Extended Data Figure 2.

The squamate hemipenis mesenchyme initiates with limb-like cellular dynamics from the coelomic epithelium through a epithelial-to-mesenchymal transition (EMT) a) Injection of GFP-expressing lentiviruses into the coelom of chicken embryos at HH14 labels cells emerging from the epithelium that contribute to the hindlimb mesenchyme (arrowhead). b) In lizards, labeled cells leaving the coelomic epithelium contribute to the hemipenis mesenchyme (arrowheads). c) Dorsal view of the hindlimb region of an E10.0 mouse embryo. d) Transversal section of a limb bud, showing EMT of the coelomic epithelium (diffuse Laminin staining, empty arrowhead), as cells contribute to the limb-bud mesenchyme. e) Dorsal view of the budding hemipenis of a snake embryo, one day after egg deposition. f) Transversal section of the hemipenis region. The basement membrane of the coelomic epithelium is breaking down (empty arrowhead), while it is intact for both the nephric duct and the surface ectoderm (arrowheads). g-o) Expression of genitalia and limb genes during hemipenis initation. g-i) Tbx4 is expressed early and late during hemipenis initiation, in both the coelomic epithelium (arrowhead) and the hemipenis mesenchyme (arrow). j-l) Tbx5 is only expressed later, in the mesenchyme (arrow), but is absent from the coelomic epithelium (empty arrowheads). m-o) Limb marker gene Lhx9 (see also Fig. 4e) is absent from both epithelium (empty arrowhead) and mesenchyme (empty arrow), but can de detected in dI1 neurons (asterisk). All gene expression assessed in at least n=3. Scale bars, 50 μm. co: coelom; lb: limb; hp: hemipenis; nd: nephric duct; cl: cloaca.