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MicroRNA-124-3p Plays a Crucial Role in Cleft Palate Induced by Retinoic Acid.

ABSTRACT: Cleft lip with/without cleft palate (CL/P) is one of the most common congenital birth defects, showing the complexity of both genetic and environmental contributions [e.g., maternal exposure to alcohol, cigarette, and retinoic acid (RA)] in humans. Recent studies suggest that epigenetic factors, including microRNAs (miRs), are altered by various environmental factors. In this study, to investigate whether and how miRs are involved in cleft palate (CP) induced by excessive intake of all-trans RA (atRA), we evaluated top 10 candidate miRs, which were selected through our bioinformatic analyses, in mouse embryonic palatal mesenchymal (MEPM) cells as well as in mouse embryos treated with atRA. Among them, overexpression of miR-27a-3p, miR-27b-3p, and miR-124-3p resulted in the significant reduction of cell proliferation in MEPM cells through the downregulation of CP-associated genes. Notably, we found that excessive atRA upregulated the expression of miR-124-3p, but not of miR-27a-3p and miR-27b-3p, in both in vivo and in vitro. Importantly, treatment with a specific inhibitor for miR-124-3p restored decreased cell proliferation through the normalization of target gene expression in atRA-treated MEPM cells and atRA-exposed mouse embryos, resulting in the rescue of CP in mice. Taken together, our results indicate that atRA causes CP through the induction of miR-124-3p in mice.

SUBMITTER: Yoshioka H

PROVIDER: S-EPMC8219963 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:During embryogenesis, a complex interplay between extracellular matrix (ECM) molecules, regulatory molecules, and growth factors mediates morphogenetic processes involved in palatogenesis. Transforming growth factor-beta (TGF-beta), retinoic acid (RA), and gamma-aminobutyric acid (GABA)ergic signaling systems are also potentially involved. Using [3H]glucosamine and [35S]methionine incorporation, anion exchange chromatography, semiquantitative radioactive RT-PCR, and a TGF-beta binding assay, we aimed to verify the presence of phenotypic differences between primary cultures of secondary palate (SP) fibroblasts from 2-year-old subjects with familial nonsyndromic cleft lip and/or palate (CLP-SP fibroblasts) and age-matched normal SP (N-SP) fibroblasts. The effects of RA--which, at pharmacologic doses, induces cleft palate in newborns of many species--were also studied. We found an altered ECM production in CLP-SP fibroblasts that synthesized and secreted more glycosaminoglycans (GAGs) and fibronectin (FN) compared with N-SP cells. In CLP-SP cells, TGF-beta3 mRNA expression and TGF-beta receptor number were higher and RA receptor-alpha (RARA) gene expression was increased. Moreover, we demonstrated for the first time that GABA receptor (GABRB3) mRNA expression was upregulated in human CLP-SP fibroblasts. In N-SP and CLP-SP fibroblasts, RA decreased GAG and FN secretion and increased TGF-beta3 mRNA expression but reduced the number of TGF-beta receptors. TGF-beta receptor type I mRNA expression was decreased, TGF-beta receptor type II was increased, and TGF-beta receptor type III was not affected. RA treatment increased RARA gene expression in both cell populations but upregulated GABRB3 mRNA expression only in N-SP cells. These results show that CLP-SP fibroblasts compared with N-SP fibroblasts exhibit an abnormal phenotype in vitro and respond differently to RA treatment, and suggest that altered crosstalk between RA, GABAergic, and TGF-beta signaling systems could be involved in human cleft palate fibroblast phenotype.

Project description:Retinoic acid (RA) is known to regulate definitive myelopoiesis but its role in vertebrate primitive myelopoiesis remains unclear. Here we report that zebrafish primitive myelopoiesis is restricted by RA in a dose dependent manner mainly before 11 hpf (hours post fertilization) when anterior hemangioblasts are initiated to form. RA treatment significantly reduces expressions of anterior hemangioblast markers scl, lmo2, gata2 and etsrp in the rostral end of ALPM (anterior lateral plate mesoderm) of the embryos. The result indicates that RA restricts primitive myelopoiesis by suppressing formation of anterior hemangioblasts. Analyses of ALPM formation suggest that the defective primitive myelopoiesis resulting from RA treatment before late gastrulation may be secondary to global loss of cells for ALPM fate whereas the developmental defect resulting from RA treatment during 10-11 hpf should be due to ALPM patterning shift. Overexpressions of scl and lmo2 partially rescue the block of primitive myelopoiesis in the embryos treated with 250 nM RA during 10-11 hpf, suggesting RA acts upstream of scl to control primitive myelopoiesis. However, the RA treatment blocks the increased primitive myelopoiesis caused by overexpressing gata4/6 whereas the abolished primitive myelopoiesis in gata4/5/6 depleted embryos is well rescued by 4-diethylamino-benzaldehyde, a retinal dehydrogenase inhibitor, or partially rescued by knocking down aldh1a2, the major retinal dehydrogenase gene that is responsible for RA synthesis during early development. Consistently, overexpressing gata4/6 inhibits aldh1a2 expression whereas depleting gata4/5/6 increases aldh1a2 expression. The results reveal that RA signaling acts downstream of gata4/5/6 to control primitive myelopoiesis. But, 4-diethylamino-benzaldehyde fails to rescue the defective primitive myelopoiesis in either cloche embryos or lycat morphants. Taken together, our results demonstrate that RA signaling restricts zebrafish primitive myelopoiesis through acting downstream of gata4/5/6, upstream of, or parallel to, cloche, and upstream of scl.

