Transcriptome Profiling of Cleft Palate inTGF-beta3 Knockout Mice Alleles: RNA-SEQ Analysis of TGF-beta3 Mice

Background: Orofacial clefts are the most common craniofacial birth defect with a complex, combinatorial etiology. Tgf-b3 regulates palatal fusion in mice; Tgf-b3 knockout mice have cleft palate (CP) lacking other major deformities. The genes downstream of Tgf-b3 during palatogenesis remain largely unexplored. Our objective was to analyze the global transcriptome changes and their contribution to CP and identify novel Tgf-b3 associated genes involved in formation of CP. We used RNA-sequencing to analyze and compare the whole transcriptome of Tgf-b3 alleles during palatal growth and fusion in mice. Results: The whole transcriptome analysis of Tgf-b3 mice (C57BL/6) alleles revealed over 6000 significantly differentially expressed genes from 14.5 and 16.5 days post coitum (dpc), in wild type (WT) and homozygous (HM) genotypes. A majority of differentially expressed genes were upregulated (WT=2421; HM=3153) compared to downregulated (WT=1694; HM= 2151) over time. With a 2.0 fold-change cut-off, downregulated genes decreased dramatically (WT=134, HM=191) compared to upregulated genes (WT=1675; HM=1936). Comparatively, gene expression differences between WT and HM were minimal. Using Ingenuity® Pathway Analysis (IPA) we identified genes which may function as primary contributors to the development of CP. Conclusions: Using RNA-seq analysis, we provided a global analysis of transcriptome changes between and within WT and HM in the Tgf-b3 mouse model system at critical stages of palate development. We identified genes that likely play key regulatory roles during palatogenesis downstream of Tgf-b3 bolstering our knowledge of CP