Developmental transcriptomics in Pristionchus reveals the logic of a plasticity gene regulatory network.

Developmental plasticity enables the production of alternative phenotypes in response to different environmental conditions. While significant advances in understanding the ecological and evolutionary implications of plasticity have been made, understanding its genetic basis has lagged. However, a decade of genetic screens in the model nematode Pristionchus pacificus has culminated in 30 genes which affect mouth-form plasticity. We also recently reported the critical window of environmental sensitivity, and therefore have clear expectations for when differential gene expression should matter. Here, we collated previous data into a gene-regulatory network (GRN), and performed developmental transcriptomics across different environmental conditions, genetic backgrounds, and mouth-form mutants to assess the regulatory logic of plasticity. We found that only two genes in the GRN (eud-1 and seud-1/sult-1) are sensitive to the environment during the critical window. Interestingly, the time points of their sensitivity differ, suggesting that they act as sequential checkpoints. We also observed temporal constraint upon the transcriptional effects of mutating the GRN and revealed unexpected feedback between mouth-form genes. Surprisingly, expression of seud-1/sult-1, but not eud-1, correlated with mouth form biases across different strains and species. Finally, a comprehensive analysis of all samples identified metabolism as a shared pathway for regulating mouth-form plasticity. These data are presented in a Shiny app to facilitate gene-expression comparisons across development in up to 14 different conditions. Collectively, our results suggest that mouth-form plasticity evolved a constrained, two-tiered logic to integrate environmental information leading up to the final developmental decision.

Newly identified nematodes from the Great Salt Lake are associated with microbialites and specially adapted to hypersaline conditions.

Extreme environments enable the study of simplified food-webs and serve as models for evolutionary bottlenecks and early Earth ecology. We investigated the biodiversity of invertebrate meiofauna in the benthic zone of the Great Salt Lake (GSL), Utah, USA, one of the most hypersaline lake systems in the world. The hypersaline bays within the GSL are currently thought to support only two multicellular animals: brine fly larvae and brine shrimp. Here, we report the presence, habitat, and microbial interactions of novel free-living nematodes. Nematode diversity drops dramatically along a salinity gradient from a freshwater river into the south arm of the lake. In Gilbert Bay, nematodes primarily inhabit reef-like organosedimentary structures built by bacteria called microbialites. These structures likely provide a protective barrier to UV and aridity, and bacterial associations within them may support life in hypersaline environments. Notably, sampling from Owens Lake, another terminal lake in the Great Basin that lacks microbialites, did not recover nematodes from similar salinities. Phylogenetic divergence suggests that GSL nematodes represent previously undescribed members of the family Monhysteridae-one of the dominant fauna of the abyssal zone and deep-sea hydrothermal vents. These findings update our understanding of halophile ecosystems and the habitable limit of animals.

Characterization of the Pristionchus pacificus "epigenetic toolkit" reveals the evolutionary loss of the histone methyltransferase complex PRC2.

Comparative approaches have revealed both divergent and convergent paths to achieving shared developmental outcomes. Thus, only through assembling multiple case studies can we understand biological principles. Yet, despite appreciating the conservation-or lack thereof-of developmental networks, the conservation of epigenetic mechanisms regulating these networks is poorly understood. The nematode Pristionchus pacificus has emerged as a model system of plasticity and epigenetic regulation as it exhibits a bacterivorous or omnivorous morph depending on its environment. Here, we determined the "epigenetic toolkit" available to P. pacificus as a resource for future functional work on plasticity, and as a comparison with Caenorhabditis elegans to investigate the conservation of epigenetic mechanisms. Broadly, we observed a similar cast of genes with putative epigenetic function between C. elegans and P. pacificus. However, we also found striking differences. Most notably, the histone methyltransferase complex PRC2 appears to be missing in P. pacificus. We described the deletion/pseudogenization of the PRC2 genes mes-2 and mes-6 and concluded that both were lost in the last common ancestor of P. pacificus and a related species P. arcanus. Interestingly, we observed the enzymatic product of PRC2 (H3K27me3) by mass spectrometry and immunofluorescence, suggesting that a currently unknown methyltransferase has been co-opted for heterochromatin silencing. Altogether, we have provided an inventory of epigenetic genes in P. pacificus to compare with C. elegans. This inventory will enable reverse-genetic experiments related to plasticity and has revealed the first loss of PRC2 in a multicellular organism.

Histone 4 lysine 5/12 acetylation enables developmental plasticity of Pristionchus mouth form.

Development can be altered to match phenotypes with the environment, and the genetic mechanisms that direct such alternative phenotypes are beginning to be elucidated. Yet, the rules that govern environmental sensitivity vs. invariant development, and potential epigenetic memory, remain unknown. Here, we show that plasticity of nematode mouth forms is determined by histone 4 lysine 5 and 12 acetylation (H4K5/12ac). Acetylation in early larval stages provides a permissive chromatin state, which is susceptible to induction during the critical window of environmental sensitivity. As development proceeds deacetylation shuts off switch gene expression to end the critical period. Inhibiting deacetylase enzymes leads to fixation of prior developmental trajectories, demonstrating that histone modifications in juveniles can carry environmental information to adults. Finally, we provide evidence that this regulation was derived from an ancient mechanism of licensing developmental speed. Altogether, our results show that H4K5/12ac enables epigenetic regulation of developmental plasticity that can be stored and erased by acetylation and deacetylation, respectively.

