During development, biomechanical forces contour the body and provide shape to internal organs. We previously demonstrated that a regulatory network involving the conserved proteins SYM-3/FAM102A, SYM-4/WDR44, and MEC-8/RBPMS are required to maintain epidermal integrity in response to biomechanical forces in C. elegans embryos. Embryos and larvae that are doubly mutant for mec-8; sym-3 or mec-8; sym-4 have defective anterior morphology, called the Pharynx Ingressed or Pin phenotype. We showed that MEC-8 regulates the alternative splicing of multiple structural proteins including a fibrillin-like protein, FBN-1, which acts specifically in epidermal cells and is a component of the apical extracellular matrix (aECM or sheath). FBN-1 functions to withstand a variety of biomechanical forces acting on the epidermis, including an internally directed pulling force exerted by the pharynx as it undergoes stretch and elongation. Our current studies indicate that FBN-1 may attach directly to epidermal cells via integrins, which have not previously been reported to act within the epidermal aECM of embryos. Consistent with this, deletion of RGD (integrin-binding) sites in FBN-1 leads to induction of the Pin phenotype. In addition, mutations in fbn-1, sym-3 and sym-4 can enhance the “notched-head” phenotype (seen in integrin mutants) and inhibition of integrin subunits enhances Pin in sym-3 and sym-4 mutant backgrounds.
Our studies further indicate that SYM-3 and SYM-4 act in a parallel pathway to MEC-8 and may promote the trafficking of structurally important proteins in epidermal cells. Functional SYM-3 and SYM-4 fluorescent reporters localize to vesicle-like structures at or near the plasma membrane in epidermal cells. Furthermore, SYM-3 and SYM-4 act in a common genetic pathway with the RAB-11 GTPase, a known regulator of endocytic recycling and exocytosis, which has been shown to regulate integrin localization in other systems. Interestingly, sym-4 is in an operon with vha-20/(ATP6-ap2/(P)RR, a multifunctional protein involved in vesicle acidification and planar cell polarity. Consistent with a previous study, we find that loss of vha-20 perturbs RAB-11 localization and vha-20(RNAi) enhances Pin in sym-4 mutants. In other studies, we have identified novel components of the aECM that function with FBN-1 to stabilize epidermal architecture during embryogenesis. We have also implicated roles for several cytoskeletal and cell-cell adhesion proteins in maintaining normal epidermal structure. Taken together, we have identified a network of genes that are required to maintain epidermal architecture in response to a variety of biomechanical forces during embryogenesis.