Innovations in metazoan development arise from evolutionary modification of gene regulatory networks (GRNs). We report widespread cryptic variation in the requirement for two key regulatory inputs, SKN-1/Nrf2 and MOM-2/Wnt, into the C. elegans endoderm GRN. While some natural isolates show a nearly absolute requirement for these two regulators, in others, most embryos differentiate endoderm in their absence. GWAS and analysis of recombinant inbred lines reveal multiple genetic regions underlying this broad phenotypic variation. We observe a reciprocal trend, in which genomic variants, or knockdown of endoderm regulatory genes, that result in a high SKN-1 requirement often show low MOM-2/Wnt requirement and vice-versa, suggesting that cryptic variation in the endoderm GRN may be tuned by opposing requirements for these two key regulatory inputs. These findings reveal that while the downstream components in the endoderm GRN are common across metazoan phylogeny, initiating regulatory inputs are remarkably plastic even within a single species.

Gene regulatory networks (GRNs) that direct animal embryogenesis must respond to varying environmental and physiological conditions to ensure robust construction of organ systems. While GRNs are evolutionarily modified by natural genomic variation, the roles of epigenetic processes in shaping plasticity of GRN architecture are not well understood. The endoderm GRN inCaenorhabditis elegansis initiated by the maternally supplied SKN-1/Nrf2 bZIP transcription factor; however, the requirement for SKN-1 in endoderm specification varies widely among distinctC. eleganswild isotypes, owing to rapid developmental system drift driven by accumulation of cryptic genetic variants. We report here that heritable epigenetic factors that are stimulated by transient developmental diapause also underlie cryptic variation in the requirement for SKN-1 in endoderm development. This epigenetic memory is inherited from the maternal germline, apparently through a nuclear, rather than cytoplasmic, signal, resulting in a parent-of-origin effect (POE), in which the phenotype of the progeny resembles that of the maternal founder. The occurrence and persistence of POE varies between different parental pairs, perduring for at least 10 generations in one pair. This long-perduring POE requires piwi-interacting RNA (piRNA) function and the germline nuclear RNA interference (RNAi) pathway, as well as MET-2 and SET-32, which direct histone H3K9 trimethylation and drive heritable epigenetic modification. Such nongenetic cryptic variation may provide a resource of additional phenotypic diversity through which adaptation may facilitate evolutionary changes and shape developmental regulatory systems.

Developmental gene regulatory networks (GRNs) underpin metazoan embryogenesis and have undergone substantial modification to generate the tremendous variety of animal forms present on Earth today. The nematode Caenorhabditis elegans has been a central model for advancing many important discoveries in fundamental mechanistic biology and, more recently, has provided a strong base from which to explore the evolutionary diversification of GRN architecture and developmental processes in other species. In this short review, we will focus on evolutionary diversification of the GRN for the most ancient of the embryonic germ layers, the endoderm. Early embryogenesis diverges considerably across the phylum Nematoda. Notably, while some species deploy regulative development, more derived species, such as C. elegans, exhibit largely mosaic modes of embryogenesis. Despite the relatively similar morphology of the nematode gut across species, widespread variation has been observed in the signaling inputs that initiate the endoderm GRN, an exemplar of developmental system drift (DSD). We will explore how genetic variation in the endoderm GRN helps to drive DSD at both inter-and intraspecies levels, thereby resulting in a robust developmental system. Comparative studies using divergent nematodes promise to unveil the genetic mechanisms controlling developmental plasticity and provide a paradigm for the principles governing evolutionary modification of an embryonic GRN.

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19Gene regulatory networks (GRNs) that direct animal embryogenesis must respond to varying 20 environmental and physiological conditions to ensure robust construction of organ systems. While GRNs 21 are evolutionarily modified by natural genomic variation, the roles of epigenetic processes in shaping 22 plasticity of GRN architecture are not well-understood. The endoderm GRN in C. elegans is initiated by the 23 maternally supplied SKN-1/Nrf2 bZIP transcription factor; however, the requirement for SKN-1 in endoderm 24 specification varies widely among distinct C. elegans wild isolates owing to rapid developmental system 25 drift driven by accumulation of cryptic genetic variants. We report here that heritable epigenetic factors that 26 are stimulated by transient developmental diapause also underlie cryptic variation in the requirement for 27 SKN-1 in endoderm development. This epigenetic memory is inherited from the maternal germline, 28 apparently through a nuclear, rather than cytoplasmic, signal, resulting in a parent-of-origin effect (POE), 29in which the phenotype of the progeny resembles that of the maternal founder. The occurrence and 30 persistence of POE varies between different parental pairs, perduring for at least ten generations in one 31 pair. This long-perduring POE requires piwi-piRNA function and the germline nuclear RNAi pathway, as 32 well as MET-2 and SET-32, which direct histone H3K9 trimethylation and drive heritable epigenetic 33 modification. Such non-genetic cryptic variation between wild isolates may provide a resource of additional 34 phenotypic diversity through which adaptation may facilitate evolutionary change and shape developmental 35 regulatory systems.36 42 disruption of this process often leads to lethal consequences (reviewed in refs. 3, 4). In C. elegans, aberrant 43 reprogramming of epigenetic memory can result in transgenerational accumulation of inappropriate 44 epigenetic marks and a progressive sterile mortal germline (Mrt) phenotype (5, 6). In many cases, the Mrt 45 phenotype is exacerbated by heat stress, demonstrating that environmental factors may influence 46 epigenetic reprogramming in the germline, and that these epigenetic modifications may be passed to 47 subsequent generations (7, 8). Interestingly, C. elegans wild isolates, each carrying a unique haplotype, 48 exhibit variation in the temperature-induced Mrt phenotype, suggesting differential stress response and 49 distinct epigenetic landscapes in natural populations of the species (9). 50Many of the documented instances of epigenetic inheritance in mammals are parental or 51 intergenerational effects (less than three generations for female transmission and two generations for male 52 transmission), which can be attributed to direct exposure of the developing embryos in utero to the triggers 53 that alter epigenetic states (2). Parental traumatic experience can trigger heritable behavioral changes and 54 nutritional status of the parents can cause metabolic remodeling in the offspring, which often lasts for o...