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Ethylene thiourea (ETU), a metabolite of the fungicide ethylene bisdithiocarbamate (EBDC), has received great concern because of its harmful effects. ETU-induced anorectal malformations (ARMs) in rat models have been reported and widely used in the study of ARMs embryogenesis. Dysplasia of the lumbosacral spinal cord (LSSC), pelvic floor muscles (PFMs), and hindgut (HG) during intrauterine life affects postoperative defecation in patients with ARMs. However, the underlying toxic effects of ETU and pathological mechanisms in the three defecation-related tissues of fetuses with ARMs have not been reported. Thus, this study aimed to elucidate the molecular mechanisms involved in ARMs, with a focus on the dysregulation of miR-200b-3p and its downstream target tropomodulin 3 (TMOD3). The mRNA and protein levels of miR-200b-3p and TMOD3 in LSSC, PFMs, and HG of fetal rats with ARMs were evaluated by reverse transcription quantitative polymerase chain reaction and Western blotting (WB) on embryonic day 17 (E17). Further, a dual-luciferase reporter assay confirmed their targeting relationship. Gene silencing and overexpression of miR-200b-3p and TMOD3 were performed to verify their functions in HEK-293 T cells. Fetal rats with ARMs also received intra-amniotic microinjection of Ad-TMOD3 on E15, and key molecules in nuclear factor kappa (NF-κB) signaling and apoptosis were evaluated by WB on E21. Abnormally high levels of miR-200b-3p inhibited TMOD3 expression by binding with its 3′-untranslated region, leading to the activation of the non-canonical NF-κB signaling pathway, which is critical in the maldevelopment of LSSC, PFMs, and HG in ARMs rats. Furthermore, miR-200b-3p triggered apoptosis by directly targeting TMOD3. Notably, intra-amniotic Ad-TMOD3 microinjection revealed that the upregulation of TMOD3 expression mitigates the effects of miR-200b-3p on the activation of non-canonical NF-κB signaling and apoptosis in fetal rat model of ARMs. A novel miR-200b-3p/TMOD3/non-canonical NF-κB signaling axis triggered the massive apoptosis in LSSC, PFMs, and HG of ARMs, which was restored by the intra-amniotic injection of Ad-TMOD3 during embryogenesis. Our results indicate the potential of TMOD3 as a treatment target to restore defecation.

Anorectal malformations (ARMs) are congenital diseases that lead to postoperative fecal incontinence, constipation, and soiling, despite improvements in surgery; however, their pathological mechanisms remain unclear. Here, we report the role of microRNA-141-3p in maintaining homeostasis between apoptosis and autophagy in the lumbosacral defecation center of fetal rats with ARMs. Elevated microRNA-141-3p expression inhibited YIN-YANG-1 expression by binding its 3′ UTR, and repressed autophagy and triggered apoptosis simultaneously. Then, adenylate cyclase 3 was screened to be the downstream target gene of YIN-YANG-1 by chromatin immunoprecipitation sequencing experiments, and Yin Yang 1 could positively activate the transcription of adenylate cyclase 3 by directly interacting with the motif GAGATGG and ATGG in its promoter. Intraamniotic microinjection of adeno-rno-microRNA-141-3p-sponge-GFP in fetal rats with ARMs on embryonic day 15 restored apoptosis-autophagy homeostasis. These findings reveal that microRNA-141-3p upregulation impaired homeostasis between apoptosis and autophagy by inhibiting the YIN-YANG-1/adenylate cyclase 3 axis, and that intraamniotic injection of anti-microRNA-141-3p helped maintain homeostasis in the lumbosacral defecation center of ARMs during embryogenesis.

The discovery of N6-methyladenosine (m6A) methylation and its role in translation has led to the emergence of a new field of research. Despite accumulating evidence suggesting that m6A methylation is essential for the pathogenesis of cancers and aging diseases by influencing RNA stability, localization, transformation, and translation efficiency, its role in normal and abnormal embryonic development remains unclear. An increasing number of studies are addressing the development of the nervous and gonadal systems during embryonic development, but only few are assessing that of the immune, hematopoietic, urinary, and respiratory systems. Additionally, these studies are limited by the requirement for reliable embryonic animal models and the difficulty in collecting tissue samples of fetuses during development. Multiple studies on the function of m6A methylation have used suitable cell lines to mimic the complex biological processes of fetal development or the early postnatal phase; hence, the research is still in the primary stage. Herein, we discuss current advances in the extensive biological functions of m6A methylation in the development and maldevelopment of embryos/fetuses and conclude that m6A modification occurs extensively during fetal development. Aberrant expression of m6A regulators is probably correlated with single or multiple defects in organogenesis during the intrauterine life. This comprehensive review will enhance our understanding of the pivotal role of m6A modifications involved in fetal development and examine future research directions in embryogenesis.