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First published online 13 February 2008
doi: 10.1242/dev.013086

Development 135, 1097-1106 (2008)
Published by The Company of Biologists 2008
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Notochord-derived Shh concentrates in close association with the apically positioned basal body in neural target cells and forms a dynamic gradient during neural patterning

Chester E. Chamberlain*, Juhee Jeong{dagger}, Chaoshe Guo{ddagger}, Benjamin L. Allen and Andrew P. McMahon§

Department of Molecular and Cellular Biology, The Biolabs, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA.


Figure 1
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  		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 ShhGFP/+, ShhGFP/GFP and ShhGFP/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 ShhGFP/+ 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).

 

Figure 2
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  		Fig. 2. 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 (ShhGFP:::Cre) (Harfe et al., 2004Go) 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., 2005Go) 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).

 

Figure 3
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  		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 ShhGFP/GFP neural tube at this stage are displayed along the x-axis. Scale bars: 5 µm in G-I.

 

Figure 4
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  		Fig. 4. 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 x 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].

 

Figure 5
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  		Fig. 5. 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), {gamma}-tubulin ({gamma}-tub, red), polaris (turquoise). Images are single confocal slices. Association of Shh::GFP ligand, {gamma}-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.

 

Figure 6
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  		Fig. 6. 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 {alpha}-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.

 

Figure 7
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  		Fig. 7. Quantitation of Shh::GFP ligand puncta and basal bodies in early ventral neural tubes. Eight-somite-stage mouse neural tubes cut at the level of the heart were analyzed at three distinct volumes (V1, V2, V3) along the D-V axis. (A) The relative positions of V1, V2 and V3 in the xy-plane. Each volume is 9 µm wide (x-axis), 9 µm long (y-axis) and 2.1 µm deep (a stack of six confocal images taken at 0.35 µm steps along the z-axis). Nuclei (blue), Shh::GFP ligand (green), {gamma}-tubulin (red). (B) Quantification of Shh::GFP ligand puncta and basal bodies within each apical volume. A Shh::GFP punctum is basal-body-associated if it is within 1 µm of a {gamma}-tubulin punctum. A nucleus is associated with an apical volume if it is laterally positioned to that volume. Ventral midline nuclei are associated with V3. Note that the percentage of Shh::GFP puncta associated with the basal body decreases ventrally.

 

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© The Company of Biologists Ltd 2008