Fig. 5. Gene expression and patterns of cell death in developing 4C-BAC16 limbs. (A) Identical pattern of Shh expression in 4C-BAC16 mutant and wild-type limb buds. White arrow indicates the anterior part of mutant hind limb. (B-E) Expression patterns of Hoxd13 in the right hind limbs of 2C (B,D) and 4C (C,E) BAC16 mutants. 2C-BAC16 expression patterns resemble wild type (not shown). Dashed lines (B,C) represent the expected normal posterioranterior limit of Hoxd13 expression in developing hind limb buds; limb anterior is towards the bottom of photograph. (F) Medial and (G,H) anterior views of 2C (G) and 4C (H) E11.5 hind limb buds, showing a reduction in Dkk1 expression in the anterior two-thirds of the AER (region between the open arrowhead and black arrowhead) in the 4C-BAC16 limb. The 2C pattern resembles wild type (not shown) and `I' marks the anterior side of each limb corresponding to where digit I develops. Arrowheads point to the ANZ, where Dkk1 expression is completely quenched in the 4C limb bud. (I) Dorsal view of left and right hind limbs (LHL and RHL) from a 4C-BAC16 mutant stained with Neutral Red. The cell death pattern in this 4C-LHL resembles that of wild type (not shown). `I' marks the position of developing digit I condensates and the arrow indicates the absence of Neutral Red-positive cells in the abnormal (larger digit I) right hind limb, compared with the presence of such cells in the corresponding region in the normal left hind limb. Also note the similar, unperturbed, patterns of Neutral Red staining at the posterior margins of each limb. (J) Comparison of Wnt5a expression in wild-type and 4C-mutant embryos shows a higher Wnt5a signal in both fore and hind limbs of 4C-mutants, when compared with the wild type, particularly in the region generating digit I in the hind limb buds (arrows). Scale bars: 400 µm in F,I; 600 µm in B,D; 1.0 mm in J; 1.2 mm in A.