Figure 1. A transient Shh activity pulse suffices to specify all digits with enforced cell survival.

(A) Shh expression timeline in wildtype mouse hindlimb (Shh/ZPA, purple). Limb axis orientation indicated by compasses (upper left) in all figures. (B) Summary of recombination timelines (orange; see Methods for somite (so) staging/range) for tamoxifen (Tam)-induced Shh deletion at different times relative to Shh expression onset and subsequent digit outcomes (at right); KO, Shh null phenotype; ND, not done. At E9.5 Tam, deletion precedes Shh expression and all limbs have a Shh null phenotype. By E9.75 and later, deletion occurs after the Shh role in maintaining cell survival has commenced and both Bax[+] (apoptosis competent) and Bax[-] (cell survival enforced) embryos display some rescue of digit formation (see Table S1); >E10 data summarized from Zhu et al (2008). (C) Shh activity assayed by Ptch1 RNA (arrows) at times after Tam as indicated on timeline. Shh activity was first detected at 6hr (29 so) after injection in a subset of both control Shh^+/C;Bax-CKO (7/12+), and Shh-CKO;Bax-CKO (7/15+) embryos, and became robust by 8hr (30 so) in control (12/12+), but was absent in all Shh-CKO;Bax-CKO embryos (0/10+) from t=8hrs (30 so) and later. (D) Skeletal staining (E16.5) after Bax/Bak and Shh removal by Tam (treated at E9.5+3h, as in C). In hindlimbs with Bax/Bak present (Bax^c/+) all Shh-CKO embryos (28/28) have Shh null phenotype. In hindlimbs with Bax/Bak absent (Bax-CKO), about 50% of the Shh-CKO embryos (18/31) have 3–5 normal digits (5-digit phenotype shown in right-most panel) and normal zeugopod bones (tibia, Ti; fibula, Fi), but 100% Shh null embryos (Shh^−/−; 18/18) with Bax/Bak removed still retain the null mutant phenotype. Related to Figure S1 and Table S1.