Forward Genetics Identifies ptr-18 and Other Genes as Developmental Regulators of the Four-Cell Tail Tip in C. elegans

In C. elegans , the epidermis and its overlying extracellular matrix form a primary protective barrier, functioning as the first line of defense against environmental factors. To properly develop those cellular boundaries, a tightly controlled interaction of many molecules and pathways is needed. Mutant alleles of paqr-2 and iglr-2 (lipid homeostasis), dpy-21 (membrane trafficking), and sma-1 (actin-binding spectrin) result in hermaphrodite tail tip defects suggesting that this simple four-cell structure can serve as a sensitive model for the identification of pathways responsible for the establishment of cellular boundaries. With this in mind, we performed a small forward genetics screen of ∼800 ethyl methanesulfonate-mutagenized haploid genomes and identified 21 mutants with a tail end defective (Ted) phenotype. Whole genome sequencing of these mutants identified mutations in genes encoding either structural constituents of the cuticle itself (mostly collagen genes) or protein with regulatory functions. By using CRISPR/Cas9 we confirmed six novel alleles of ptr-18, paqr-2, nab-1, ncam-1, vab-9 and efn-4. We further characterized the loss of function allele ptr-18(et70) , which encodes a patch domain-containing (PTCHD) protein homologous to human PTCHD1. ptr-18(et70) has a significant effect on growth and development of the worms, while also increasing membrane permeability. Lipidomics analysis revealed no major alterations in membrane lipid composition, implicating cuticle defects as the primary cause of the observed permeability phenotype.