This Was Unexpected Given the Functional Evidence That Hh Signals Control Enteric Neuron

Supplementary Information 2

Supplementary Figure 2. Enteric neurons do not transduce Hh signals. Intestinal sections from Ptch1LacZ/+ (A), Gli2 LacZ/+ (B) and Gli1 LacZ/+ (C) mice at E16.5 exhibit no staining of enteric neurons (arrows). (D) A section from a Gli1LacZ/+ mouse, stained with anti-peripherin (Per, green), is overlaid with the image of an adjacent section, stained with X-gal (Gli1-expressing nuclei are blue). (E,F) Sections from Ptch1LacZ/+ mice at P10 (E) and adult (F) reveal enteric nuclei that are expressing Ptch1 (arrows). (G,H) Adjacent sections of an adult Gli1LacZ/+ mouse. The section in (G) is stained with X-gal. In H, the section was immunostained with anti-peripherin (green) and the X-gal stained image is overlaid onto this image. X-gal positive nuclei are false-colored red. The white dotted line in G outlines the peripherin-staining region seen in H.

Discussion:

Hh signaling plays a crucial role in the development of the enteric nervous system1-4. Both Shh and Ihh knockout mouse embryos have deficiencies in enteric neuron patterning 1, and manipulations of Hh signaling lead to mispatterned neurons in chicken explants 2 and zebrafish embryos4. Since there is still some debate as to whether the regulation of neuronal patterning by Hh is direct or indirect 4, we looked closely at nerve plexes in E14.5, E16.5 and adult Gli1LacZ/+ and Ptch1LacZ/+ animals. Antibodies to peripherin, an intermediate filament marker of mature neurons and glia was used to confirm the position of these plexes, which are quite morphologically distinct even in the absence of nerve-specific immunostains. In the fetal tissues examined, we found no Ptch1, Gli1 or Gli2 positive cells in the neuronal plexes. However, in P10 samples, weak staining for Ptch1 is present in a few of the cells of Auerbach’s plexus. In adults, this staining is more robust, but only a subset of cells is stained. At no stage were Gli1 positive neurons detected. Though more investigation is necessary using markers for more immature neuronal cells, our data appear to support the contention that the effect of Hh signaling on neuronal differentiation in fetal life is indirect, as suggested by data in both chicken2 and zebrafish 4. Of course, these data do not rule out a direct effect of Hh on neural crest precursors prior to their migration to gut plexes.

1.Ramalho-Santos M, Melton DA, McMahon AP. Hedgehog signals regulate multiple aspects of gastrointestinal development. Development 2000;127:2763-72.

2.Sukegawa A, Narita T, Kameda T, Saitoh K, Nohno T, Iba H, Yasugi S, Fukuda K. The concentric structure of the developing gut is regulated by Sonic hedgehog derived from endodermal epithelium. Development 2000;127:1971-80.

3.Fu M, Lui VC, Sham MH, Pachnis V, Tam PK. Sonic hedgehog regulates the proliferation, differentiation, and migration of enteric neural crest cells in gut. J Cell Biol 2004;166:673-84.

4.Reichenbach B, Delalande JM, Kolmogorova E, Prier A, Nguyen T, Smith CM, Holzschuh J, Shepherd IT. Endoderm-derived Sonic hedgehog and mesoderm Hand2 expression are required for enteric nervous system development in zebrafish. Dev Biol 2008;318:52-64.