Systematic analysis of palatal transcriptome to identify cleft palate genes within TGFß3-knockout mice alleles: RNA-Seq analysis of TGFß3 Mice.

ABSTRACT

BACKGROUND: In humans, cleft palate (CP) accounts for one of the largest number of birth defects with a complex genetic and environmental etiology. TGFß3 has been established as an important regulator of palatal fusion in mice and it has been shown that TGFß3-null mice exhibit CP without any other major deformities. However, the genes that regulate cellular decisions and molecular mechanisms maintained by the TGFß3 pathway throughout palatogenesis are predominantly unexplored. Our objective in this study was to analyze global transcriptome changes within the palate during different gestational ages within TGFß3 knockout mice to identify TGFß3-associated genes previously unknown to be associated with the development of cleft palate. We used deep sequencing technology, RNA-Seq, to analyze the transcriptome of TGFß3 knockout mice at crucial stages of palatogenesis, including palatal growth (E14.5), adhesion (E15.5), and fusion (E16.5). RESULTS: The overall transcriptome analysis of TGFß3 wildtype mice (C57BL/6) reveals that almost 6000 genes were upregulated during the transition from E14.5 to E15.5 and more than 2000 were downregulated from E15.5 to E16.5. Using bioinformatics tools and databases, we identified the most comprehensive list of CP genes (n = 322) in which mutations cause CP either in humans or mice, and analyzed their expression patterns. The expression motifs of CP genes between TGFß3+/- and TGFß3-/- were not significantly different from each other, and the expression of the majority of CP genes remained unchanged from E14.5 to E16.5. Using these patterns, we identified 8 unique genes within TGFß3-/- mice (Chrng, Foxc2, H19, Kcnj13, Lhx8, Meox2, Shh, and Six3), which may function as the primary contributors to the development of cleft palate in TGFß3-/- mice. When the significantly altered CP genes were overlaid with TGFß signaling, all of these genes followed the Smad-dependent pathway. CONCLUSIONS: Our study represents the first analysis of the palatal transcriptome of the mouse, as well as TGFß3 knockout mice, using deep sequencing methods. In this study, we characterized the critical regulation of palatal transcripts that may play key regulatory roles through crucial stages of palatal development. We identified potential causative CP genes in a TGFß3 knockout model, which may lead to a better understanding of the genetic mechanisms of palatogenesis and provide novel potential targets for gene therapy approaches to treat cleft palate.