High-Throughput Cloning of Temperature-Sensitive Caenorhabditis elegans Mutants with Adult Syncytial Germline Membrane Architecture Defects

Abstract

The adult Caenorhabditis elegans hermaphrodite gonad consists of two mirror-symmetric U-shaped arms, with germline nuclei located peripherally in the distal regions of each arm. The nuclei are housed within membrane cubicles that are open to the center, forming a syncytium with a shared cytoplasmic core called the rachis. As the distal germline nuclei progress through meiotic prophase, they move proximally and eventually cellularize as their compartments grow in size. The development and maintenance of this complex and dynamic germline membrane architecture are relatively unexplored, and we have used a forward genetic screen to identify 20 temperature-sensitive mutations in 19 essential genes that cause defects in the germline membrane architecture. Using a combined genome-wide SNP mapping and whole genome sequencing strategy, we have identified the causal mutations in 10 of these mutants. Four of the genes we have identified are conserved, with orthologs known to be involved in membrane biology, and are required for proper development or maintenance of the adult germline membrane architecture. This work provides a starting point for further investigation of the mechanisms that control the dynamics of syncytial membrane architecture during adult oogenesis.

Evolutionary dynamics of the SKN-1 → MED → END-1,3 regulatory gene cascade inCaenorhabditisendoderm specification

“…with an AGTATAC core (BROITMAN- LOWRY et al 2015). Despite the high divergence of 749 the MED factors as a group, indicating relaxed selection, there is nonetheless maintenance of their 750 binding site sequence over evolutionary time.…”

MIP-MAP: High Throughput Mapping ofCaenorhabditis elegansTemperature Sensitive Mutants via Molecular Inversion Probes

“…Such genes encompass a range of classes that include roles in cell division, development and fertility – key components to all multicellular organisms. Temperature-sensitive (TS) lethal alleles can facilitate the genetic analysis of essential genes through the conditional modulation of their function without the complications that balancer chromosomes can introduce when present in non-conditional lethal strains (Golden et al 2000; O’Rourke et al 2011a; Lowry et al 2015). There are several reported screens for TS lethal alleles in C. elegans but to date, there are only a small portion of genes with TS alleles identified (Zonies et al 2010; Ehmke et al 2014; Lowry et al 2015).…”

Lysosomal activity regulatesCaenorhabditis elegansmitochondrial dynamics through vitamin B12 metabolism

“…drp-1(or1393) is a temperature-sensitive allele with a conserved glycine to glutamic acid (G39E) substitution mutation in the N-terminal dynamin domain. This mutation causes high penetrance lethality at the non-permissive temperature (12) (Figs. 1A, 1B and S1A).…”