TY - JOUR
T1 - SHH E176/E177-Zn2+ conformation is required for signaling at endogenous sites
AU - Himmelstein, Diana S.
AU - Cajigas, Ivelisse
AU - Bi, Chunming
AU - Clark, Brian S.
AU - Van Der Voort, Grant
AU - Kohtz, Jhumku D.
N1 - Funding Information:
We thank E.A. Garber (Biogen) for the human Shh-E176A and ShhN-E176A cDNA constructs, D. Baker (Biogen) for uSHHN protein, A.McMahon (Harvard) for ShhN/+ mice, T. Caspary (Emory) for Wim mice, D.S.H.'s thesis committee (J. Kessler, A. Chenn, and R. Miller, Northwestern University) for helpful comments throughout the course of this work, N. Copeland (National Cancer Institute) for EL250 cells and recombineering plasmids PL253 and PL452, D. Epstein (U Penn) for Shh in situ probe, J. Rubenstein (UCSF) for Dlx2 in situ probe, and the following lab members for technical expertise and advice: M. VanGompel (recombineering and plasmid design), J.C. Savage (initial characterization of α-SHHCL/P), and J. Feng (early charactrerization of SHH-E176A and SHHN-E176A proteins, including the Western in Fig. S1). Antibodies shown in Fig. 5M-a and E1 anti-SHH were obtained from the Developmental Studies Hybridoma Bank. This work is funded by NIMH R01MH094653 to J.D.K. The manuscript has been approved by all authors.
Publisher Copyright:
© 2017 Elsevier Inc.
PY - 2017/4/15
Y1 - 2017/4/15
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85014255697&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85014255697&partnerID=8YFLogxK
U2 - 10.1016/j.ydbio.2017.02.006
DO - 10.1016/j.ydbio.2017.02.006
M3 - Article
C2 - 28263766
AN - SCOPUS:85014255697
SN - 0012-1606
VL - 424
SP - 221
EP - 235
JO - Developmental biology
JF - Developmental biology
IS - 2
ER -