Overcoming Redundancy: An RNAi Enhancer Screen for Morphogenesis Genes inCaenorhabditis elegans

Abstract

Morphogenesis is an important component of animal development. Genetic redundancy has been proposed to be common among morphogenesis genes, posing a challenge to the genetic dissection of morphogenesis mechanisms. Genetic redundancy is more generally a challenge in biology, as large proportions of the genes in diverse organisms have no apparent loss of function phenotypes. Here, we present a screen designed to uncover redundant and partially redundant genes that function in an example of morphogenesis, gastrulation in Caenorhabditis elegans. We performed an RNA interference (RNAi) enhancer screen in a gastrulation-sensitized double-mutant background, targeting genes likely to be expressed in gastrulating cells or their neighbors. Secondary screening identified 16 new genes whose functions contribute to normal gastrulation in a nonsensitized background. We observed that for most new genes found, the closest known homologs were multiple other C. elegans genes, suggesting that some may have derived from rounds of recent gene duplication events. We predict that such genes are more likely than single copy genes to comprise redundant or partially redundant gene families. We explored this prediction for one gene that we identified and confirmed that this gene and five close relatives, which encode predicted substrate recognition subunits (SRSs) for a CUL-2 ubiquitin ligase, do indeed function partially redundantly with each other in gastrulation. Our results implicate new genes in C. elegans gastrulation, and they show that an RNAi-based enhancer screen in C. elegans can be used as an efficient means to identify important but redundant or partially redundant developmental genes.

Identifying Regulators of Morphogenesis Common to Vertebrate Neural Tube Closure andCaenorhabditis elegansGastrulation

“…elegans strains and worm maintenance C. elegans were cultured and handled as described in Brenner (1974). Experiments were performed using the following strains: wild-type Bristol N2, LP77 end-3(ok1448); ced-5(n1812) (Sawyer et al 2011 (Wehman et al 2011), end-1(ok558), end-3(ok1448), LP54 PH::mCherry; NMY-2::GFP, MT2547 ced-4(n1162), MT2551 ced-4(n1162), dpy-17(e164), MT5287 ced-4(n1894), MT5816 ced-4(n2273), MT5851 ced-4(n2274), WM43 gex-3(zu196), OX232 wve-1(zu469) unc-101(m1)/hin1 (gift from the Soto laboratory), OX309 wve-1(zu469) unc-101(m1)/hin1 (gift from the Soto laboratory), VC1180 gex-2(ok1603)/dpy-9(e12), LP163 unc-119(ed3) III; nuo-3(cp14[nuo-3aD + unc-119(+)]) IV/nT1[qIs51], LP418 sptf-3(cp154[mNG^SEC^3xFlag::sptf-3]) I, LP419 spft-3(cp155[mNG^3xFlag::sptf-3]) I, and LP362 gex-3(cp114[mNG^3xFlag::gex-3]) IV (Dickinson et al 2015).…”

“…Primary screen: RNAi-by-feeding: RNAi-by-feeding was performed by a protocol outlined previously (Sawyer et al 2011) with minor modifications. Three to five L 4 larvae from a wildtype background or LP77 end-3(ok1448); ced-5(n1812) gastrulation-sensitized background were placed on RNAi plates seeded with double-stranded RNA (dsRNA)-producing bacterial strains (Timmons and Fire 1998;Kamath et al 2001).…”

“…For plasmid PCR, we used a primer recognizing the T7 promoter sequence; for cDNA PCR, we used a two-step PCR strategy outlined previously (Sawyer et al 2011). All PCR products were gel purified and used as a template in an in vitro transcription reaction (T7 RiboMAX Express RNAi System, Promega) according to the manufacturer's instructions.…”

“…Here we report the screening of a large set of C. elegans NTD gene homologs for roles in gastrulation using a two-step RNA interference (RNAi) enhancer screen developed previously (Sawyer et al 2011). Our screen succeeded in identifying four NTD homologs with roles in C. elegans gastrulation.…”

Development and Cell Polarity of the C. elegans Intestine

“…The E-cell is born at the 8-cell stage (A) and with the beginning of gastrulation (24-cell stage), 2 E-cells (E 2 ) ingress into the embryo (B) where they further undergo cell divisions (C, 4 E-cells, E 4 ). The ingression of Ea and Ep cells depends on correct cell fate specification and polarization of the machinery that orchestrates cell shape changes and cell migration (Lee and Goldstein, 2003;Sawyer et al, 2011). Among these, PAR-3 and PAR-6 proteins regulate apical accumulation of myosin heavy chain, and a Wnt-Frizzled signaling pathway modulates contraction of the actomyosin network that drives apical constriction and finally leads to correct ingression of endodermal precursor cells (Cabello et al, 2010;Grana et al, 2010;Lee et al, 2006).…”