A relative shift in cloacal location repositions external genitalia in amniote evolution

The move of vertebrates to a terrestrial lifestyle required major adaptations in their locomotory apparatus and reproductive organs. While the fin-to-limb transition has received considerable attention(1,2), little is known about the developmental and evolutionary origins of external genitalia. Similarities in gene expression have been interpreted as a potential evolutionary link between the two anatomical structures(3-6), yet without providing any underlying developmental mechanism. Here, we have reexamined this question using micro-Computed Tomography (μCT), lineage tracing in three amniote clades and RNA-Seq transcriptional profiling. We show that the developmental origin of external genitalia has shifted through evolution, and in some taxa limbs and genitals share a common primordium. In squamates, the genitalia develop directly from the budding hindlimbs, or the remnants thereof, whereas in mice the genital tubercle originates from the ventral and tail bud mesenchyme. The recruitment of different cell populations for genital outgrowth follows a change in the relative position of the cloaca, the genitalia organizing center. Ectopic grafting of the cloaca demonstrates the conserved ability of different mesenchymal cells to respond to these genitalia-inducing signals. Our results support a limb-like developmental origin of external genitalia as the ancestral condition. Moreover, it suggests that a change in the relative position of the cloacal signaling center during evolution has led to an altered developmental route of external genitalia in mammals, while preserving parts of the ancestral limb molecular circuitry due to a common evolutionary origin.