Abstract
The study objective was to investigate the role of fibroblast growth factor 9 (Fgf9) in normal and anorectal malformation (ARM) embryos during the development of the anorectum. Fgf9 expression was assayed in both normal rat embryos and embryos with ARM induced by exposure to ethylenethiourea (ETU) on embryonic day 10 (E10). Fgf9 expression was assayed by immunohistochemical staining, Western blotting, and real-time quantitative polymerase chain reaction (qRT-PCR). Immunohistochemical staining revealed spatiotemporal changes in Fgf9 expression between E13 and E16. Fgf9-positive cells predominated in the mesenchyme of the cloaca on E13 and E14 and at the fusion site of the urorectal septum and cloacal membrane, rectal epithelium, and anal membrane on E15. Fgf9-positive cells were obviously decreased after the anal membrane ruptured on E16. Fgf9-positive staining was significantly decreased in embryos with ARM compared with normal embryos from E13 to E15. The results of Western blots and qRT-PCR were consistent, with significantly increased Fgf9 expression in the hindgut and rectum of normal embryos than in embryos with ARM from E13 to E15. However, there was no difference between the two groups on E16. These results suggested that the anorectal embryogenesis might depend on the induction of Fgf9 signal. The expression of Fgf9 was downregulated in ETU-induced ARM embryos, which might be related to the development of ARM.
Highlights
Anorectal malformations (ARM) are congenital malformations that occur in approximately 1 in 2500–3500 live births worldwide [1, 2] and are usually associated with multiple malformations
Abundant fibroblast growth factor 9 (Fgf9)-immunopositive cells were present in the cloacal mesenchyme, and some positive cells were expressed in the epithelium of the hindgut (Figures 1(a) and 1(b))
There are heated debates between scholars who hold different views, it is generally believed that ARM is associated with dysplasia of cloaca and the failure of fusion of urorectal septum (URS) with cloacal membrane (CM) [14, 16]
Summary
Anorectal malformations (ARM) are congenital malformations that occur in approximately 1 in 2500–3500 live births worldwide [1, 2] and are usually associated with multiple malformations. Fgfs comprise a 22-member gene family encoding heparan sulfate-binding proteins and regulator of cell growth and embryonic development following binding to fibroblast growth factor receptors (Fgfrs). Previous studies have documented the abnormal expression of the Fgf and Fgfr2b genes in rat embryos with ARM [10, 11], but the roles of the other members of the Fgf gene family in the formation of ARM have not been studied. In order to reveal the regulation effect of Fgf on ARM, we analyzed the distribution and the expression level of Fgf from E13 to E16 in rat embryos with ethylenethiourea- (ETU-) induced ARM by immunohistochemistry, Western blotting, and real-time quantitative polymerase chain reaction (qRT-PCR)
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