Gas1 is a receptor for sonic hedgehog to repel PNAS PLUS enteric axons Shiying Jina, David C. Martinellia,1, Xiaobin Zhenga, Marc Tessier-Lavigneb, and Chen-Ming Fana,2 aDepartment of Embryology, Carnegie Institution of Washington, Baltimore, MD 21218; and bRockefeller University, New York, NY 10065 Edited by Kathryn V. Anderson, Sloan-Kettering Institute, New York, NY, and approved November 26, 2014 (received for review September 26, 2014) The myenteric plexus of the enteric nervous system controls the neurons (1, 2). Germ-line and endoderm-specific conditional mouse movement of smooth muscles in the gastrointestinal system. They mutants for Shh and/or Ihh display reduced mesenchyme and extend their axons between two peripheral smooth muscle layers smooth muscle mass (6, 8). Shh and Ihh directly act on the gut to form a tubular meshwork arborizing the gut wall. How a tubular mesoderm, as inactivation of smoothened (Smo), the obligatory Hh axonal meshwork becomes established without invading centrally signaling component, in gut mesoderm causes the same defect (8). toward the gut epithelium has not been addressed. We provide Mice mutant for growth arrest specific gene 1 (Gas1), which encodes evidence here that sonic hedgehog (Shh) secreted from the gut an Hh surface binding receptor (9, 10), also share this defect (11). epithelium prevents central projections of enteric axons, thereby The role of Hh signaling in enteric neuron development is less forcing their peripheral tubular distribution. Exclusion of enteric defined. Shh mutant intestines contained more punctuated Tuj1 central projections by Shh requires its binding partner growth (an antibody recognizing neuronal βΙΙΙ-tubulin) staining signals er- arrest specific gene 1 (Gas1) and its signaling component smooth- roneously located near the base of the villi and were interpreted to ened (Smo) in enteric neurons. Using enteric neurons differentiated have a substantial number of mislocalized enteric neurons, whereas from neurospheres in vitro, we show that enteric axon growth is Ihh mutants had no enteric neurons (6). However, endoderm- not inhibited by Shh. Rather, when Shh is presented as a point specific conditional Shh;Ihh mutants were reported to have enteric source, enteric axons turn away from it in a Gas1-dependent man- neurons (8). Using the enteric progenitor/neuron marker P75 to ner. Of the Gαi proteins that can couple with Smo, G protein α Z better assess the cell body, we showed that Gas1 mutant intestines + (Gnaz) is found in enteric axons. Knockdown and dominant negative had quantitatively more P75-positive (P75 ) enteric progenitors/ inhibition of Gnaz dampen the axon-repulsive response to Shh, and neurons due to increased proliferation, and a small fraction of NEUROSCIENCE Gnaz mutant intestines contain centrally projected enteric axons. them were abnormally located near the base of the villi (11). Together, our data uncover a previously unsuspected mechanism However, the enteric abnormalities of Shh and Gas1 mutants underlying development of centrifugal tubular organization and may arise as a secondary patterning defect (12) due to reduced identify a previously unidentified effector of Shh in axon guidance. mesenchyme/smooth muscle mass or reduced levels of BMP4 in the mesenchyme (6), as ectopic expression of BMP4 could enteric neuron | axon guidance | chemorepellent | Hedgehog | Gas1 negatively influence enteric progenitor positioning and prolifer- ation (12). Conversely, recombinant Shh can inhibit enteric pro- he enteric nervous system is the largest peripheral nervous genitor differentiation and migration in vitro (7). Single nucleotide Tsubsystem, often likened to a second brain (1). Embedded in polymorphisms (SNPs) of the PTCH1 gene,aconservedHhpath- the gastrointestinal tract, it controls various aspects of digestive way inhibitory component, are associated with a high risk of function ranging from movement and secretion to absorption. Hirschsbrung’s disease (13, 14). Furthermore, Ptch1 conditional Despite their final location, enteric neurons are of neural crest origin, and the majority of enteric progenitors are derived from Significance vagal neural crest cells at midgestation stages of the mouse embryo (1, 2). Under the control of glial cell-derived neuro- The enteric nervous system is the largest peripheral nervous trophic factor (GDNF) and Endothelin 3 (END3) signaling, they subsystem. Enteric axons form a tubular meshwork arborizing migrate into the anterior gut mesenchyme. Once there, they the smooth muscles of the digestive tract to control gut move- continue to propagate and migrate posteriorly through most of ment. Deficiencies of enteric neurons cause megacolon and the length of the gut. The first appearing