Identification of Osr2 Transcriptional Target Genes in Palate Development

Abstract

Previous studies have identified the odd-skipped related 2 (Osr2) transcription factor as a key intrinsic regulator of palatal shelf growth and morphogenesis. However, little is known about the molecular program acting downstream of Osr2 in the regulation of palatogenesis. In this study, we isolated palatal mesenchyme cells from embryonic day 12.5 (E12.5) and E13.5 Osr2RFP/+ and Osr2RFP/- mutant mouse embryos and performed whole transcriptome RNA sequencing analyses. Differential expression analysis of the RNA sequencing datasets revealed that expression of 70 genes was upregulated and expression of 61 genes was downregulated by >1.5-fold at both E12.5 and E13.5 in the Osr2RFP/- palatal mesenchyme cells, in comparison with Osr2RFP/+ littermates. Gene ontology analysis revealed enrichment of signaling molecules and transcription factors crucial for skeletal development and osteoblast differentiation among those significantly upregulated in the Osr2 mutant palatal mesenchyme. Using quantitative real-time polymerase chain reaction (RT-PCR)and in situ hybridization assays, we validated that the Osr2-/- embryos exhibit significantly increased and expanded expression of many osteogenic pathway genes, including Bmp3, Bmp5, Bmp7, Mef2c, Sox6, and Sp7 in the developing palatal mesenchyme. Furthermore, we demonstrate that expression of Sema3a, Sema3d, and Sema3e, is ectopically activated in the developing palatal mesenchyme in Osr2-/- embryos. Through chromatin immunoprecipitation, followed by RT-PCR analysis, we demonstrate that endogenous Osr2 protein binds to the promoter regions of the Sema3a and Sema3d genes in the embryonic palatal mesenchyme. Moreover, Osr2 expression repressed the transcription from the Sema3a and Sema3d promoters in cotransfected cells. Since the Sema3 subfamily of signaling molecules plays diverse roles in the regulation of cell proliferation, migration, and differentiation, these data reveal a novel role for Osr2 in regulation of palatal morphogenesis through preventing aberrant activation of Sema3 signaling. Together, these data indicate that Osr2 controls multiple molecular pathways, including BMP and Sema3 signaling, in palate development.