Fig. 3. Shh::GFP ligand forms a gradient at the apical region that can be modified by Smo and Skn activity. (A-I) Sections of 14- to 15-somite-stage mouse neural tubes of indicated genotypes. (A-C) Immunostaining for Nkx6.1, whose expression is activated by Shh signaling, and for Pax6, whose expression is repressed by Shh signaling in the ventral neural tube. (D-F) Shh::GFP ligand distribution directly visualized by confocal microscopy. (G-I) High magnification of the apical, ventral neural tube. Images are a projection of six confocal images taken sequentially along the z-axis compressed into one to provide a better sampling of the total Shh::GFP ligand distribution. Note that Shh::GFP ligand forms a large punctum near the ventral midline (G, red arrow) at this stage that is presumably made up of several smaller puncta. White arrows indicate approximate dorsal limit of detectable Shh::GFP ligand. (J) Profiles of apical Shh::GFP ligand along the D-V axis of indicated genotypes. Profile represents an average based on two sections per embryo across three embryos. Approximate positions of Shh-dependent progenitors p3, pMN and p2 in a Shh^GFP/GFP neural tube at this stage are displayed along the x-axis. Scale bars: 5 µm in G-I.