Single-cell and spatial transcriptomics of vertebrate stomachs reveal the molecular and cellular basis of rumination in ruminants

The stomach, an essential digestive organ in vertebrates, underwent significant changes in its morphology, chamber numbers, and physiology, in response to dietary diversity. However, its cellular and molecular basis underlying the 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 structure, 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. In response to strong selection forces by fermentation and dietary challenges, the rapid evolution of enteroendocrine cells and associated genes (e.g., TSPYL4 and CD47) was observed particularly in ruminants. Meanwhile, ruminants have evolved chamber-specific cell types with enhanced expressions of relevant functional genes (e.g., KRT6A in spinous cells of forestomach, LUC7L in smooth muscle cells (SMCs) of abomasum, and TSPYL4 in enteroendocrine cells of abomasum), which were validated by fluorescence in situ hybridization and spatial 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, while Luc7l knock-out in mice results in decreased rumination symptoms such as reduced gastric emptying and impaired gastric motility. These findings elucidate the cellular and molecular adaptations underlying stomach evolution, and provide potential cellular and gene targets for engineering monogastric animals to acquire functional rumination digestive capabilities.