Figure 4.

The cloacal signaling center can recruit different mesenchymal cell populations for the outgrowth of external genitalia. a) Schematic of the hindlimb grafting procedure in chicken embryos. GFP-transgenic cloacae are transplanted into the proximal-ventral portion wild-type hindlimbs. b,c) Ectopic outgrowth (arrowheads) on limbs with cloacal grafts (n=30/118). d) Venn diagram of pairwise differential expression analysis results (log(fold change) > 1.5, p-value < 0.05) of limbs versus genital tissues, for early and late budding stages in anole and mouse. e) Heat map of Z-score normalized values of core 25 genes showing consistent differential expression between limbs and genitalia. Mouse row-based hierarchical clustering was re-used for anole samples. f-h) Analysis of limb- and genital-specific markers. Expression of limb markers Lhx9 (f) and Tbx18 (g) is down-regulated near the GFP-positive cloacal graft (empty arrowheads), while genital marker Isl1 (h) is expressed ectopically (arrowhead). i) Schematic of the tail bud grafting procedure in chicken embryos. GFP-transgenic cloacae are transplanted into ventral wild-type tailbuds. j) Ectopic outgrowth on tails with cloacal grafts (n=16/87). k) Genital marker Isl1 is up-regulated ectopically in the tail, close to the GFP-positive cloacal graft (arrowhead). All gene expression assessed in at least n=3. Scale bars, 200 μm.