Expression profiling the developing mammalian enteric nervous system identifies marker and candidate Hirschsprung disease genes

Abstract

The enteric nervous system (ENS) is composed of neurons and glial cells, organized as interconnected ganglia within the gut wall, which controls peristalsis of the gut wall and secretions from its glands. The Ret receptor tyrosine kinase is expressed throughout enteric neurogenesis and is required for normal ENS development; humans with mutations in the RET locus have Hirschsprung disease (HSCR, an absence of ganglia in the colon), and mice lacking Ret have total intestinal aganglionosis. The Ret mutant mouse provides a tool for identifying genes implicated in development of the ENS. By using RNA from WT and Ret mutant (aganglionic) gut tissue and DNA microarrays, we have conducted a differential screen for ENS-expressed genes and have identified hundreds of candidate ENS-expressed genes. Forty-seven genes were selected for further analysis, representing diverse functional classes. We show that all of the analyzed genes are expressed in the ENS and that the screen was sensitive enough to identify genes marking only subpopulations of ENS cells. Our screen, therefore, was reliable and sensitive and has identified many previously undescribed genes for studying ENS development. Moreover, two of the genes identified in our screen Arhgef3 and Ctnnal1, have human homologues that map to previously identified HSCR susceptibility loci, thus representing excellent candidates for HSCR genes. This comprehensive profile of ENS gene expression refines our understanding of ENS development and serves as a resource for future developmental, biochemical, and human genetic studies.

Fig. 1.

Expression profile of selected genes in the E15.5 mouse gut. RNA in situ hybridization of the genes identified in Table 1 is on transverse cryosections of E15.5 mouse small intestine. At E15.5, the enteric nervous system is organized into the myenteric plexus (mp), situated between the developing muscle layers (ml). Comparison of gene expression of known ENS markers reveals that all genes analyzed have equivalent expression within the myenteric region (mp in Ret).

Fig. 2.

Mapk10 and Mab21l1 are expressed in Ret^+/+ but not Ret^k−/k− intestines. Representative comparison of gene expression in Ret^+/+ versus Ret^k−/k− embryos by RNA in situ hybridization: Mapk10 (A, B, E, and F) and Mab21l1 (C, D, G, and H). Both genes are expressed in the myenteric layer of the Ret^+/+ intestine (A and C), but expression is lost in the Ret^k−/k− intestine (B and D). We see expression throughout the myenteric plexus of the Ret^+/+ stomach for both Mapk10 (E) and Mab21l1 (G) but only a small number of Mapk10- or Mab21l1-expressing cells in the equivalent region of the Ret^k−/k− stomach (arrows in F and H).

Fig. 3.

VIP and Cart are expressed in a subpopulation of Ret-expressing enteric neurons. Comparisons of gene expression profiles of Ret (A) to VIP (B), and Cart (C) by RNA in situ hybridization suggest that VIP and Cart are expressed in a smaller proportion of the ENS than Ret. A direct comparison of Ret expression (brown in D) and Cart expression reveals that Cart expression is found in only a small proportion of Ret-expressing cells (coexpression of Cart and Ret is seen as black; arrowhead in D).

Fig. 4.

Analysis of Cart, Dpysl3, and Gng3 gene expression from E10 to P0. RNA in situ hybridization on E11.5 whole embryos (A–C) and on transverse sections of P0 intestines (D–F). Cart is expressed in a subset of enteric neurons at E11.5 (arrowhead in A) and at P0 (arrowhead in D). In the ENS, Dpysl3 expression can be seen at E11.5 (arrowhead in B) and P0 (arrowhead in E). Gng3 is expressed in the ENS at E11.5 (arrowhead in C) and P0 (arrowhead in F).