Seifert et al Fgf8 Dev2009

Seifert et al Fgf8 Dev2009 - RESEARCH ARTICLE 2643...

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2643 RESEARCH ARTICLE INTRODUCTION The genital tubercle is the developmental precursor to the male and female external genitalia. Its outgrowth and patterning have been likened to the vertebrate limb bud, but unlike the limb, the genital tubercle is composed of cells from all three germ layers (Seifert et al., 2008). Initiation of genital outgrowth begins at approximately E10.5, when paired genital swellings appear on either side of the cloacal membrane (Perriton et al., 2002). The paired swellings are joined by an anterior swelling a day later and collectively these form the genital tubercle. Signaling between the endoderm of the embryonic cloaca (which gives rise to the urethral epithelium) and the surrounding mesoderm is necessary for outgrowth and patterning of the genital tubercle. Mice lacking both copies of the sonic hedgehog ( Shh ) gene initiate outgrowth of the paired swellings, but these fail to form a genital tubercle and the mice exhibit complete agenesis of the external genitalia (Haraguchi et al., 2001; Perriton et al., 2002). Although Shh is required for the maintenance of genital outgrowth, it is not the early initiation signal. In the vertebrate limb bud, fibroblast growth factors (Fgfs) 8 and 10 mediate the initiation of budding, and sustained outgrowth of the limb is controlled by Fgf signaling from the apical ectodermal ridge (AER) (Mariani and Martin, 2003; Ng et al., 1999). In the genital tubercle, Fgf8 is expressed in the distal urethral epithelium (Haraguchi et al., 2000; Perriton et al., 2002). Previous work identified Fgf8 as a genital outgrowth signal and compared its role to that during limb development (Haraguchi et al., 2000). It was reported that removal of the distal urethral epithelium causes the arrest of genital outgrowth in organ culture, and that application of Fgf8-loaded beads can restore (or at least augment) gene expression and tubercle outgrowth (Haraguchi et al., 2000). Furthermore, treatment of cultured tubercles with Fgf8- neutralizing antibody may inhibit development (Haraguchi et al., 2000). This and subsequent studies led to the suggestion that Fgf8 expression in the distal urethral epithelium is required for outgrowth of the genitalia (Haraguchi et al., 2000; Haraguchi et al., 2001; Haraguchi et al., 2007; Morgan et al., 2003; Ogino et al., 2001; Perriton et al., 2002; Satoh et al., 2004; Suzuki et al., 2003; Suzuki et al., 2008; Yamada et al., 2006). Although Fgf8 is widely held to be the outgrowth signal, its function in genital development has not been examined genetically, in part because Fgf8 null embryos die prior to genital tubercle initiation (Meyers et al., 1998). Here we provide a direct test of the hypotheses that Fgf8 is required for initiation, outgrowth and normal development of the external genitalia. MATERIALS AND METHODS Mice Mouse strains used in this study have been described previously: Shh GFPcre (Harfe et al., 2004); Fgf8 fl/fl (Lewandoski et al., 2000), Fgf4 fl/fl (Sun et al., 2000) and Wnt5a –/– (Yamaguchi et al., 1999). We generated mice lacking Fgf8 in the genital tubercle by crossing Shh GFPcre ;Fgf8 fl/+ males to Fgf8 fl/fl females, and denote these mice as Fgf8 cKO
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