Project description:BACKGROUND:Cleft lip (CL), one of the most common congenital birth defects, shows considerable geographic and ethnic variation, with contribution of both genetic and environmental factors. Mouse genetic studies have identified several CL-associated genes. However, it remains elusive how these CL-associated genes are regulated and involved in CL. Environmental factors may regulate these genes at the post-transcriptional level through the regulation of non-coding microRNAs (miRNAs). In this study, we sought to identify miRNAs associated with CL in mice. RESULTS:Through a systematic literature review and a Mouse Genome Informatics (MGI) database search, we identified 55 genes that were associated with CL in mice. Subsequent bioinformatic analysis of these genes predicted that a total of 33 miRNAs target multiple CL-associated genes, with 20 CL-associated genes being potentially regulated by multiple miRNAs. To experimentally validate miRNA function in cell proliferation, we conducted cell proliferation/viability assays for the selected five candidate miRNAs (miR-124-3p, let-7a-5p, let-7b-5p, let-7c-5p, and let-7d-5p). Overexpression of miR-124-3p, but not of the others, inhibited cell proliferation through suppression of CL-associated genes in cultured mouse embryonic lip mesenchymal cells (MELM cells) isolated from the developing mouse lip region. By contrast, miR-124-3p knockdown had no effect on MELM cell proliferation. This miRNA-gene regulatory mechanism was mostly conserved in O9-1 cells, an established cranial neural crest cell line. Expression of miR-124-3p was low in the maxillary processes at E10.5, when lip mesenchymal cells proliferate, whereas it was greatly increased at later developmental stages, suggesting that miR-124-3p expression is suppressed during the proliferation phase in normal palate development. CONCLUSIONS:Our findings indicate that upregulated miR-124-3p inhibits cell proliferation in cultured lip cells through suppression of CL-associated genes. These results will have a significant impact, not only on our knowledge about lip morphogenesis, but also on the development of clinical approaches for the diagnosis and prevention of CL.

Project description:As the developing zebrafish pancreas matures, hormone-producing endocrine cells differentiate from pancreatic Notch-responsive cells (PNCs) that reside within the ducts. These new endocrine cells form small clusters known as secondary (2°) islets. We use the formation of 2° islets in the pancreatic tail of the larval zebrafish as a model of β-cell neogenesis. Pharmacological inhibition of Notch signaling leads to precocious endocrine differentiation and the early appearance of 2° islets in the tail of the pancreas. Following a chemical screen, we discovered that blocking the retinoic acid (RA)-signaling pathway also leads to the induction of 2° islets. Conversely, the addition of exogenous RA blocks the differentiation caused by Notch inhibition. In this report we characterize the interaction of these two pathways. We first verified that signaling via both RA and Notch ligands act together to regulate pancreatic progenitor differentiation. We produced a transgenic RA reporter, which demonstrated that PNCs directly respond to RA signaling through the canonical transcriptional pathway. Next, using a genetic lineage tracing approach, we demonstrated these progenitors produce endocrine cells following inhibition of RA signaling. Lastly, inhibition of RA signaling using a cell-type specific inducible cre/lox system revealed that RA signaling acts cell-autonomously in PNCs to regulate their differentiation. Importantly, the action of RA inhibition on endocrine formation is evolutionarily conserved, as shown by the differentiation of human embryonic stem cells in a model of human pancreas development. Together, these results revealed a biphasic function for RA in pancreatogenesis. As previously shown by others, RA initially plays an essential role during embryogenesis as it patterns the endoderm and specifies the pancreatic field. We reveal here that later in development RA is involved in negatively regulating the further differentiation of pancreatic progenitors and expands upon the developmental mechanisms by which this occurs.

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MicroRNA-124-3p Plays a Crucial Role in Cleft Palate Induced by Retinoic Acid.

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