Three-dimensional-mapping of smooth muscle morphogenesis in the vertebrate gastrointestinal tract

Gastrointestinal (GI) motility depends on smooth muscle contractions to propel contents through the digestive tract. GI muscle is organized into two perpendicular layers: the inner circular smooth muscle (CSM) and the outer longitudinal smooth muscle (LSM) layers. This organization is important for proper motility. Despite several investigations, GI smooth muscle development remains incompletely understood. Most of previous studies, which mainly relied on αSMA detection in tissue sections, focused on a single region of the GI tract or a specific developmental stage. Other smooth muscle markers, such as CALPONIN1 and γSMA, were less frequently used, and the LSM layer was not systematically analyzed.In this study, we combined three-dimensional confocal imaging of whole-mount cleared embryonic chick guts with heatmap analysis to generate the first spatial atlas of smooth muscle development. We characterized the expression patterns of αSMA, γSMA and CALPONIN1, thereby mapping the dynamics of the formation and differentiation of CSM and LSM layers along the antero-posterior axis. This study provides a framework for future investigations into the regulatory mechanisms governing smooth muscle patterning and maturation along the GI tract.