Notochord-derived Shh::GFP ligand concentrates at the apical region of the neural tube. Neural tube sections of embryos of genotypes and stages indicated. (A-C) Shh::gfp expression visualized by RNA in situ hybridization. (D-F) Shh::GFP distribution directly visualized by confocal microscopy. Shh::GFP concentrated in an apical region of ventral midline cells overlying the notochord (white arrows in D-F) and basolateral region (red arrow, E). (G-I) Immunostaining showing Shh-dependent neural progenitor domains p3 (Nkx2.2) and pMN (Olig2). (J) Immunostaining for Shh in wild-type mouse embryos using an Shh antibody. Note apical signal (arrow). (K,L) A Shh-driven GFP::Cre transgene (Shh^GFP:::Cre) (Harfe et al., 2004) is only detected in the Shh-expressing notochord (arrow in K). Wnt1::GFP, following ShhGFP::Cre-mediated activation of a ubiquitous Wnt1::gfp expression allele (Carroll et al., 2005) specifically in the notochord, is detected in the neural tube (arrow in L). Note that the most dorsally positioned Shh::GFP ligand signal in D is punctate and is adjacent to the most dorsally positioned pMN cell in G (blue arrows).

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.

Shh::GFP ligand distribution during the emergence of ventral pattern. (A-F) Sections of 6- to 12-somite-stage mouse ventral neural tubes were analyzed at three developmental time points as determined by the appearance of distinct ventral cell types at the ventral midline (T1, p2; T2, pMN; T3, p3). (G) Schematic showing the changes in domain state across three distinct regions (R1-R3) of ventral neural tube. Each region represents a 9 μm × 9 μm square along the D-V axis with R3 beginning at the apical region of ventral midline cells. Red, p3; white, pMN; blue, p2. (H-J) Single confocal sections of ventral neural tube at the apical region (Shh::GFP ligand, green; nuclei, blue). (K,L) Quantitation of Shh::GFP ligand intensity (y-axis, average pixel intensity) at different time points and regions. Each time point represents four sections from three embryos. Significant differences from R3 are indicated by asterisks [P-values: T3(R3,R2), P<0.0298; T3(R2,R1), P<0.1275; T3(R1,R3), P<0.0125; R3(T3,T2), P<0.0417; R3(T2,T1), P<0.0775; R3(T3,T1), P<0.0458].

Subcellular distribution of concentrated Shh::GFP ligand in apical region. (A-I) High magnification of apical region in 8-somite-stage mouse neural tubes of indicated genotypes. Nuclei (blue), Shh::GFP ligand (green), γ-tubulin (γ-tub, red), polaris (turquoise). Images are single confocal slices. Association of Shh::GFP ligand, γ-tubulin and polaris is shown by arrows. Shh::GFP ligand continues to associate with the basal body region in Smo and Skn mutants. (J) Three-dimensional surface rendering of six confocal images taken sequentially along the z-axis. Note the adjacent, non-overlapping co-localization of the three proteins.

Association of Shh::GFP ligand with lysosomes and stabilized microtubules. (A-C) High magnification of apical compartment of 8-somite-stage mouse neural tubes of indicated genotype. Nuclei (blue), Shh::GFP ligand (green), Lamp1 (red). The association of Shh::GFP ligand with the lysosome is indicated by the arrows. The bulk of Shh::GFP ligand apical accumulation does not co-segregate with the lysosome. (D-F) Immunostaining for acetylated α-tubulin reveals a network of stabilized microtubules (red) that span between the apical and basal surfaces of neural progenitors. Shh::GFP (green) is present in the notochord (asterisk). The boxed region in D is shown at high magnification in E,F. Arrows in E indicate a string of Shh::GFP ligand puncta.