RNA-seq analysis of palatal transcriptome changes in all-trans retinoic acid-induced cleft palate of mice

• Published in: Environmental toxicology and pharmacology 2020-11, Vol.80, p.103438, Article 103438
• Main Authors: Peng, Yao, Wang, Xin-huan, Su, Chao-nan, Qiao, Wei-wei, Gao, Qian, Sun, Xue-fei, Meng, Liu-yan
• Format: Article
• Language: English
• Subjects: All-trans retinoic acid; Animals; Bone growth; Cleft lip/palate; Cleft palate; Cleft Palate - chemically induced; Cleft Palate - genetics; Crystallin; Developmental stages; Embryos; Extracellular matrix; Female; Gene expression; Gene expression profile; Gene Expression Regulation, Developmental - drug effects; Gene Ontology; Genes; Gestational Age; Immunohistochemistry; Maxillofacial; Metabolic pathways; Mice; Mice, Inbred C57BL; Neonates; Osteogenesis; Palate - drug effects; Palate - embryology; Pregnancy; Prenatal Exposure Delayed Effects - chemically induced; Prenatal Exposure Delayed Effects - genetics; Proteins; Real-Time Polymerase Chain Reaction; Retinoic acid; Ribonucleic acid; RNA; RNA - genetics; RNA-Seq; Shelves; Toxicity; Transcription; Transcriptome - drug effects; Transcriptomes; Tretinoin - toxicity
• ISSN: 1382-6689; 1872-7077
• DOI: 10.1016/j.etap.2020.103438

•Multiple metabolic pathways may be involved in atRA-induced cleft palate.•AtRA may dysregulated ECM and crystallin components to control the elevation.•AtRA may down-regulated Spp1to influence palatal bone development. Cleft palate is a common congenital maxillofacial malformation in newborns. All-trans retinoic acid (atRA) is an ideal exogenous stimulus to construct a mouse cleft palate model. However, the precise pathogenic mechanism remains to be elucidated. In our study, to explore the toxicity of atRA on palatal shelves during different stages of palate development, a total of 100 mg/kg atRA was administered to C57BL/6 mice at embryonic day 10.5 (E10.5). Mouse embryonic palatal shelves at E13.5, E14.5, E15.5, and E16.5 were collected for RNA extraction and histological treatment. Changes in gene expression were tested through RNA-seq. Selected differentially expressed genes (DEGs) related to metabolic pathways, such as Ptgds, Ttr, Cyp2g1, Ugt2a1 and Mgst3, were validated and analyzed by Quantitative real-time PCR (qRT-PCR). In addition, Gene Oncology analysis showed that transcriptional changes of genes from extracellular matrix (ECM) components, such as Spp1, and crystallin family might play important role in palatal shelves elevation (E13.5-E14.5). Therefore, the protein expression level of Ttr and Spp1 from E13.5 to E16.5 were tested by immunohistochemistry (IHC). Besides, the mRNA level of Spp1, were down-regulated at E16.5 and the protein were down-regulated at E15.5 and E16.5 in all-trans retinoic acid group, suggesting that atRA may involve in palatal bone formation by regulating Spp1. Overall, gene transcriptional profiles were obviously different at each time point of palate development. Thus, this study summarized some pathways and genes that may be related to palatogenesis and cleft palate through RNA-seq, to provide a direction for subsequent studies on the mechanism and targeted therapy of cleft palate.