Functional and phylogenetic analysis shows that Fgf8 is a marker of genital induction in mammals but is not required for external genital development

In mammalian embryos, male and female external genitalia develop from the genital tubercle. Outgrowth of the genital tubercle is maintained by the urethral epithelium, and it has been reported that Fgf8 mediates this activity. To test directly whether Fgf8 is required for external genital development, we conditionally removed Fgf8 from the cloacal/ urethral epithelium. Surprisingly, Fgf8 is not necessary for initiation, outgrowth or normal patterning of the external genitalia. In early genital tubercles, we found no redundant Fgf expression in the urethral epithelium, which contrasts with the situation in the apical ectodermal ridge (AER) of the limb. Analysis of Fgf8 pathway activity showed that four putative targets are either absent from early genital tubercles or are not regulated by Fgf8. We therefore examined the distribution of Fgf8 protein and report that, although it is present in the AER, Fgf8 is undetectable in the genital tubercle. Thus, Fgf8 is transcribed, but the signaling pathway is not activated during normal genital development. A phylogenetic survey of amniotes revealed Fgf8 expression in genital tubercles of eutherian and metatherian mammals, but not turtles or alligators, indicating that Fgf8 expression is neither a required nor a conserved feature of amniote external genital development. The results indicate that Fgf8 expression is an early readout of the genital initiation signal rather than the signal itself. We propose that induction of external genitalia involves an epithelial-epithelial interaction at the cloacal membrane, and suggest that the cloacal ectoderm may be the source of the genital initiation signal.