Figure 6. The schlei mutation partially rescues digit number in Shh mutant limbs.

Sox9 marks condensing digits in e13.5 forelimbs (Akiyama et al., 2002), revealing five digits in wild type (A), an ectopic anterior digit in schlei mutants (B), a single digit in Shh mutants (C), and at least four digits in the double mutant (D). The direct positive target of Shh, Gli1, is not activated in Shh (G) or the double mutant (H), compared with the anterior expansion of expression observed in schlei (F) relative to wild type (E). However, expression of Gremlin, which is anteriorly expanded in schlei (arrow in J) as compared to wild type (I), is partially restored in the double mutant (L) despite its complete absence in the Shh mutant (K), suggestive of a loss of Gli3R function. The rescue of digit number in schlei-Shh double mutants may be due in part to a rescue of cell death; TUNEL (green staining) in e10.5 forelimb sections demonstrates that, similar to wild type (M) and schlei (N), double mutant limbs (P) lack the extensive distal cell death observed in Shh mutants (O). Hoxd11 is expressed in the presumptive digits two through five in wild type (Q), and the ectopic digit in schlei variably expresses Hoxd11, indicating posterior identity in the example shown (R). Hoxd11 is absent in both the Shh mutant (S) and the double mutant (T). Blue staining in M–P = DAPI. Control and schlei mutant images are shown at the same magnification.