Synthetic lethal analysis of Caenorhabditis elegans posterior embryonic patterning genes identifies conserved genetic interactions

Abstract

Phenotypic robustness is evidenced when single-gene mutations do not result in an obvious phenotype. It has been suggested that such phenotypic stability results from 'buffering' activities of homologous genes as well as non-homologous genes acting in parallel pathways. One approach to characterizing mechanisms of phenotypic robustness is to identify genetic interactions, specifically, double mutants where buffering is compromised. To identify interactions among genes implicated in posterior patterning of the Caenorhabditis elegans embryo, we measured synthetic lethality following RNA interference of 22 genes in 15 mutant strains. A pair of homologous T-box transcription factors (tbx-8 and tbx-9) is found to interact in both C. elegans and C. briggsae, indicating that their compensatory function is conserved. Furthermore, a muscle module is defined by transitive interactions between the MyoD homolog hlh-1, another basic helix-loop-helix transcription factor, hnd-1, and the MADS-box transcription factor unc-120. Genetic interactions within a homologous set of genes involved in vertebrate myogenesis indicate broad conservation of the muscle module and suggest that other genetic modules identified in C. elegans will be conserved.

Figure 1

A synthetic lethal matrix of genes implicated in posterior patterning. (a) A heat map of survival (1 - % embryonic lethality) for RNAi of 13 genes (and a soaking buffer control) in 16 different genetic backgrounds. Values plotted are the average of two measurements (see Additional data file 1 for raw data). (b) A heat map of P-values corresponding to each test of synthetic lethality. A t-test was used with the null hypothesis that the survival of a given combination of RNAi and mutation is equal to the product of survivals of the RNAi in wild type and the mutation without RNAi (P-values can be found in Additional data file 1). A dashed gray line outlines the square (symmetric) part of the matrix and its diagonal in (a) and (b). RNAi of genes resulting in 100% embryonic lethality in wild type are not included in the matrix (pal-1, elt-1, lin-26, and unc-62). Mutant genotypes are listed in Table 1.

Figure 2

Synthetic lethal phenotypes of the homologous transcription factors tbx-8 and tbx-9. Recently hatched L1 larvae or an arrested embryo are shown for (a) wild-type, (b) tbx-9(RNAi), (c) tbx-8(ok656), and (d, e) tbx-8(ok656); tbx-9(RNAi). Panels (b) to (e) reflect the increasing severity of the phenotype, with (b) and (c) providing examples of the posterior morphological defects observed at low frequency after disruption of tbx-8 or tbx-9. (d, e) Examples of the severe morphological defects observed at high frequency after disruption of both tbx-8 and tbx-9; (d) shows an individual that has hatched but will arrest as a larva, and (e) shows an embryo that has arrested before hatching (embryonic lethal). The anterior is cropped in (b) and (c). The scale bar in (a) equals 10 μm and applies to (a-e).

Figure 3

Modular genetic interactions between three transcription factors controlling differentiation of muscle. A recently hatched L1 larva is shown for (a) wild type and (b) hlh-1(cc561), and (c) an arrested Pat embryo is shown for hlh-1(cc561); unc-120(RNAi). The scale bar in (a) equals 10 μm and applies to (a-c). Mutation of hlh-1, hnd-1 or unc-120 alone results in dumpy, uncoordinated larvae and Pat embryos (as in (b) and (c)) at low frequency. Disruption of function of any two of these three genes by mutation and RNAi significantly elevates both frequencies so that in the most potent combinations 100% Pat embryos result. The Pat phenotype has been shown to result specifically from the disruption of muscle differentiation and function [26,27]. (d) A summary of the proportion hatching (green) and proportion arresting as Pat embryos (black) for each single mutant and each of the six genetic interactions tested.