Fig. 1. Shh::GFP activity and distribution in the mouse neural tube. (A) Diagram of full-length and processed Shh::GFP. The Shh processing site is disrupted by GFP insertion and a new processing site is added after the GFP (red) so that processing releases an N-terminal fragment that is GFP tagged. (B-H) Shh::GFP specifies all Shh-dependent ventral cell identities. (B-D) Analysis of Shh-dependent neural progenitors in E10.5 spinal cord regions of embryos with indicated genotypes. Immunostaining with ventral-specific markers to identify ventral progenitor domains: Nkx2.2 (p3, lower green), Olig2 (pMN, red), and Pax7 (dorsal progenitors, upper green). (E-G) In situ hybridization for Shh RNA in E10.5 neural tube sections of embryos with indicated genotypes at the spinal cord level. (H) Quantification of the size of each progenitor domain (p3, pMN, p2, p1/p0, d) along the D-V axis in cell diameters in Shh^GFP/+, Shh^GFP/GFP and Shh^GFP/GFP;Ptch1^+/- neural tube sections at the level of the forelimb (E10.5). Values represent the average from two sections per embryo across three embryos of a given genotype. (I-L)Neural tube cross-sections of Shh^GFP/+ embryos at E10.5 (dorsal is up and ventral down). (I) Shh and Shh::gfp expression visualized by RNA in situ hybridization. (J) Shh::GFP distribution directly visualized by confocal microscopy. Shh::GFP protein at the expressing floor plate (^*), responding progenitor (below the arrowhead) and non-responding mantle (arrow) domains. (K) Immunostaining of Shh-dependent neural progenitor domains p3 (Nkx2.2, green) and pMN (Olig2, red) in the ventricular region. Pax7 is repressed by low-level Shh signaling and is restricted to dorsal neural precursors (green). (L) Immunostaining of post-mitotic neurons (β3-tubulin, red).