Abstract
Development of the palate comprises sequential stages of growth, elevation, and fusion of the palatal shelves. The mesenchymal component of palates plays a major role in early phases of palatogenesis, such as growth and elevation. Failure in these steps may result in cleft palate, the second most common birth defect in the world. These early stages of palatogenesis require precise and chronological orchestration of key physiological processes, such as growth, proliferation, differentiation, migration, and apoptosis. There is compelling evidence for the vital role of TGFβ-mediated regulation of palate development. We hypothesized that the isoforms of TGFβ regulate different cellular biofunctions of the palatal mesenchyme to various extents. Human embryonic palatal mesenchyme (HEPM) cells were treated with TGFβ1, β2, and β3 for microarray-based gene expression studies in order to identify the roles of TGFβ in the transcriptome of the palatal mesenchyme. Following normalization and modeling of 28,869 human genes, 566 transcripts were detected as differentially expressed in TGFβ-treated HEPM cells. Out of these altered transcripts, 234 of them were clustered in cellular biofunctions, including growth and proliferation, development, morphology, movement, cell cycle, and apoptosis. Biological interpretation and network analysis of the genes active in cellular biofunctions were performed using IPA. Among the differentially expressed genes, 11 of them are known to be crucial for palatogenesis (EDN1, INHBA, LHX8, PDGFC, PIGA, RUNX1, SNAI1, SMAD3, TGFβ1, TGFβ2, and TGFβR1). These genes were used for a merged interaction network with cellular behaviors. Overall, we have determined that more than 2% of human transcripts were differentially expressed in response to TGFβ treatment in HEPM cells. Our results suggest that both TGFβ1 and TGFβ2 orchestrate major cellular biofunctions within the palatal mesenchyme in vitro by regulating expression of 234 genes.
Highlights
Cleft lip and/or palate is one of the most prevalent birth defects worldwide (1 in 800 live births; Schutte and Murray, 1999; Spritz, 2001), and is caused by failures in palate development
We found that expression of only 566 genes, which corresponds to >2% of the overall human transcriptome, were differentially expressed in transforming growth factor β (TGFβ)-treated Human embryonic palatal mesenchyme (HEPM) cells with statistical significance; including candidate genes recognized as inducers of cleft palate either in human or mouse (EDN1, INHBA, LHX8, PDGFC, PIGA, RUNX1, SNAI1, SMAD3, TGF β1, TGF β2, and TGF βR1)
We explored the alterations in gene expression in HEPM cells extracted from human embryonic palatal shelves in response to 10 ng/ml TGFβ1, TGFβ2, and TGFβ3 for 24 h
Summary
Cleft lip and/or palate is one of the most prevalent birth defects worldwide (1 in 800 live births; Schutte and Murray, 1999; Spritz, 2001), and is caused by failures in palate development. The formation of a continuous palate is a complex process composed of multiple steps, including palatal shelf growth, elevation, attachment, and fusion. Palatogenesis in the human spans from approximately gestational day 48 to 59 and the outgrowth of the secondary palate can generally be detected around day 49. During day 54–55, the palatine processes rapidly elevate, assuming a horizontal position which allows them to grow toward each other, attach, and fuse (Wyszynski, 2002). With slight variation among strains, the stages of palatogenesis in mice [12.5–16.5 days post coitum (dpc)] are extremely similar and comparable to that of humans; mice have been used as a model to study human palate development (Ferguson, 1988). The failure of palatal shelves to Abbreviations: FC, fold change; HEPM, human embryonic palatal mesenchyme; IKB, ingenuity knowledge base; IPA, ingenuity pathway analysis; MEE, medial edge epithelium; TGFβ, transforming growth factor β
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