Fig. 5. Hindlimb (HL) anteroposterior markers reveal apparent loss of anterior bud mesenchyme. Dorsal views of whole-mount in situ hybridization embryos are shown. (A) The anterior border of Hoxc11 mRNA is revealed at the junction between somites 26 and 27. In mutant embryos, the proportion of Hoxc11-negative anterior mesenchyme relative to Hoxc11-positive mesenchyme is reduced, consistent with the loss of anterior bud mesenchyme revealed in Fig. 5A. (B) The zone of polarizing activity (ZPA) is revealed by hybridization for sonic hedgehog (Shh). Whereas in wild-type and Pitx^-/- embryos the ZPA occupies the posterior quadrant of the HL bud, this structure extends all the way up to half the HL buds in Pitx1^-/-,Pitx2^+/- embryos. (C) Expression of Gli3 in anterior hindlimb buds is present in embryos of the three genotypes. (D) Expression of Fgf4 in AER. The extent of Fgf4 expression appears anteriorized in mutant embryos compared with wild type, again in agreement with the loss of anterior mesenchyme. (E) Expression of the posterior limb bud mesenchyme marker, Hand2 (dHand in figure) is also extended anteriorly. Two examples at different developmental stages are shown with LR differences in the anterior extension of Hand2 expression. (Bottom row) Anterior bud expression of Hand2 is shown in the right HL bud of an E11.5 Pitx1^-/- embryo, and in the right HL of a E12.5 Pitx1^-/-,Pitx2^+/- embryo.

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