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

Systematic analysis of palatal transcriptome to identify cleft palate genes within TGFβ3-knockout mice alleles: RNA-Seq analysis of TGFβ3 Mice Ozturk et al. Ozturk et al. BMC Genomics 2013, 14:113 http://www.biomedcentral.com/1471-2164/14/113 Ozturk et al. BMC Genomics 2013, 14:113 http://www.biomedcentral.com/1471-2164/14/113 RESEARCH ARTICLE Open Access Systematic analysis of palatal transcriptome to identify cleft palate genes within TGFβ3-knockout mice alleles: RNA-Seq analysis of TGFβ3 Mice Ferhat Ozturk1,4, You Li2, Xiujuan Zhu1, Chittibabu Guda2,3 and Ali Nawshad1* Abstract Background: In 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). Results: The 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. Conclusions: Our 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. Keywords: RNA-Seq, Next-generation sequencing, TGFβ3, Knockout, Transcriptome, Cleft palate, Palatogenesis, Palate, Craniofacial * Correspondence: 1 Department of Oral Biology, College of Dentistry, University of Nebraska Medical Center, 40th and Holdrege St, Lincoln, NE 68583, USA Full list of author information is available at the end of the article © 2013 Ozturk et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Ozturk et al. BMC Genomics 2013, 14:113 http://www.biomedcentral.com/1471-2164/14/113 Background Cleft palate (CP) is the second most common birth defect (1/800 live births) in humans, and is caused by the lack of fusion of the embryonic palatal shelves early in gestation (6–10 weeks) [1]. The formation of a continuous palate is a complex process involving multiple steps, including: palatal shelf growth, elevation, attachment, and fusion. The stages of palatogenesis are regulated by numerous genes that are essential for normal palate development. Our laboratory has a long-standing interest in identifying the genetic and molecular mechanisms that regulate palatogenesis in order to understand the factors involved in the development of orofacial clefts [2-11]. It has been shown that both genetic and environmental elements contribute to the development of cleft palate [12,13]. We previously presented that Transforming Growth Factor (TGF)-β isoforms play essential roles in the regulation of palatal morphogenesis, including the finding that TGFβ3 mediates palatal fusion both in primary cells and organ culture [5,14,15]. According to several population-based mutation screening studies, TGFβ3 is considered a candidate gene for non-syndromic CP in humans [16-22]. Furthermore, TGFβ3 knockout mice are born with CP but lack other major defects [23-25]. In our earlier studies [26], we examined gene expression during palatal fusion in normal mice using microarray analysis and detected several genes essential for completion of palatal development. As genome sequencing technologies advanced, it has become feasible to systematically analyze global transcriptomal changes and identify the key molecular components in the developing palate during crucial stages of palatogenesis. Failure to regulate functional or structural genes during these stages may result in cleft palate. Next-generation sequencing (NGS) technologies, or RNA-Seq, have recently emerged as a revolutionary tool of transcriptomics [27] by revealing the complex landscape of the transcriptome with high-throughput at an incomparable level of sensitivity and accuracy [27,28]. The results of RNA-Seq demonstrate high levels of reproducibility for both technical and biological replicates [29,30]. The analysis of TGFβ3 knockout mice by RNA-Seq provides a valuable resource to facilitate our understanding of the complex genetic and molecular mechanisms of palatogenesis. This technology also allows us to detect allelic and splice variants of some of the genes involved in palate development in TGFβ3 knockout mice, which is currently beyond the scope of this article. Evidence suggests that TGFβ3 is essential to palatogenesis, especially during embryonic days (E) E14.5 to E16.5. However, the cellular mechanisms maintained and regulated by TGFβ3 signaling have not been extensively explored to enable an understanding of those genes functionally regulated by TGFβ3 during normal palatogenesis or those genes deregulated in TGFβ3-null Page 2 of 18 mice. Our objective in this study was to analyze transcriptome changes and their contribution to the development of CP among different gestational ages and TGFβ3 knockout alleles. In this study, we (...truncated)