enteric arbor is the constipation—that is, Hirschsprung’s disease. How enteric axons myenteric plexus, composed of islands of neurons interconnected are kept in the smooth muscle layers without projecting toward by longitudinal and circumferential axons between the inner the gut epithelium is unknown. We provide, to our knowledge, circular and outer longitudinal smooth muscle layers in the the first insights into how these axons are confined to the pe- stomach, intestine, and colon to effect contraction. In cross- riphery and, in the process, define a novel mechanism of sonic section, this plexus presents a simple centrifugal ring structure, hedgehog signaling for axon repulsion involving the guidance but when viewed superficially, it is organized as an extensive receptor growth arrest specific gene 1 and the signaling G mesh-like tubular arborization encasing the gut wall. Axonal protein α z. We believe this to be a conceptual advance in un- arborization along the gut smooth muscles is controlled by over- derstanding a key topological problem in axonal arborization lapping and sequential actions of GDNF and Neuturin, a GDNF and to have important health implications. family member acting mainly postnatally (3, 4). Recently, the planar polarity pathway has also been implicated in enteric connectivity Author contributions: S.J., M.T.-L., and C.-M.F. designed research; S.J., D.C.M., and C.-M.F. (5). Patients carrying mutations that cause enteric neuron de- performed research; S.J. and D.C.M. contributed new reagents/analytic tools; S.J. and X.Z. ficiency, including in genes of the GDNF signaling pathway, present analyzed data; and S.J., M.T.-L., and C.-M.F. wrote the paper. with megacolon and constipation associated with poor bowel The authors declare no conflict of interest. ’ movement, a condition known as Hirschsbrung s disease (1, 2). This article is a PNAS Direct Submission. Growth of the gut mucosal mesenchyme and smooth muscle, on Freely available online through the PNAS open access option. the other hand, depends on Hedgehog (Hh) signaling during em- 1Present address: School of Medicine, Stanford University, Stanford, CA 94305. – Sonic Hh Shh Indian Hh bryonic development (6 8). Both ( )and 2To whom correspondence should be addressed. Email: [email protected]. Ihh ( ) are expressed in the gut endoderm (6), which is radially sur- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. rounded by mucosa mesenchyme, smooth muscles, and enteric 1073/pnas.1418629112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1418629112 PNAS Early Edition | 1of8 Downloaded by guest on October 2, 2021 mutant mouse enteric progenitors have a reduced propensity for Tuj1 signals along the gut wall than wild-type intestines (6). In neurogenesis in vitro (13). Thus, Shh signaling may play a direct contrast to the study that interpreted punctuated Tuj1 signals role in enteric neuron development. near the base of the villi as mislocalized enteric neurons (6), we Whether the gut epithelium directly acts on the myenteric found abundant long thin Tuj1 signals extending deep into the axons to confine their periphery connectivity within the smooth villi, indicating an additional phenotype of erroneously projec- muscles is not known. During our continuous investigation of ting axons (Fig. 1B). The difference between these studies may Shh and Gas1 mutant gut defects, we found that their intestines lie in sample preparations, staining protocols, or planes of sec- contained Tuj1 staining signals deep inside the villi, almost tion. Upon quantification, 68 ± 14% of the villi examined con- reaching the gut epithelium, suggesting a previously unap- tain axons projecting toward the central region where the gut preciated axonal projection defect. Given that Shh is an axon epithelium lies (Fig. 1B; quantification in Fig. 1I). Erroneously guidance molecule in the central nervous system (CNS) (15, 16), projecting axons were also found in the stomach, but not in the we were motivated to investigate whether Shh in the gut epi- colon (Fig. S1 A and B). These data encouraged further in- thelium acts to prevent erroneous entry of enteric axons into the vestigation into whether the Shh signaling pathway plays a direct villi. Below, we provide evidence for a previously unknown en- role in enteric axon guidance. teric axon repulsion mechanism mediated directly by Shh via its receptor Gas1 and signaling component Smo. We further Gas1, but Not Cdo or Boc, Mutants Show Centrally Projecting Enteric implicateauniqueGαi protein, the pertussis toxin (PTX)- Exons. We next sought to define which of the three mammalian insensitive Gnaz (17), in mediating Shh-directed chemorepulsion. Hh binding proteins on the plasma membrane—Gas1, Cdo, or Boc (9, 10,