Determinants of orofacial clefting I: Effects of 5-Aza-2′-deoxycytidine on cellular processes and gene expression during development of the first branchial arch

Abstract

In this study, we identify gene targets and cellular events mediating the teratogenic action(s) of 5-Aza-2′-deoxycytidine (AzaD), an inhibitor of DNA methylation, on secondary palate development. Exposure of pregnant mice (on gestation day (GD) 9.5) to AzaD for 12 hrs resulted in the complete penetrance of cleft palate (CP) in fetuses. Analysis of cells of the embryonic first branchial arch (1-BA), in fetuses exposed to AzaD, revealed: 1) significant alteration in expression of genes encoding several morphogenetic factors, cell cycle inhibitors and regulators of apoptosis; 2) a decrease in cell proliferation; and, 3) an increase in apoptosis. Pyrosequencing of selected genes, displaying pronounced differential expression in AzaD-exposed 1-BAs, failed to reveal significant alterations in CpG methylation levels in their putative promoters or gene bodies. CpG methylation analysis suggested that the effects of AzaD on gene expression were likely indirect.

Determinants of orofacial clefting II: Effects of 5-Aza-2′-deoxycytidine on gene methylation during development of the first branchial arch

“…The palatal processes in AzaD-exposed fetuses were comparatively smaller and remained unreoriented and unfused relative to those found in control fetuses at GD15.5. These observations are consistent with the decreased cellular proliferation and increased apoptosis observed in the 1-BA of in utero AzaD-exposed fetuses (see accompanying study [26]). Moreover, significant alterations in the expression of numerous genes associated with cellular proliferation and apoptosis were observed in 1-BAs 12-hr after in utero AzaD-exposure.…”

“…Animal maintenance, developmental staging of embryos, AzaD dosing regimen and identification of the CP phenotype are provided in detail in the accompanying study [26]. Briefly, pregnant mice (ICR strain) were injected with either AzaD (1mg/kg) (Sigma Chemical Co., St. Louis, MO) or PBS (the vehicle) on GD9.5 and 1-BA tissues isolated 12-hr later for extraction of both RNA and genomic DNA.…”

“…cDNAs were synthesized, using the SuperScript III First Strand Synthesis kit (Life Technologies), from 1 μg of mRNA. Expression levels were determined by quantitative real-time PCR (qRT-PCR) using cDNA samples obtained from in utero AzaD- or vehicle-exposed 1-BA tissues (see accompanying study [26]) and AzaD-treated or untreated MEMM cells. qRT-PCR was undertaken using TaqMan assay primers (Applied Biosystems) in a ViiA7 Real-time PCR System (Applied Biosystems).…”

“…Moreover, significant alterations in the expression of numerous genes associated with cellular proliferation and apoptosis were observed in 1-BAs 12-hr after in utero AzaD-exposure. Also included were genes associated with the Fgf, Wnt, SHH and Tgfβ signaling pathways and transcriptional regulation, all of which are known to play key roles in palatal morphogenesis (see accompanying study [26]. A number of these genes (82%) were found to be upregulated after AzaD exposure, presumably due to the effects of promoter demethylation.…”

“…A number of these genes (82%) were found to be upregulated after AzaD exposure, presumably due to the effects of promoter demethylation. However, a preliminary analysis of CpG methylation levels in 10 selected genes exhibiting some of the most pronounced changes in gene expression in AzaD-exposed 1-BAs indicated that CpG methylation levels were not substantially altered when compared to unexposed, control tissues (see accompanying study [26]). To assess in greater depth the demethylating effects of AzaD on the observed changes in gene expression, we sought to identify differentially methylated regions (DMRs) in the DNA isolated from in utero AzaD exposed 1-BAs.…”