Delineating a Conserved Genetic Cassette Promoting Outgrowth of Body Appendages

Abstract

The acquisition of the external genitalia allowed mammals to cope with terrestrial-specific reproductive needs for internal fertilization, and thus it represents one of the most fundamental steps in evolution towards a life on land. How genitalia evolved remains obscure, and the key to understanding this process may lie in the developmental genetics that underpins the early establishment of the genital primordium, the genital tubercle (GT). Development of the GT is similar to that of the limb, which requires precise regulation from a distal signaling epithelium. However, whether outgrowth of the GT and limbs is mediated by common instructive signals remains unknown. In this study, we used comprehensive genetic approaches to interrogate the signaling cascade involved in GT formation in comparison with limb formation. We demonstrate that the FGF ligand responsible for GT development is FGF8 expressed in the cloacal endoderm. We further showed that forced Fgf8 expression can rescue limb and GT reduction in embryos deficient in WNT signaling activity. Our studies show that the regulation of Fgf8 by the canonical WNT signaling pathway is mediated in part by the transcription factor SP8. Sp8 mutants elicit appendage defects mirroring WNT and FGF mutants, and abolishing Sp8 attenuates ectopic appendage development caused by a gain-of-function β-catenin mutation. These observations indicate that a conserved WNT-SP8-FGF8 genetic cassette is employed by both appendages for promoting outgrowth, and suggest a deep homology shared by the limb and external genitalia. Mammalian limbs and external genitalia are body appendages specialized for locomotion and internal fertilization, respectively. Despite their marked anatomical and functional differences, development of the limb and external genitalia appears to involve similar genetic controls, and some have suggested that regulatory mechanisms common to both might be evolutionarily linked. One essential aspect for appendage development is the establishment and maintenance of a separated proximodistal developmental axis apart from the main body axis, which is often instructed by a distal signaling epithelium. Herein, we adopted comprehensive mouse genetic approaches to investigate regulatory mechanisms underlying the distal signaling center in the limb and the GT, and uncovered a conserved genetic cassette that is utilized by both paired and unpaired appendages to establish a distal signaling center in the epithelium that mediates subsequent proximodistal outgrowth. Our results further suggested that the evolution of the external genital organ involved co-option of the same genetic program underpinning limb development.