Figure 3. Formation of Digit Condensations in Wild-Type and Shh^flox/flox;Cre+ Embryos.

(A) Normal condensation sequence of d4, d2, d5, d3 in fore and hindlimb buds visualized by LacZ activity in Shh^+/+;Noggin^LacZ/+ embryos at E11.25–E12.5. Digit 1 was not obvious in either wild-type or mutant condensations until very late (due to small size) and was not included in evaluating order.

(B–E) Condensation formation visualized by LacZ activity in Shh^flox/flox;Cre+;Noggin^LacZ/+ and control sibling (Cre−) limbs after tamoxifen given at the times indicated ([B] and [C] show forelimb; [D] and [E] show hindlimb). Note gap for d3 (arrow in [B]) and attenuated d5 (asterisk in [C]) upon progressively earlier Shh removal in mutants. The order of condensation loss was the same as in skeletal elements (Figure 2). In severer phenotypes, narrowing of the space between long bone condensations and conversion to a single condensation were also seen frequently ([D] and not shown).

(F and G) Double staining for posterior marker Tbx2 and condensations (LacZ activity) in Shh^flox/flox;Cre+;Noggin^LacZ/+ embryos demonstrates Tbx2 RNA (arrowheads mark anterior margin) in mesenchyme surrounding the distal posterior condensation of mutant embryos with two remaining digit precursors (F), and with a single, remaining long-digit precursor (G). In control siblings (Cre−), Tbx2 RNA extends anteriorly up to d3 (arrowheads) but is completely absent from d2.