We have analyzed the well-described gene regulatory network (GRN) for endoderm specification in C. elegans to investigate the basis for evolutionary plasticity in GRN architecture. Deployment of the endoderm GRN is initiated by the combined action of the SKN-1 transcription factor and an inductive signal mediated in part by MOM-2/Wnt. While removal of either maternal SKN-1 or MOM-2 in the N2 strain results in a partially penetrant loss of gut, simultaneous removal of both effectively eliminates endoderm. We found that the requirement for SKN-1 and MOM-2/Wnt in endoderm development varies dramatically across the 97 haplotypes: while RNAi knockdown of
skn-1 or
mom-2 results in fully penetrant lethality, we observed broad variation in penetrance across the spectrum of strains, ranging from <1% to 100% of arrested embryos with differentiated gut. The extreme variation in requirement for SKN-1 reflects bona fide differences in GRN function, as introgression of a
skn-1(-) chromosomal mutation in selected isolates yield quantitatively the same effect as for RNAi. The penetrance of the
skn-1(-) vs.
mom-2(-) phenotypes did not correlate across the isolates, indicating that this variation does not arise from complementarity in the relative strengths of the SKN-1 and Wnt inputs into the GRN. Genome-wide association studies (GWAS) and Efficient Mixed Model Analysis (EMMA) revealed that much of the variation across the strains is attributable to genomic regions on LG IV and V. Further, analysis of recombinant inbred lines (RILs) with large differences in SKN-1 requirement by bulk segregant analysis (BSA) and whole-population genotyping (WPG) revealed two major contributing regions, including one identified by GWAS, as was also confirmed in introgressed lines. While these studies revealed that a multiplicity of genes and genetic interactions are responsible for the quantitative differences in GRN regulatory inputs, there also appear to be very substantial epigenetic contributions to the variation that follows the maternal line, as shown by reciprocal crosses of four different pairs of isotypes. This striking parent-of-origin effect persisted through at least five generations, suggesting a parental imprinting phenomenon. These findings demonstrate that the GRN for embryonic endoderm specification has undergone unexpectedly rapid evolutionary modification during radiation of the C. elegans species and reveal that some divergence between isotypes has a non-genetic origin.