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.

Development of the External Genitalia and Their Sexual Dimorphic Regulation in Mice

“…Recently, FGF8 is suggested to be the signal output of WNT signaling during GT budding [Lin et al, 2013]. The loss of CTNNB results in GT agenesis which is partially rescued by overexpression of Fgf8 [Lin et al, 2013]. This confirms that FGF8 still serves as a significant indicator of protrusion and sustained outgrowth of GT.…”

“…Its importance during GT development is evident as early loss of Shh results in GT agenesis [Haraguchi et al, 2001;Perriton et al, 2002;Lin et al, 2009;Miyagawa et al, 2009a]. WNT/CTNNB (beta-catenin) signaling is also observed in the ectoderm, endoderm, and the mesoderm of the GT, and mutations of Ctnnb are associated with protrusion defects [Lin et al, 2008[Lin et al, , 2013Miyagawa et al, 2009a].…”

“…However, the functional relevance of FGF8 still needs to be elucidated. Recently, FGF8 is suggested to be the signal output of WNT signaling during GT budding [Lin et al, 2013]. The loss of CTNNB results in GT agenesis which is partially rescued by overexpression of Fgf8 [Lin et al, 2013].…”

Deep homology in the age of next-generation sequencing

“…Combining next-generation transcriptomic analyses with comparative embryological and functional experiments can inform us about the developmental mechanisms that lead to the appearance of deep homology. For example, such was the motivation in studying the evolution of genital bud transcriptomes in amniotes, an embryonic structure that was known to share substantial gene expression similarities with developing limbs [56,57]. Comparative lineage tracing experiments uncovered considerable variation in the embryonic origins of amniote external genitalia [52].…”

Battle of sex hormones in genitalia anomalies

“…By E11.5, the two swellings have merged and formed a single GT (4,5). The GT and limb bud use a common genetic cassette to promote their outgrowth, which involves a signaling center containing Wnt and Fgf activities (6). However, the signaling center in the GT is the endodermally derived distal urethral epithelium, in contrast to the ectodermally derived apical ectodermal ridge in limbs (4).…”