cells. This type of cellular self-killing has long been assumed to be essential for early development. In the nematode Caenorhabditis elegans, however, the core apoptotic cell death pathway appears to be dispensable for embryogenesis when most developmental cell deaths take place: mutant nematodes defective for apoptosis develop into adulthood, with superficially normal morphology and behavior. Accumulating evidence indicates a similar situation in mammalian systems as well. For example, apoptosis-deficient mice can grow as healthy, fertile adults. These observations raise the possibility that alternative cell death mechanisms may compensate for the lack of apoptotic machinery in developing embryos. Interestingly, C. elegans embryogenesis can also occur without autophagy, an alternative form of cellular self-destruction (also called autophagic cell death). In an upcoming paper we report that simultaneous inactivation of the autophagic and apoptotic gene cascades in C. elegans arrests development at early stages, and the affected embryos exhibit severe morphological defects. Double-mutant nematode embryos deficient in both autophagy and apoptosis are unable to undergo body elongation or to arrange several tissues correctly. This novel function of autophagy genes in morphogenesis indicates a more fundamental role for cellular self-digestion in tissue patterning than previously thought.     

Form of the worm: genetics of epidermal morphogenesis in C. elegans

The development of the epidermis of the nematode worm Caenorhabditis elegans illustrates many common processes of epithelial morphogenesis. In the worm, these morphogenetic movements have been described with single-cell resolution, and the roles of individual cells have been probed in laser killing experiments. Genetic dissection is yielding insights into the molecular mechanisms of these complex morphogenetic processes.     

Autophagy genes are required for normal lipid levels in C. elegans

Autophagy is a cellular catabolic process in which various cytosolic components are degraded. For example, autophagy can mediate lipolysis of neutral lipid droplets. In contrast, we here report that autophagy is required to facilitate normal levels of neutral lipids in C. elegans. Specifically, by using multiple methods to detect lipid droplets including CARS microscopy, we observed that mutants in the gene bec-1 (VPS30/ATG6/BECN1), a key regulator of autophagy, failed to store substantial neutral lipids in their intestines during development. Moreover, loss of bec-1 resulted in a decline in lipid levels in daf-2 [insulin/IGF-1 receptor (IIR) ortholog] mutants and in germline-less glp-1/Notch animals, both previously recognized to accumulate neutral lipids and have increased autophagy levels. Similarly, inhibition of additional autophagy genes, including unc-51/ULK1/ATG1 and lgg-1/ATG8/MAP1LC3A/LC3 during development, led to a reduction in lipid content. Importantly, the decrease in fat accumulation observed in animals with reduced autophagy did not appear to be due to a change in food uptake or defecation. Taken together, these observations suggest a broader role for autophagy in lipid remodeling in C. elegans.     

C. elegans ankyrin repeat protein VAB-19 is a component of epidermal attachment structures and is essential for epidermal morphogenesis

Elongation of the epidermis of the nematode Caenorhabditis elegans involves both actomyosin-mediated changes in lateral epidermal cell shape and body muscle attachment to dorsal and ventral epidermal cells via intermediate-filament/hemidesmosome structures. vab-19 mutants are defective in epidermal elongation and muscle attachment to the epidermis. VAB-19 is a member of a conserved family of ankyrin repeat-containing proteins that includes the human tumor suppressor Kank. In epidermal cells, VAB-19::GFP localizes with components of epidermal attachment structures. In vab-19 mutants, epidermal attachment structures form normally but do not remain localized to muscle-adjacent regions of the epidermis. VAB-19 localization requires function of the transmembrane attachment structure component Myotactin. vab-19 mutants also display aberrant actin organization in the epidermis. Loss of function in the spectrin SMA-1 partly bypasses the requirement for VAB-19 in elongation, suggesting that VAB-19 and SMA-1/spectrin might play antagonistic roles in regulation of the actin cytoskeleton.     

PlexinD1 and semaphorin signaling are required in endothelial cells for cardiovascular development

The identification of new signaling pathways critical for cardiac morphogenesis will contribute to our understanding of congenital heart disease (CHD), which remains a leading cause of mortality in newborn children worldwide. Signals mediated by semaphorin ligands and plexin receptors contribute to the intricate patterning of axons in the central nervous system. Here, we describe a related signaling pathway involving secreted class 3 semaphorins, neuropilins, and a plexin receptor, PlexinD1, expressed by endothelial cells. Interruption of this pathway in mice results in CHD and vascular patterning defects. The type of CHD caused by inactivation of PlexinD1 has previously been attributed to abnormalities of neural crest. Here, we show that this form of CHD can be caused by cell-autonomous endothelial defects. Thus, molecular programs that mediate axon guidance in the central nervous system also function in endothelial cells to orchestrate critical aspects of cardiac morphogenesis.     

Directed cell invasion and asymmetric adhesion drive tissue elongation and turning in C. elegans gonad morphogenesis

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Components of the SWI/SNF complex are required for asymmetric cell division in C. elegans

Asymmetric cell division is a fundamental process that produces cellular diversity during development. We have identified two mutants in C. elegans (psa-1 and psa-4) in which the asymmetry of T cell division is disrupted. psa-1 and psa-4 encode homologs of yeast SWI3 and SWI2/SNF2, respectively, which are components of the SWI/SNF complex. We show by RNA interference assay that homologs of other components of SWI/SNF are also involved in T cell division. psa-1 and psa-4 are likely to be required in the T cell during mitosis to cause asymmetric cell division. Because the SWI/SNF complex is required for asymmetric division in S. cerevisiae, these results demonstrate that at least some aspects of the mechanism of asymmetric cell division are conserved between yeast and a multicellular organism.     

SMA-1 spectrin has essential roles in epithelial cell sheet morphogenesis in C. elegans

During Caenorhabditis elegans development, the embryo acquires its vermiform shape due to changes in the shape of epithelial cells, a process that requires an apically localized actin cytoskeleton. We show that SMA-1, an ortholog of beta(H)-spectrin required for normal morphogenesis, localizes to the apical membrane of epithelial cells when these cells are rapidly elongating. In spc-1 alpha-spectrin mutants, SMA-1 localizes to the apical membrane but its organization is altered, consistent with the hypothesis these proteins act together to form an apically localized spectrin-based membrane skeleton (SBMS). SMA-1 is required to maintain the association between actin and the apical membrane; sma-1 mutant embryos fail to elongate because actin, which provides the driving force for cell shape change, dissociates from the apical membrane skeleton during morphogenesis. Analysis of sma-1 expression constructs and mutant strains indicates SMA-1 maintains the association between actin and the apical membrane via interactions at its N-terminus and this activity is independent of alpha-spectrin. SMA-1 also preserves dynamic changes in the organization of the apical membrane skeleton. Taken together, our results show the SMA-1 SBMS plays a dynamic role in converting changes in actin organization into changes in epithelial cell shape during C. elegans embryogenesis.