Figure 6. Increased SHH^Cl/P cilia BB association and SHH cross-linking in Shh^E177A/− embryonic spinal cord.

A–F′. Confocal analysis of immunofluorescence staining using H160 (A–C′, green) and α-SHH^CL/P (D–F′, green), on E9.5 Shh^+/+ (A, A′, D, D′), Shh^E177A/− (B, B′, E, E′), and Shh^−/− (C, C′, F, F′) lumbar spinal cord sections. Scale bars 10μm (A–F), 5μm (A′–F′). G. Schematic of the spinal cord highlighting the regions where SHH^Cl/P puncta are counted. Bar graph on the left shows the total number of puncta per 2000μm² area; bar graph on the right shows the %SHH^CL/P associated with γ-tubulin. Counting is performed in ventral (Vent) and intermediate (Int) regions of the Shh^+/+ (black bars) and Shh^E177A/− (gray bars) E9.5 lumbar spinal cord. In Shh^+/+, the dorsal boundary of the intermediate region is 200 μm from the dorsal edge of the spinal cord; the ventral boundary of the intermediate region is 320 μm from the ventral edge of the spinal cord. A corresponding region is used for Shh^E177A/− embryos. Data represented as mean ± SD, Student’s t-test, total number of puncta: ^*p=0.02.(ventral region) and ^*p= 0.01, (intermediate region), %SHH^CL/P associated with γ-tubulin ^*p=0.002 (ventral region). n=3 embryos for each region. H. Western analysis of membrane extracts isolated from anterior region of Shh^+/+, Shh^E177A/−, and Shh^−/− E10.5 embryos. The top of the blot was probed with α-SHH^CL/P and bottom was probed with H160. The lower part of the blot was reprobed with anti-β-actin for a loading control. A long gel (14 cm) was run for better protein separation. α-SHH^Cl/P identifies SHH-E177A^CL in Shh^E177A/−, indicated by the arrow. H160 identifies SHH monomers migrating as multiple bands between 19–29 kD (SHH^M). There is a shift from the 20kD monomer to the cross-linked form in Shh^+/+ compared to Shh^E177A/−. Shh^−/− extracts are loaded as a negative control. I. Ventral spinal cord schematics show that Shh^E177A/− mutants lack diffuse Shh staining (light green) in floorplate, with increased puncta (dark green) expression in the spinal cord ventricle. J. Models proposing possible roles of SHH-E177, Zn²⁺ and SHH/^CL/P in SHH signaling. I. Pre-signaling E176/E177-Zn²⁺ activation model of SHH signaling at cilia BBs. II. Post-signaling model of SHH^CL/P formation at cilia BBs. While the pre-signaling model is preferred based on the findings listed below, it is also possible that SHHCL/P can function in both pre and post-signaling. The model is based on the following findings:

1. E176A/E177A modulates Zn²⁺-mediated conformational change, detected by increased formation of cross-linked dimers.
2. E176A/E177A is active at ectopic, but not endogenous sites in vivo.
3. In Shh^E177A/− mutant spinal cord, increased accumulation of SHH-E177A occurs near cilia BBs.
4. SHH^CL/P is still present in the ventral spinal cord even in the absence of the SHH receptor PTC1, supporting a pre-signaling role of SHH^CL/P.

We propose that SHH signaling is regulated by a Zn²⁺-induced cross-linked/conformational change that occurs at cilia BBs. The E176/E177 site is required for this cross-linked/conformational change at cilia BBs, explaining why E177A mutation eliminates SHH signaling at endogenous sites. Co-factors that regulate the availability/concentration of Zn²⁺, pH, the C-terminal cholesterol modification, and PTC1 are likely to contribute to SHH signaling at endogenous sites, an environment that is not recapitulated in ectopic signaling events. The dotted arrow indicates the possibility of a dynamic state of conversion between inactive and active states. Proof of such conversions will rely on the development of tools to study these events in vivo.