Cellular transcriptomics reveals evolutionary adaptation and rumination of vertebrate stomachs

SUMMARY Stomach, an essential digestive organ in vertebrates, has undergone significant changes in its morphology, chamber numbers, and physiology, in response to dietary diversity. However, its cellular and molecular basis underlying evolutionary adaptation remains largely uncharacterized. Here we report a single-cell and spatial transcriptomic atlas of stomachs from 23 vertebrate species with diverse feeding habits (e.g., omnivores, carnivores, and herbivores) and gastric architectures, including monogastric (e.g., primates), two- (e.g., aves), three- (e.g., camel) and four-chambered stomachs (e.g., sheep). We reveal conservation and divergence in cell-type composition, developmental trajectory, evolutionary origins and forces, spatial distribution, gene regulatory network and effect, metabolic signature and disease susceptibility associated with the feeding habits and stomach chamber. The diversification of plant based diets, particularly the expansion of coarse fibrous plants that required enhanced mechanical processing, driving the rapid evolution of specific cells and associated genes. Ruminants have evolved chamber-specific cell types with enhanced expressions of relevant functional genes (e.g., KRT6A in spinous cells of forestomach, LUC7L in SMCs of abomasum, and TSPYL4 in abomasal enteroendocrine cells), which were validated by fluorescence in situ hybridization and spatial transcriptomic profiling. The three cell-specific expressed genes showed significant effects on cell proliferation and migration by RNA interference to knock down their expressions. In particular, the knockdown of LUC7L in SMCs promotes a transition from a contractile to a synthetic phenotype, and Luc7l knock-out in mice delays gastric emptying and impairs gastric motility, demonstrating that this gene is essential for the coordinated multi-chambered gastric motility that underlies rumination in ruminants. These findings elucidate the cellular and molecular adaptations underlying the evolution of stomach coupled with the emergence of diet land plants. Our results provide potential cellular and gene targets for engineering monogastric animals to acquire functional rumination digestive capabilities as well as for medication of gastric mobility.