A Strategy To Isolate Modifiers ofCaenorhabditis elegansLethal Mutations: Investigating the Endoderm Specifying Ability of the Intestinal Differentiation GATA Factor ELT-2

Abstract

The ELT-2 GATA factor normally functions in differentiation of the C. elegans endoderm, downstream of endoderm specification. We have previously shown that, if ELT-2 is expressed sufficiently early, it is also able to specify the endoderm and to replace all other members of the core GATA-factor transcriptional cascade (END-1, END-3, ELT-7). However, such rescue requires multiple copies (and presumably overexpression) of the end-1p::elt-2 cDNA transgene; a single copy of the transgene does not rescue. We have made this observation the basis of a genetic screen to search for genetic modifiers that allow a single copy of the end-1p::elt-2 cDNA transgene to rescue the lethality of the end-1end-3 double mutant. We performed this screen on a strain that has a single copy insertion of the transgene in an end-1end-3 background. These animals are kept alive by virtue of an extrachromosomal array containing multiple copies of the rescuing transgene; the extrachromosomal array also contains a toxin under heat shock control to counterselect for mutagenized survivors that have been able to lose the rescuing array. A screen of ∼14,000 mutagenized haploid genomes produced 17 independent surviving strains. Whole genome sequencing was performed to identify genes that incurred independent mutations in more than one surviving strain. The C. elegans gene tasp-1 was mutated in four independent strains. tasp-1 encodes the C. elegans homolog of Taspase, a threonine-aspartic acid protease that has been found, in both mammals and insects, to cleave several proteins involved in transcription, in particular MLL1/trithorax and TFIIA. A second gene, pqn-82, was mutated in two independent strains and encodes a glutamine-asparagine rich protein. tasp-1 and pqn-82 were verified as loss-of-function modifiers of the end-1p::elt-2 transgene by RNAi and by CRISPR/Cas9-induced mutations. In both cases, gene loss leads to modest increases in the level of ELT-2 protein in the early endoderm although ELT-2 levels do not strictly correlate with rescue. We suggest that tasp-1 and pqn-82 represent a class of genes acting in the early embryo to modulate levels of critical transcription factors or to modulate the responsiveness of critical target genes. The screen’s design, rescuing lethality with an extrachromosomal transgene followed by counterselection, has a background survival rate of <10−4 without mutagenesis and should be readily adapted to the general problem of identifying suppressors of C. elegans lethal mutations.

Evolutionary Change in Gut Specification in Caenorhabditis Centers on the GATA Factor ELT-3 in an Example of Developmental System Drift

“…Other weaker regulatory inputs into gut specification have been found that result from more global effects on embryonic gene expression. These include the Sp1 orthologue SPTF-3, the histone deacetylase HDA-1, the p300-like factor CBP-1, the Pur-alpha orthologue PLP-1, the endopeptidase TASP-1, the Prion-like-(Q/N-rich)-domain-bearing protein PQN-82, and other factors that can modulate gene expression epigenetically [ 39 , 40 , 41 , 42 , 43 ]. Finally, other factors have been identified that are important for restricting the activity of SKN-1 to MS and E, such as the RNA-binding proteins MEX-1 and PIE-1 [ 44 , 45 , 46 ].…”

“…This ability is not a widespread property of GATA factors, as overexpression of ELT-1 and ELT-3A, two hypodermis-specific factors, does not promote gut fate [50,[65][66][67]. Interestingly, all of end-1, end-3, and elt-7 can be replaced with a single multicopy transgene that fuses the end-1 promoter to the coding region of either elt-2 or elt-7 [42,59,65].…”

Evolution and Developmental System Drift in the Endoderm Gene Regulatory Network of Caenorhabditis and Other Nematodes

“…In E, Wnt signaling further represses tbx-35 expression (Broitman- Maduro et al, 2006). studies revealed many novel regulators implied in endoderm development and embryogenesis (Witze et al, 2009;Du et al, 2014;Sullivan-Brown et al, 2016;Tintori et al, 2016;Dineen et al, 2018;Wiesenfahrt et al, 2018). The elucidation of C. elegans endoderm GRN provides a strong foundation from which to explore the diversification of endoderm GRN in other organisms.…”

Model Organism Modifier (MOM): a user-friendly Galaxy workflow to detect modifiers from genome sequencing data using Caenorhabditis elegans

“…reduced embryonic lethality which allows strain propagation despite the presence of the original lethal variant ( Geister et al . 2018 ; Wiesenfahrt et al . 2018 ; Jean et al .…”