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Canonical models of intestinal regeneration emphasize the critical role of the crypt stem cell niche to generate enterocytes that migrate to villus ends. Burmese pythons possess extreme intestinal regenerative capacity yet lack crypts, thus providing opportunities to identify noncanonical but potentially conserved mechanisms that expand our understanding of regenerative capacity in vertebrates, including humans. Here, we leverage single-nucleus RNA sequencing of fasted and postprandial python small intestine to identify the signaling pathways and cell–cell interactions underlying the python’s regenerative response. We find that python intestinal regeneration entails the activation of multiple conserved mechanisms of growth and stress response, including core lipid metabolism pathways and the unfolded protein response in intestinal enterocytes. Our single-cell resolution highlights extensive heterogeneity in mesenchymal cell population signaling and intercellular communication that directs major tissue restructuring and the shift out of a dormant fasted state by activating both embryonic developmental and wound healing pathways. We also identify distinct roles of BEST4+ enterocytes in coordinating key regenerative transitions via NOTCH signaling. Python intestinal regeneration shares key signaling features and molecules with mammalian gastric bypass, indicating that conserved regenerative programs are common to both. Our findings provide different insights into cooperative and conserved regenerative programs and intercellular interactions in vertebrates independent of crypts which have been otherwise obscured in model species where temporal phases of generative growth are limited to embryonic development or recovery from injury. 

Abstract Understanding and predicting the relationships between genotype and phenotype is often challenging, largely due to the complex nature of eukaryotic gene regulation. A step towards this goal is to map how phenotypic diversity evolves through genomic changes that modify gene regulatory interactions. Using the Prairie Rattlesnake (Crotalus viridis) and related species, we integrate mRNA-seq, proteomic, ATAC-seq and whole genome resequencing data to understand how specific evolutionary modifications to gene regulatory network components produce differences in venom gene expression. Through comparisons within and between species, we find a remarkably high degree of gene expression and regulatory network variation across even a shallow level of evolutionary divergence. We use these data to test hypotheses about the roles of specific trans-factors and cis-regulatory elements, how these roles may vary across venom genes and gene families, and how variation in regulatory systems drive diversity in venom phenotypes. Our results illustrate that differences in chromatin and genotype at regulatory elements play major roles in modulating expression. However, we also find that enhancer deletions, differences in transcription-factor expression, and variation in activity of the insulator protein CTCF also likely impact venom phenotypes. Our findings provide insight into the diversity and gene-specificity of gene regulatory features and highlight the value of comparative studies to link gene regulatory network variation to phenotypic variation. 

Understanding how regulatory mechanisms evolve is critical for understanding the processes that give rise to novel phenotypes. Snake venom systems represent a valuable and tractable model for testing hypotheses related to the evolution of novel regulatory networks, yet the regulatory mechanisms underlying venom production remain poorly understood. Here, we use functional genomics approaches to investigate venom regulatory architecture in the prairie rattlesnake and identify cis -regulatory sequences (enhancers and promoters), trans -regulatory transcription factors, and integrated signaling cascades involved in the regulation of snake venom genes. We find evidence that two conserved vertebrate pathways, the extracellular signal-regulated kinase and unfolded protein response pathways, were co-opted to regulate snake venom. In one large venom gene family (snake venom serine proteases), this co-option was likely facilitated by the activity of transposable elements. Patterns of snake venom gene enhancer conservation, in some cases spanning 50 million yr of lineage divergence, highlight early origins and subsequent lineage-specific adaptations that have accompanied the evolution of venom regulatory architecture. We also identify features of chromatin structure involved in venom regulation, including topologically associated domains and CTCF loops that underscore the potential importance of novel chromatin structure to coevolve when duplicated genes evolve new regulatory control. Our findings provide a model for understanding how novel regulatory systems may evolve through a combination of genomic processes, including tandem duplication of genes and regulatory sequences, cis -regulatory sequence seeding by transposable elements, and diverse transcriptional regulatory proteins controlled by a co-opted regulatory cascade.