Bmp4 is an essential growth factor for the initiation of genital tubercle (GT) outgrowth

The external genitalia are appendage organs outgrowing from the posterior body trunk. Murine genital tubercle (GT), anlage of external genitalia, initiates its outgrowth from embryonic day (E) 10.5 as a bud structure. Several growth factors such as fibroblast growth factor (FGF), Wnt and Sonic hedgehog (Shh) are essential for the GT outgrowth. However, the mechanisms of initiation of GT outgrowth are poorly understood. We previously identified bone morphogenetic protein (Bmp) signaling as a negative regulator for GT outgrowth. We show here novel aspects of Bmp4 functions for GT outgrowth. We identified the Bmp4 was already expressed in cloaca region at E9.5, before GT outgrowth. To analyze the function of Bmp4 at early stage for the initiation of GT outgrowth, we utilized the Hoxa3-Cre driver and Bmp4 ^flox/flox mouse lines. Hoxa3 ^Cre/+ ; Bmp4 ^flox/flox mutant mice showed the hypoplasia of GT with reduced expression of outgrowth promoting genes such as Wnt5a, Hoxd13 and p63, whereas Shh expression was not affected. Formation of distal urethral epithelium (DUE) marked by the Fgf8 expression is essential for controlling mesenchymal genes expression in GT and subsequent its outgrowth. Furthermore, Fgf8 expression was dramatically reduced in such mutant mice indicating the defective DUE formation. Hence, current results indicate that Bmp4 is an essential growth factor for the initiation of GT outgrowth independent of Shh signaling. Thus, Bmp4 positively regulates for the formation of DUE. The current study provides new insights into the function of Bmp signaling at early stage for the initiation of GT outgrowth.