Figure 4. Effect of Timed Shh Removal on Cell Survival and Proliferation.
(A) Apoptosis (red fluorescence) in whole-mount forelimb (FL) and hindlimb (HL) buds of ShhΔ/flox;Cre+ embryos and control siblings (as in Figure 1) was analyzed at the ages indicated (left), following tamoxifen (Tam) injection at the times indicated (top). Dotted white lines indicate limb bud borders. Note the low level of increased apoptosis in Shh+/flox;Cre+ embryos over Shh+/Δ;Cre− controls (Cre+ controls are shown for all E9.5 embryos), which was negligible compared to apoptosis in Shh mutants. By E11.5, apoptosis in mutant FLs (after Tam at E9.5 or E10) was declining (data not shown), but still prominent in HLs.
(B) Mitotic cell indices (mean ± σ) determined by anti-pH3 staining of Shh mutant and sibling controls (see Experimental Procedures and Figure S4).
(C) Cell cycle analysis of limb bud cells from Shh mutant and sibling controls at 24 hr after Tam at E10. Dot plots show bivariate flow cytometry analysis of BrdU/DNA-stained cells from sibling control (7,148 cells) or mutant (13,187 cells) embryos (1 hr BrdU pulse). G1 and S phase distributions were altered, but cell size indices were unchanged in Shh mutant compared to control limb cells (data not shown).
(D) Schematic showing dual roles of Shh in relation to formation of digit condensations, and to other signals regulating digit identity downstream of Shh. Digit 1, which is Shh independent (Chiang et al., 2001), is not shown. Panels below wild-type (WT) show phenotypes resulting when only the duration of Shh function is shortened during the expansion phase (this report), compared to quantitatively reduced Shh activity or altered Shh lipid modification during both phases. Condensations shown below different Shh-duration times (arrows) indicate the number of condensations ultimately forming after limb bud expansion, not already-formed elements.