Nematode biphasic 'boom and bust' dynamics are dependent on host bacterial load while linking dauer and mouth-form polyphenisms.

Cross-kingdom interactions involve dynamic processes that shape terrestrial ecosystems and represent striking examples of co-evolution. The multifaceted relationships of entomopathogenic nematodes with their insect hosts and symbiotic bacteria are well-studied cases of co-evolution and pathogenicity. In contrast, microbial interactions in soil after the natural death of insects and other invertebrates are minimally understood. In particular, the turnover and succession of nematodes and bacteria during insect decay have not been well documented - although it represents a rich ecological niche with multiple species interactions. Here, we utilize developmentally plastic nematode Pristionchus pacificus and its associated scarab beetles as models. On La Réunion Island, we collected rhinoceros beetle Oryctes borbonicus, induced death, and placed carcasses in cages both on the island and in a mock-natural environment in the laboratory controlling for high spatial and temporal resolution. Investigating nematode population density and dispersal dynamics, we were able to connect two imperative plasticities, dauer and mouth form. We observed a biphasic 'boom and bust' dispersal dynamic of dauer larvae that corresponds to bacterial load on carcasses but not bacterial type. Strikingly, all post-dauer adults have the predatory mouth form, demonstrating novel intricate interactions on decaying insect hosts. Thus, ecologically relevant survival strategies incorporate critical plastic traits.

Geometric morphometrics of microscopic animals as exemplified by model nematodes.

While a host of molecular techniques are utilized by evolutionary developmental (evo-devo) biologists, tools for quantitative evaluation of morphology are still largely underappreciated, especially in studies on microscopic animals. Here, we provide a standardized protocol for geometric morphometric analyses of 2D landmark data sets using a combination of the geomorph and Morpho R packages. Furthermore, we integrate clustering approaches to identify group structures within such datasets. We demonstrate our protocol by performing exemplary analyses on stomatal shapes in the model nematodes Caenorhabditis and Pristionchus. Image acquisition for 80 worms takes 3-4 d, while the entire data analysis requires 10-30 min. In theory, this approach is adaptable to all microscopic model organisms to facilitate a thorough quantification of shape differences within and across species, adding to the methodological toolkit of evo-devo studies on morphological evolution and novelty.

Adult Influence on Juvenile Phenotypes by Stage-Specific Pheromone Production.

Many animal and plant species respond to population density by phenotypic plasticity. To investigate if specific age classes and/or cross-generational signaling affect density-dependent plasticity, we developed a dye-based method to differentiate co-existing nematode populations. We applied this method to Pristionchus pacificus, which develops a predatory mouth form to exploit alternative resources and kill competitors in response to high population densities. Remarkably, adult, but not juvenile, crowding induces the predatory morph in other juveniles. High-performance liquid chromatography-mass spectrometry of secreted metabolites combined with genetic mutants traced this result to the production of stage-specific pheromones. In particular, the P. pacificus-specific di-ascaroside#1 that induces the predatory morph is induced in the last juvenile stage and young adults, even though mouth forms are no longer plastic in adults. Cross-generational signaling between adults and juveniles may serve as an indication of rapidly increasing population size, arguing that age classes are an important component of phenotypic plasticity.

Young genes have distinct gene structure, epigenetic profiles, and transcriptional regulation.

Species-specific, new, or "orphan" genes account for 10%-30% of eukaryotic genomes. Although initially considered to have limited function, an increasing number of orphan genes have been shown to provide important phenotypic innovation. How new genes acquire regulatory sequences for proper temporal and spatial expression is unknown. Orphan gene regulation may rely in part on origination in open chromatin adjacent to preexisting promoters, although this has not yet been assessed by genome-wide analysis of chromatin states. Here, we combine taxon-rich nematode phylogenies with Iso-Seq, RNA-seq, ChIP-seq, and ATAC-seq to identify the gene structure and epigenetic signature of orphan genes in the satellite model nematode Pristionchus pacificus Consistent with previous findings, we find young genes are shorter, contain fewer exons, and are on average less strongly expressed than older genes. However, the subset of orphan genes that are expressed exhibit distinct chromatin states from similarly expressed conserved genes. Orphan gene transcription is determined by a lack of repressive histone modifications, confirming long-held hypotheses that open chromatin is important for new gene formation. Yet orphan gene start sites more closely resemble enhancers defined by H3K4me1, H3K27ac, and ATAC-seq peaks, in contrast to conserved genes that exhibit traditional promoters defined by H3K4me3 and H3K27ac. Although the majority of orphan genes are located on chromosome arms that contain high recombination rates and repressive histone marks, strongly expressed orphan genes are more randomly distributed. Our results support a model of new gene origination by rare integration into open chromatin near enhancers.

