Background & Aims Infantile hypertrophic pyloric stenosis (IHPS) is a common birth anomaly characterized by obstruction of the pyloric lumen. A genome-wide association study implicated NKX2-5, which encodes a transcription factor that is expressed in embryonic heart and pylorus, in the pathogenesis of IHPS. However, the function of the NKX2-5 in pyloric smooth muscle development has not been directly examined. We investigated the pattern of Nkx2-5 during the course of murine pyloric sphincter development and examined co-expression of Nkx2-5 with Gata3 and Sox9—other transcription factors with pyloric-specific mesenchymal expression. We also assessed pyloric sphincter development in mice with disruption of Nkx2-5 or Gata3. Methods We used immunofluorescence analysis to compare levels of NKX2-5, GATA3, and SOX9 in different regions of smooth muscle cells. Pyloric development was assessed in mice with conditional or germline deletion of Nkx2-5 or Gata3, respectively. Results Gata3, Nkx2-5, and Sox9 were co-expressed in differentiating smooth muscle cells of a distinct fascicle of the pyloric outer longitudinal muscle (OLM). Expansion of this fascicle coincided with development of the pyloric sphincter. Disruption of Nkx2-5 or Gata3 caused severe hypoplasia of this fascicle and alters pyloric muscle shape. Although expression of Sox9 required Nkx2-5 and Gata3, there was no apparent hierarchical relationship between Nkx2-5 and Gata3 during pyloric OLM development. Conclusions Nkx2-5 and Gata3 are independently required for the development of a pyloric OLM fascicle, which required for pyloric sphincter morphogenesis, in mice. These data indicate that regulatory changes that alter Nkx2-5 or Gata3 expression could contribute to pathogenesis of IHPS.

The gastric ligaments are superficial cord-like structures, located on the lesser curvature of the stomach, that extend from the pylorus to the esophagus. These ligaments have been documented in a wide variety of mammalian species, including humans, but their composition and ontogeny is unexplored. Here, we demonstrate that, during ontogeny, the gastric ligaments are first visible as extensions from a C-shaped domain of Gata3-expressing cells that surround the future pylorus; this domain will later give rise to the pyloric outer longitudinal muscle (OLM). The open ends of the C are located ventrally, and, beginning at embryonic day (E) 13.5, the ligaments grow anteriorly from these points. Whereas most other ligaments of the stomach are neurovascular in nature, the gastric ligaments are composed of smooth muscle cells that mature between E14.5 and E16.5. The gastric ligaments coexpress the transcription factors Gata3, Nkx2-5, and Sox9, and germline loss of Gata3 or conditional deletion of Nkx2-5 abrogates Sox9 expression and impairs gastric ligament smooth muscle development; similar phenotypes were previously seen in the OLM. In accord with this molecular contiguity between the OLM and gastric ligaments, three-dimensional image reconstruction highlights physical contiguity between these smooth muscle structures, suggesting that they may work together as a unit to control flexure of the pyloric region, a function similar to the ligament of Treitz at the duodenojejunal junction. These findings may have implications for our understanding of normal pyloric sphincter function, as well as the common human congenital pathology idiopathic hypertrophic pyloric stenosis.