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. 2014 Jun;21(6):978-89.
doi: 10.1038/cdd.2014.20. Epub 2014 Mar 14.

Dysregulation of Wnt inhibitory factor 1 (Wif1) expression resulted in aberrant Wnt-β-catenin signaling and cell death of the cloaca endoderm, and anorectal malformations

R C-L Ng  1 , D Matsumaru  2 , A S-H Ho  3 , M-M Garcia-Barceló  1 , Z-W Yuan  4 , D Smith  5 , L Kodjabachian  6 , P K-H Tam  1 , G Yamada  2 , V C-H Lui  1
Affiliations

Dysregulation of Wnt inhibitory factor 1 (Wif1) expression resulted in aberrant Wnt-β-catenin signaling and cell death of the cloaca endoderm, and anorectal malformations

R C-L Ng et al. Cell Death Differ. 2014 Jun.

Abstract

In mammalian urorectal development, the urorectal septum (urs) descends from the ventral body wall to the cloaca membrane (cm) to partition the cloaca into urogenital sinus and rectum. Defective urs growth results in human congenital anorectal malformations (ARMs), and their pathogenic mechanisms are unclear. Recent studies only focused on the importance of urs mesenchyme proliferation, which is induced by endoderm-derived Sonic Hedgehog (Shh). Here, we showed that the programmed cell death of the apical urs and proximal cm endoderm is particularly crucial for the growth of urs during septation. The apoptotic endoderm was closely associated with the tempo-spatial expression of Wnt inhibitory factor 1 (Wif1), which is an inhibitor of Wnt-β-catenin signaling. In Wif1(lacZ/lacZ) mutant mice and cultured urorectum with exogenous Wif1, cloaca septation was defective with undescended urs and hypospadias-like phenotypes, and such septation defects were also observed in Shh(-/-) mutants and in endodermal β-catenin gain-of-function (GOF) mutants. In addition, Wif1 and Shh were expressed in a complementary manner in the cloaca endoderm, and Wif1 was ectopically expressed in the urs and cm associated with excessive endodermal apoptosis and septation defects in Shh(-/-) mutants. Furthermore, apoptotic cells were markedly reduced in the endodermal β-catenin GOF mutant embryos, which counteracted the inhibitory effects of Wif1. Taken altogether, these data suggest that regulated expression of Wif1 is critical for the growth of the urs during cloaca septation. Hence, Wif1 governs cell apoptosis of urs endoderm by repressing β-catenin signal, which may facilitate the protrusion of the underlying proliferating mesenchymal cells towards the cm for cloaca septation. Dysregulation of this endodermal Shh-Wif1-β-catenin signaling axis contributes to ARM pathogenesis.

