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Fig. 1. Experimental strategy for identifying Hoxd-regulated genes in developing genitalia and forelimbs. (A) In situ hybridization of a wild-type E12.5 embryo stained with a Hoxd13 probe illustrates the tissues dissected for this study. Hoxd13 is strongly and broadly expressed in the genital bud (right) and distal forelimb (left). Genital buds and distal forelimb buds were dissected (arrows indicate cut points) from E12.5 wild-type (WT) embryos and from homozygous mutant embryos in which all nine Hoxd genes had been deleted (HoxD^Del1-13). (B) Gene expression was analyzed with Affymetrix microarrays (each array is represented schematically as a circle; see Materials and methods). (C) Microarray analyses yielded two sets of initially non-overlapping differentially-expressed candidate genes from limbs and genitals. A subset of the candidates (16 genes) was validated by quantitative real-time RT-PCR analysis. (D) Expression of the 16 candidate genes was measured by real-time RT-PCR over developmental time from secondary samples collected from E11.5 to E14.5. Nine out of 14 confirmed genes also had highly significant differential expression in the other tissue on at least one day of development. (E) The transcript profiles of the 14 genes were visualized by whole-mount in situ hybridization (WISH), in several cases further validating their differential expression.

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