Systematic analysis of palatal transcriptome to identify cleft palate genes within TGFβ3-knockout mice alleles: RNA-Seq analysis of TGFβ3 Mice

Abstract

BackgroundIn humans, cleft palate (CP) accounts for one of the largest number of birth defects with a complex genetic and environmental etiology. TGFβ3 has been established as an important regulator of palatal fusion in mice and it has been shown that TGFβ3-null mice exhibit CP without any other major deformities. However, the genes that regulate cellular decisions and molecular mechanisms maintained by the TGFβ3 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 TGFβ3 knockout mice to identify TGFβ3-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 TGFβ3 knockout mice at crucial stages of palatogenesis, including palatal growth (E14.5), adhesion (E15.5), and fusion (E16.5).ResultsThe overall transcriptome analysis of TGFβ3 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 TGFβ3+/− and TGFβ3−/− 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 TGFβ3−/− mice (Chrng, Foxc2, H19, Kcnj13, Lhx8, Meox2, Shh, and Six3), which may function as the primary contributors to the development of cleft palate in TGFβ3−/− mice. When the significantly altered CP genes were overlaid with TGFβ signaling, all of these genes followed the Smad-dependent pathway.ConclusionsOur study represents the first analysis of the palatal transcriptome of the mouse, as well as TGFβ3 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 TGFβ3 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.

Nicotine Exposure During Pregnancy Results in Persistent Midline Epithelial Seam With Improper Palatal Fusion

“…These, in parallel with the transcription factors Snai1 and Snai2, promote MES apoptosis and disintegration. 6,21,[39][40][41][42][43] Our results demonstrated that not only the Tgfβ-pathway genes but also other pathway-related genes might interact each other to regulate medial edge epithelium disintegration and complete palatogenesis (Supplementary Figure 2) In several studies, it has been suggested that there is a gene-environment interaction exists between smoking and TGFα expression for the induction of cleft palate. 8,25,[44][45][46][47][48][49] By using our microarray analysis and filtering cleft palate-related genes, we determined that nicotine is the highly susceptible element of smoking to induce down-regulation of TGFα, which may explain the palatal size abnormality observed in nicotine-treated pups.…”

“…22 Gene targeting to knock-out specific genes in mice has generated, by the date, 84 lossof-function mutants that exhibits cleft palate. Information about these knockout models was collected from a literature review 4,21,23,50 and genes were clustered into families of cytokine/receptor, homeobox genes, and miscellaneous (Supplementary Table 1). Out of 84 cleft palate-susceptible genes, expression of 19 of them was up-regulated, and 20 of them were down-regulated significantly in response to consistent nicotine uptake during pregnancy.…”

“…21 The molecules involved in cleft palate formation in knockout models include cytokines, receptors, growth factors, ion channels, kinases, extracellular matrix proteins, homeobox genes, and transcription regulators 22,23 (Supplementary Table 1). According to the findings of this study, 46% of the cleft palate-causing genes (39 out of 84) were found to be affected by nicotine exposure.…”

Long non-coding RNA H19-mediated mouse cleft palate induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin

“…Several lncRNAs have been found to serve a critical role in embryonic development (4), while the dysfunction of lncRNAs is associated with widespread conditions, such as birth defects (5). Cleft palate (CP) is the most common congenital malformation in the oral and craniofacial region, and may occur at any stage of the palate development, including the palatal shelf growth, elevation or fusion (6). In addition, the loss of medial edge epithelial cells or failure of mesenchymal consolidation can cause CP.…”

“…The identification of lncRNAs has provided novel opportunities for the investigation of the precise etiology and pathogenesis of CP, which remain unclear. However, although a great number of studies have investigated the underlying mechanisms of TCDD-induced CP (8,14,(16)(17)(18), the involvement of lncRNAs in TCDD-induced CP has been rarely reported (6). lncRNA H19 is a 2,300 bp non-coding RNA (ncRNA), which is transcribed from the maternal allele, with a high expression observed prenatally and a low expression observed postnatally.…”