Identification of essential genes in Caenorhabditis elegans through whole-genome sequencing of legacy mutant collections

Abstract

It has been estimated that 15%-30% of the ∼20,000 genes in C. elegans are essential, yet many of these genes remain to be identified or characterized. With the goal of identifying unknown essential genes, we performed whole-genome sequencing on complementation pairs from legacy collections of maternal-effect lethal and sterile mutants. This approach uncovered maternal genes required for embryonic development and genes with apparent sperm-specific functions. In total, 58 putative essential genes were identified on chromosomes III-V, of which 52 genes are represented by novel alleles in this collection. Of these 52 genes, 19 (40 alleles) were selected for further functional characterization. The terminal phenotypes of embryos were examined, revealing defects in cell division, morphogenesis, and osmotic integrity of the eggshell. Mating assays with wild-type males revealed previously unknown male-expressed genes required for fertilization and embryonic development. The result of this study is a catalog of mutant alleles in essential genes that will serve as a resource to guide further study toward a more complete understanding of this important model organism. As many genes and developmental pathways in C. elegans are conserved and essential genes are often linked to human disease, uncovering the function of these genes may also provide insight to further our understanding of human biology.

Keywords: C. elegans; embryogenesis; essential genes; fertilization; legacy mutants; maternal-effect; whole-genome sequencing.

Figure 1

Schematic of gene assignments and deficiency mapping. Genes and deficiencies are shown with their relative positions on chromosomes III–V (coordinates listed in Supplementary File S2). Approximate boundaries of each deficiency were determined by the coordinates of the closest gene known to lie outside of the deletion, when possible (indicated by a faded edge). If no such genes with physical coordinates are known, the outermost gene known to lie inside the deletion was used as the boundary (indicated by a sharp edge). Gene names are colored according to the deficiency under which the alleles were mapped. Genes names assigned to alleles that did not map under any of the tested deficiencies are highlighted in gray. top-3 and bckd-1A on chromosome III are represented by multiple complementation groups with conflicting results from deficiency mapping.

Figure 2

Biological process GO terms overrepresented in the set of 58 putative essential genes. Bar length represents the number of genes in the set associated with each GO term. Overrepresentation was analyzed using PANTHER version 16.0 (Thomas et al. 2003) and P-values were adjusted with the Bonferroni multiple testing correction. Results were filtered to include terms with adjusted P < 0.05 and edited to exclude redundant terms. A list of overrepresented GO terms and associated genes can be found in Supplementary File S3.

Figure 3

Embryonic arrest visualized with DIC microscopy for select maternal-effect lethal mutants. Eggs were dissected from homozygous mutants and imaged immediately (A) or incubated in distilled water overnight before imaging (B–D). (A) Eggs dissected from dgtr-1(t2043) homozygotes exhibit signs of an osmotic integrity defect, by filling the eggshell completely. (B) dlat-1(t2035) embryos exhibit early embryonic arrest, with most embryos consisting of four cells or less. (C) ZK688.9(t1433) embryos arrest with approximately 100 cells. (D) Terminal embryos of nstp-2(t1835) have a lumpy body wall morphology and constricted nose; most animals were moving inside the eggshell but did not hatch. All scale bars represent 10 μm.