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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. 

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. 

Abstract BackgroundSnakes exhibit extreme intestinal regeneration following months-long fasts that involves unparalleled increases in metabolism, function, and tissue growth, but the specific molecular control of this process is unknown. Understanding the mechanisms that coordinate these regenerative phenotypes provides valuable opportunities to understand critical pathways that may control vertebrate regeneration and novel perspectives on vertebrate regenerative capacities. ResultsHere, we integrate a comprehensive set of phenotypic, transcriptomic, proteomic, and phosphoproteomic data from boa constrictors to identify the mechanisms that orchestrate shifts in metabolism, nutrient uptake, and cellular stress to direct phases of the regenerative response. We identify specific temporal patterns of metabolic, stress response, and growth pathway activation that direct regeneration and provide evidence for multiple key central regulatory molecules kinases that integrate these signals, including major conserved pathways like mTOR signaling and the unfolded protein response. ConclusionCollectively, our results identify a novel switch-like role of stress responses in intestinal regeneration that forms a primary regulatory hub facilitating organ regeneration and could point to potential pathways to understand regenerative capacity in vertebrates.