Fig. 1. Gli3 and Hoxd12 interact genetically during limb development. E17.5-18.5 limb skeletons (left and middle two columns), and E11.5-12.5 hindlimb bud Shh expression (right column) of (A) weak Tg-Hoxd12 line (identical to wild type, +/+), (B) Gli3^+/-, (C) Tg-Hoxd12;Gli3^+/-, (D) Gli3^-/- and (E) Tg-Hoxd12;Gli3^-/- embryos. Hindlimb long bones (fe, femur; ti, tibia; fi, fibula) and digits (I-V) are marked for Tg-Hoxd12. Extra digits (^*) with distinct identities are marked for Gli3^+/- and Tg-Hoxd12;Gli3^+/-. Anterior is top, posterior bottom, for all panels except column 2 (anterior right, posterior left). Gli3^+/- (B) have only an extra digit I (arrow), whereas Tg-Hoxd12;Gli3^+/- (C) have more extensive polydactyly with posterior transformations and very distinct digit identities. By contrast, polydactyly in Tg-Hoxd12;Gli3^-/- (E) is unchanged from Gli3^-/- (D); both have 7-9 forelimb and 5-7 hindlimb digits that are all short and predominantly digit I-like (see also Fig. 2). Note that in some cases the posterior-most Gli3^-/- digits show variable cartilage staining in an otherwise clear, amorphous region that is suggestive of a rudimentary third (middle) phalanx formation (e.g. D,E). In other instances (e.g. Fig. 2F), such rudiments are completely absent from all digits. Unlike digit phenotypes, long bone shortening worsens progressively, and is severest in Tg-Hoxd12;Gli3^-/-. Normal Shh expression (E11.5-12, right column) in Tg-Hoxd12 (A) and Gli3^+/- (B) is lost by E12.5, whereas some Tg-Hoxd12;Gli3^+/- (C) have broad, deregulated Shh at E12. By contrast, Tg-Hoxd12;Gli3^-/- (D) and Gli3^-/- (E) both show only focal ectopic Shh (arrow) at E12.5.