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Figures

Figure 1
Figure 1
Descent of the urorectal septum and regression of the cloaca membrane during cloaca septation. Schematic diagram of cloaca development showing the descent of urs and regression of proximal cm leads to anal opening after cloaca septation (a–c). Normal cloaca development in rat embryos from E14.5 to E16.5 (a′–c′). Rat treated with ethylenethiourea displayed persistent cloaca at E14.5 (d; n=12), hypospadias-like phenotypes with incomplete cloaca septation at E15.5 (e; n=15) and E16.5 (f); n=9). Scale bar: 50 μm
Figure 2
Figure 2
Wif1 is crucial in urorectal development. Normally developed genital tubercle of Wif1lacZ/lacZ embryos at E12.5 (a) and E14.5 (b, c). Defective Wif1lacZ/lacZ embryos displayed perturbation of GT outgrowth and malformed genital tubercle at E12.5 (d) and E14.5 (e). Ventral view of the malformed GT at E14.5 showed the unseptated cloaca with a large hollow space and degraded ventral cloaca membrane (f)
Figure 3
Figure 3
Complementary expression pattern of Wif1 and Shh in the cloaca endoderm. Wif1 expression (brown) at the tip of the urs endoderm and the proximal cloaca membrane epithelium was increased from E11 (a) to E12.5 (b) before septation but reduced drastically at E13.5 (c). Shh expression (purple) in the cloaca endoderm and rectum endoderm showed a complementary expression pattern with Wif1. Shh was expressed at the entire cloaca endoderm by E11 (d). Shh was expressed at a very low level in urs and cloaca membrane endoderm, whereas Wif1 was expressed strongly at E12.5 (e) and E13.5 (f). Nuclear β-catenin immuno-reactivity (arrowheads) was localized in the apical urs endoderm when Wif1 expressed weak at E11 (g and g′). At E12.5 and E13.5, nuclear β-catenin immuno-reactivity was only detected in the basal urs endoderm (arrowheads) (h, h′ and i, i′) but not in the apical urs endoderm (h′′ and i′′). 3D reconstruction of Wif1 expression domains at E12.5 showed Wif1 expression in the midline of the septating cloaca (j and j′). Coronal sections of the distal and proximal region of the genital tubercle showed that Wif1 was not expressed in the distal midline ugs endoderm (k) but in the proximal midline endoderm (l). Expression of Shh was detected in the entire distal ugs endoderm (m), but its expression was absent in the proximal midline endoderm (n). White arrowheads indicate the plane of coronal sections. Regions highlighted were magnified as shown. Scale bar: 50 μm
Figure 4
Figure 4
In vivo and in vitro deletion of Shh elevated Wif1 expression. Wif1 was weakly expressed at E11.5 in control littermates (a). Deletion of Shh induced precocious Wif1 expression in the cloaca membrane endoderm and cloaca canal endoderm at E11.5 (b). By E12.5, a similar expression pattern of Wif1 was observed in both control (c) and Shh mutant embryos (d). However, the cm in Shh mutant was much thinner than the control embryo. Both cytoplasmic and nuclear β-catenin immunoreactivity decreased markedly in Shh mutant embryo (f) as compared with the control littermate (e). In vitro inhibition of Shh signaling by cyclopamine induced ectopic expression of Wif1 in the entire cloaca endoderm and surface ectoderm of the GT explant (h; n=5) compared with the control (g). Scale bar: 50 μm
Figure 5
Figure 5
Exogenous Wif1 suppressed Wnt-β-catenin signaling in the developing midline urorectum. β-gal-positive cells in TopgallacZ/+ embryos were localized at the distal urethral epithelium at E12.5 (a) and E13.5 (b). GT explant from the TopgallacZ/+ embryos at E12 were cultured for 24 h with increasing concentrations of Wif1 protein. The number of β-gal-positive cells (arrowheads) decreased in explants cultured with higher concentration of Wif1 proteins (c). GT from the E12 TopgallacZ/+ embryos cultured for 36 h with exogenous Wif1 (1.5  μg/ml) displayed cloaca membrane disintegration and un-septated cloaca (e) compared with the control (d). Scale bar: 50 μm
Figure 6
Figure 6
Wif1 colocalized with apoptotic endoderm cells during cloaca septation. AntiWif1 staining (Red) and TUNEL staining (Green) were colocalized in the urs tip endoderm and proximal cloaca membrane epithelium of the septating cloaca from E11.5 to E12.5 (a–h). The number of apoptotic cells decreased after cloaca septation together with the decrease in Wif1 expression level in the cloaca endoderm at E13.5 (i–l). Scale bar: 50 μm
Figure 7
Figure 7
Elevation of Wif1 expression increased cell apoptosis in the cloaca endoderm through Wnt-β-catenin signaling. Wif1 expression (Red) associated with cell apoptosis (Green) in wild-type embryo (a). Ectopic Wif1 expression domain (Red) in the cloaca membrane and at the tip of the urs colocalized with apoptotic cells (Green) in E11.5 ShhcreERT2/− mice (b). The cm was still intact at E13.5 in control embryos (c), whereas the cm was completely degraded at E13.5 resulting in perineal urethra in ShhcreERT2/− embryos (d). Asterisk indicates the absence of cm. Apoptotic cells (Green) in E11.5 wild-type control embryos (e), in mutant embryos with endodermal deletion of β-catenin (f) or with endodermal induction of constitutive active β-catenin (g) were localized by TUNEL. Scale bar: 50 μm
Figure 8
Figure 8
A model of signaling crosstalk regulating Wif1 and urs development. Evidence indicated that Wif1 is negatively regulated by Shh in the urs endoderm. The expression of Wif1 modulates the activity of Wnt-β-catenin signaling to control cell survival of the apical urs endoderm and cloaca membrane endoderm (a). A hypothetical mechanistic model to indicate how Wif1 facilitates the urs descent and cm degradation during cloaca septation (b–f)
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