Spatiotemporal expression of Wnt3a during striated muscle complex development in rat embryos with ethylenethiourea-induced anorectal malformations

Abstract

Numerous patients with anorectal malformations (ARMs) continue to experience fecal incontinence and constipation following surgical procedures. One of the most important factors that influences defecation is the striated muscle complex (SMC). Wnt signaling regulates the expression of myogenic regulatory factors, which serve an important role in muscle development. Therefore, the present study aimed to investigate the expression pattern of Wnt3a protein (by immunohistochemistry and western blot analysis) and mRNA [by reverse transcription‑quantitative polymerase chain reaction (RT-qPCR)] in the SMC of ARM model rats that were exposed to ethylenethiourea. Immunostaining revealed that the expression of Wnt3a exhibits space‑ and time‑dependent changes in the developing SMC of ARM model rat embryos. Immunohistochemistry demonstrated that on embryonic day 17 (E17), Wnt3a‑positive cells were observed in the SMC in normal embryos, and expression levels gradually increased as the rat embryos developed. Similar changes in Wnt3a protein expression were detected in ARM model rat embryos; however, the expression of Wnt3a was significantly reduced compared with the normal rat embryos. Western blotting and RT‑qPCR also revealed lower expression levels of Wnt3a protein and mRNA, respectively, in the SMC of ARMs model rat embryos compared with normal rat embryos. These data revealed that the expression of Wnt3a in ARM embryos was notably reduced, indicating a potential role for Wnt3a in the maldevelopment of the SMC in patients with ARMs.

Figure 1.

Wnt3a expression in the SMC of normal and ARM model rats at various embryonic developmental stages. (A) At E17, immunoreactivity specific to Wnt3a was detected in the SMC. (B) At E19, positively-stained cells were mainly localized in the SMC and were observed in the bulbocavernosus muscle. (C) At E21, the Wnt3a immunoreactivity in SMC and bulbocavernosus muscle was higher than at earlier stages. (D) Sporadic positive staining cells could be detected in the SMC on E17. (E) At E19, the SMC were developed poorly, and marginal immunoreactivity specific to Wnt3a was detected in the SMC. (F) Wnt3a-positive cells increased slowly by E21 compared with earlier days. Magnification, ×400; magnification ×40 of figures in the lower left corner. The black arrows indicate SMC; the red arrows indicate the bulbocavernosus muscle. SMC, striated muscle complex; ARM, anorectal malformation; E, embryonic day.

Figure 2.

Western blot and densitometric analysis of Wnt3a protein expression in the striated muscle complex of normal and ARM model rats at various embryonic developmental stages. Wnt3a expression was normalized to β-actin expression in the developing SMC of normal and ARMs rat embryos at E17, E19 and E21. *P<0.05 vs. corresponding control. ARM, anorectal malformation; E, embryonic day.