Differential expression of Wnt inhibitors Dickkopf-1 (Dkk-1) and Wnt inhibitory factor-1 (Wif1) in the regulation of urorectal development

Abstract

In mammals, the external genitalia, urinary tract and anorectal tract are developed from a common embryonic primordium, the urorectum. Cloaca is the hollow space inside the urorectum that connects the hindgut and the urogenital sinus. During the urorectal development, the external genitalia is formed from the outgrowth of genital tubercle (GT) protruding from the urorectum, while the future urinary tract and anorectal tract are formed by the partition of cloaca during cloacal septation. GT outgrowth and cloacal septation are important developmental events for the formation of genitourinary and anorectal system. In human, dysregulation of these developmental events results in congenital anorectal malformations (ARM).

Wnt signaling is one of the key signaling pathways that regulates urorectal development. The activity of Wnt signaling is initiated by the binding of Wnt ligands to cell surface receptors, which can be antagonized by secretory Wnt inhibitors. Dickkopf1 (Dkk1) and Wnt inhibitory factor 1 (Wif1) are secretory Wnt inhibitors implicated in urorectal development. However, the functions of other secretory Wnt inhibitors during urorectal developments remain to be elucidated.

In this study, expression analyses showed that Dkk1, Dickkopf2 (Dkk2), Dickkopf4 (Dkk4), Secreted Frizzled-related Protein 1 (Sfrp1) and Wif1 were expressed in the developing urorectum. The dynamic, overlapping and restricted expression patterns of these Wnt inhibitors were closely associated with the GT outgrowth and the cloacal septation events, implying that these Wnt inhibitors functioned in a coordinated manner in defining the field of Wnt signaling activities in the developing urorectum.

Wif1 knockout mice (〖Wif1〗^(-/-)) was used as the model to investigate the functions of and the interplay between secretory Wnt inhibitors in urorectal development. GT outgrowth and cloacal septation defects were observed in 〖Wif1〗^(-/-) embryos. Most of the 〖Wif1〗^(-/-) embryos displayed varying degrees of GT outgrowth defects, while septation defects were only occasionally observed. This suggested that GT outgrowth and cloacal septation were regulated by Wif1 via different regulatory mechanisms.

In the urorectum of 〖Wif1〗^(-/-) embryos, Dkk1 was significantly upregulated in the peri-cloacal mesenchyme. Further expression analysis suggested that Dkk1 was sufficient to rescue cloacal septation defects but not GT outgrowth defects in 〖Wif1〗^(-/-)embryos. In the 〖Wif1〗^(-/-) embryos with severe GT outgrowth defects, the Fgf8-expressing distal urethral epithelium, the signaling center in the urorectum, was absent, suggesting that the GT outgrowth defects could be contributed by the loss of dUE-expressing signals such as Fgf8. This study demonstrated the importance of secretory Wnt inhibitors in the GT outgrowth and cloacal septation and suggested that secretory Wnt inhibitors played partially overlapping roles in urorectal development. A rescue mechanism for cloacal septation performed by Dkk1 upon Wif1 deletion was proposed. Such auto-regulatory mechanism within the Wnt signaling pathway indicated that Wnt inhibitors play essential regulatory roles in the urorectal development and a balanced Wnt signaling activity modulated by Wnt inhibitors is crucial to the development of urorectum.