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Genome-wide Identification of Foxf2 Target Genes in Palate Development.

ABSTRACT: Cleft palate is among the most common structural birth defects in humans. Previous studies have shown that mutations in FOXF2 are associated with cleft palate in humans and mice and that Foxf2 acts in a Shh-Foxf-Fgf18-Shh molecular network controlling palatal shelf growth. In this study, we combined RNA-seq and ChIP-seq approaches to identify direct transcriptional target genes mediating Foxf2 function in palate development in mice. Of 155 genes that exhibited Foxf2-dependent expression in the developing palatal mesenchyme, 88 contained or were located next to Foxf2-binding sites. Through in situ hybridization analyses, we demonstrate that expression of many of these target genes, including multiple genes encoding transcription factors and several encoding extracellular matrix-modifying proteins, were specifically upregulated in the posterior region of palatal shelves in Foxf2^-/- mouse embryos. Foxf2 occupancy at many of these putative target loci, including Fgf18, in the developing palatal tissues was verified by ChIP-polymerase chain reaction analyses. One of the Foxf2 target genes, Chst2, encodes a carbohydrate sulfotransferase integral to glycosaminoglycan sulfation. Correlating with ectopic Chst2 expression, Foxf2^-/- embryos a exhibited region-specific increase in sulfated keratan sulfate and a concomitant reduction in chondroitin sulfate accumulation in the posterior palatal mesenchyme. However, expression of the core protein of versican, a major chondroitin sulfate proteoglycan important in palatal shelf morphogenesis, was increased, whereas expression of collagen I was reduced in the corresponding region of the palatal mesenchyme. These results indicate that, in addition to regulating palatal shelf growth through the Fgf18-Shh signaling network, Foxf2 controls palatal shelf morphogenesis through regulating expression of multiple transcription factors as well as through directly controlling the synthesis and processing of extracellular matrix components in the palatal mesenchyme. Our ChIP-seq and RNA-seq data sets provide an excellent resource for comprehensive understanding of the molecular network controlling palate development.

SUBMITTER: Xu J

PROVIDER: S-EPMC7088206 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.

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Genome-wide Identification of Foxf2 Target Genes in Palate Development.

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