MicroRNA expression profiling of the developing murine upper lip.

Clefts of the lip and palate are thought to be caused by genetic and environmental insults but the role of epigenetic mechanisms underlying this common birth defect are unknown. We analyzed the expression of over 600 microRNAs in the murine medial nasal and maxillary processes isolated on GD10.0-GD11.5 to identify those expressed during development of the upper lip and analyzed spatial expression of a subset. A total of 142 microRNAs were differentially expressed across gestation days 10.0-11.5 in the medial nasal processes, and 66 in the maxillary processes of the first branchial arch with 45 common to both. Of the microRNAs exhibiting the largest percent increase in both facial processes were five members of the Let-7 family. Among those with the greatest decrease in expression from GD10.0 to GD11.5 were members of the microRNA-302/367 family that have been implicated in cellular reprogramming. The distribution of expression of microRNA-199a-3p and Let-7i was determined by in situ hybridization and revealed widespread expression in both medial nasal and maxillary facial process, while that for microRNA-203 was much more limited. MicroRNAs are dynamically expressed in the tissues that form the upper lip and several were identified that target mRNAs known to be important for its development, including those that regulate the two main isoforms of p63 (microRNA-203 and microRNA-302/367 family). Integration of these data with corresponding proteomic datasets will lead to a greater appreciation of epigenetic regulation of lip development and provide a better understanding of potential causes of cleft lip.

Temporal Expression of miRNAs in Laser Capture Microdissected Palate Medial Edge Epithelium from Tgfß3(-/-) Mouse Fetuses.

Clefting of the secondary palate is the most common birth defect in humans. Midline fusion of the bilateral palatal processes is thought to involve apoptosis, epithelial to mesenchymal transition, and cell migration of the medial edge epithelium (MEE), the specialized cells of the palate that mediate fusion of the palatal processes during fetal development. Data presented in this manuscript are the result of analyses designed to identify microRNAs that are expressed and regulated by TGFß3 in developing palatal MEE. The expression of 7 microRNAs was downregulated and 1 upregulated in isolated MEE from wildtype murine fetuses on gestational day (GD) 13.5 to GD14.5 (prior to and during epithelial fusion of the palatal processes, respectively). Among this group were miRNAs linked to apoptosis (miR-378) and epithelial to mesenchymal transformation (miR-200b, miR-205, and miR-93). Tgfß3(-/-) fetuses, which present with a complete and isolated cleft of the secondary palate, exhibited marked dysregulation of distinct miRNAs both in the palatal MEE and mesenchyme when compared to comparable wild-type tissue. These included, among others, miRNAs known to affect apoptosis (miR-206 and miR-186). Dysregulation of miRNAs in the mesenchyme underlying the palatal MEE of Tgfß3(-/-) fetuses is also discussed in relation to epithelial-mesenchymal transformation of the MEE. These results are the first systematic analysis of the expression of microRNAs in isolated fetal palatal epithelium and mesenchyme. Moreover, analysis of the Tgfß3 knockout mouse model has enabled identification of miRNAs with altered expression that may contribute to the cleft palate phenotype.

Chromatin immunoprecipitation-promoter microarray identification of genes regulated by PRDM16 in murine embryonic palate mesenchymal cells.

The transcription factor PRDM16 regulates differentiation of brown adipocyte tissue in mice. Recently, however, it has been demonstrated that genetic knockout of Prdm16 in mice leads to a complete cleft of the secondary palate in offspring. To identify genes whose promoters bind PRDM16 in mouse embryonic palate/maxillary mesenchymal cells, we have conducted a chromatin immunoprecipitation-promoter microarray analysis (ChIP-Chip). One hundred and twenty-two gene promoters were identified as capable of binding PRDM16. These could be functionally grouped to include those on genes linked to muscle development, chondrogenesis and osteogenesis, in addition to many transcription factors. These results suggest that PRDM16 may play a role in differentiation of mesenchymal cells in the embryonic secondary palate that contribute to the anterior, bony palate and posterior, muscular palate.