Facile target validation in an animal model with intracellularly expressed monobodies.

Rapidly determining the biological effect of perturbing a site within a potential drug target could guide drug discovery efforts, but it remains challenging. Here, we describe a facile target validation approach that exploits monobodies, small synthetic binding proteins that can be fully functionally expressed in cells. We developed a potent and selective monobody to WDR5, a core component of the mixed lineage leukemia (MLL) methyltransferase complex. The monobody bound to the MLL interaction site of WDR5, the same binding site for small-molecule inhibitors whose efficacy has been demonstrated in cells but not in animals. As a genetically encoded reagent, the monobody inhibited proliferation of an MLL-AF9 cell line in vitro, suppressed its leukemogenesis and conferred a survival benefit in an in vivo mouse leukemia model. The capacity of this approach to readily bridge biochemical, structural, cellular characterization and tests in animal models may accelerate discovery and validation of druggable sites.

Environmental influence on Pristionchus pacificus mouth form through different culture methods.

Environmental cues can impact development to elicit distinct phenotypes in the adult. The consequences of phenotypic plasticity can have profound effects on morphology, life cycle, and behavior to increase the fitness of the organism. The molecular mechanisms governing these interactions are beginning to be elucidated in a few cases, such as social insects. Nevertheless, there is a paucity of systems that are amenable to rigorous experimentation, preventing both detailed mechanistic insight and the establishment of a generalizable conceptual framework. The mouth dimorphism of the model nematode Pristionchus pacificus offers the rare opportunity to examine the genetics, genomics, and epigenetics of environmental influence on developmental plasticity. Yet there are currently no easily tunable environmental factors that affect mouth-form ratios and are scalable to large cultures required for molecular biology. Here we present a suite of culture conditions to toggle the mouth-form phenotype of P. pacificus. The effects are reversible, do not require the costly or labor-intensive synthesis of chemicals, and proceed through the same pathways previously examined from forward genetic screens. Different species of Pristionchus exhibit different responses to culture conditions, demonstrating unique gene-environment interactions, and providing an opportunity to study environmental influence on a macroevolutionary scale.

Chromatin-enriched lncRNAs can act as cell-type specific activators of proximal gene transcription.

We recently described a new class of long noncoding RNAs (lncRNAs) that are distinguished by especially tight chromatin association and whose presence is strongly correlated to expression of nearby genes. Here, we examine the cis-enhancer mechanism of this class of chromatin-enriched RNA (cheRNA) across multiple human cell lines. cheRNAs are largely cell type specific and provide the most reliable chromatin signature to predict cis-gene transcription in every human cell type examined. Targeted depletion of three cheRNAs decreases expression of their neighboring genes, indicating potential co-activator function, and single-molecule fluorescence in situ hybridization (smFISH) of one cheRNA-distal target gene pair suggests a spatial overlap consistent with a role in chromosome looping. Additionally, the cheRNA HIDALGO stimulates the fetal hemoglobin subunit gamma 1 (HBG1) gene during erythroid differentiation by promoting contacts to a downstream enhancer. Our results suggest that multiple cheRNAs activate proximal lineage-specific gene transcription.

The genetics of phenotypic plasticity in nematode feeding structures.

Phenotypic plasticity has been proposed as an ecological and evolutionary concept. Ecologically, it can help study how genes and the environment interact to produce robust phenotypes. Evolutionarily, as a facilitator it might contribute to phenotypic novelty and diversification. However, the discussion of phenotypic plasticity remains contentious in parts due to the absence of model systems and rigorous genetic studies. Here, we summarize recent work on the nematode Pristionchus pacificus, which exhibits a feeding plasticity allowing predatory or bacteriovorous feeding. We show feeding plasticity to be controlled by developmental switch genes that are themselves under epigenetic control. Phylogenetic and comparative studies support phenotypic plasticity and its role as a facilitator of morphological novelty and diversity.

Nuclear Fractionation Reveals Thousands of Chromatin-Tethered Noncoding RNAs Adjacent to Active Genes.

A number of long noncoding RNAs (lncRNAs) have been reported to regulate transcription via recruitment of chromatin modifiers or bridging distal enhancer elements to gene promoters. However, the generality of these modes of regulation and the mechanisms of chromatin attachment for thousands of unstudied human lncRNAs remain unclear. To address these questions, we performed stringent nuclear fractionation coupled to RNA sequencing. We provide genome-wide identification of human chromatin-associated lncRNAs and demonstrate tethering of RNA to chromatin by RNAPII is a pervasive mechanism of attachment. We also uncovered thousands of chromatin-enriched RNAs (cheRNAs) that share molecular properties with known lncRNAs. Although distinct from eRNAs derived from active prototypical enhancers, the production of cheRNAs is strongly correlated with the expression of neighboring protein-coding genes. This work provides an updated framework for nuclear RNA organization that includes a large chromatin-associated transcript population correlated with active genes and may prove useful in de novo enhancer annotation.