SHH E176/E177-Zn2+ conformation is required for signaling at endogenous sites

- Conformation-specific anti-SHH antibodies distinguish between diffuse and punctate SHH in the developing spinal cord.
- SHH-E177/176, Zn2+, pH and heparin regulate SHH cross-linking.
- SHH-E177 is required for endogenous but not ectopic signaling sites in developing mice.
- SHH basal body-association remains, while diffuse SHH is absent in ShhE177A/- spinal cord.
- SHH-E177-Zn2+ maintains SHH in an activation competent conformation.

Sonic hedgehog (SHH) is a master developmental regulator. In 1995, the SHH crystal structure predicted that SHH-E176 (human)/E177 (mouse) regulates signaling through a Zn2+-dependent mechanism. While Zn2+ is known to be required for SHH protein stability, a regulatory role for SHH-E176 or Zn2+ has not been described. Here, we show that SHH-E176/177 modulates Zn2+-dependent cross-linking in vitro and is required for endogenous signaling, in vivo. While ectopically expressed SHH-E176A is highly active, mice expressing SHH-E177A at endogenous sites (ShhE177A/-) are morphologically indistinguishable from mice lacking SHH (Shh-/-), with patterning defects in both embryonic spinal cord and forebrain. SHH-E177A distribution along the embryonic spinal cord ventricle is unaltered, suggesting that E177 does not control long-range transport. While SHH-E177A association with cilia basal bodies increases in embryonic ventral spinal cord, diffusely distributed SHH-E177A is not detected. Together, these results reveal a novel role for E177-Zn2+ in regulating SHH signaling that may involve critical, cilia basal-body localized changes in cross-linking and/or conformation.

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