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The role of the T-box transcription factor TBX22 in craniofacial development
Supervisors: Dr Philip Stanier and Dr Erwin Pauws
Hypothesis: Understanding the molecular and cellular deficit in a mouse deficient for Tbx22 will allow the development of improved therapeutic intervention procedures for submucous cleft palate.
Aims and methods: Mutations in TBX22 are known to be one of the leading genetic causes of palate defects in humans (1, 2). These range from a complete cleft of the secondary palate to a closed but poorly functioning submucous cleft palate (3). We have developed a mouse model lacking Tbx22, which has a submucous cleft palate (4). This appears to result from poor palatine bone development, which in turn affects the soft palate musculature. The mouse has a distinct and reproducible phenotype that strongly mimics the human condition, therefore providing an excellent model to investigate this common phenotype. This project sets out to elucidate the fundamental role of TBX22 in craniofacial development. Preliminary studies show that Tbx22 is expressed in the paired embryonic palatal shelves but is down regulated by the time of fusion. Intramembranous bones then develop in the mid and anterior portions of the palate, forming the supportive structures of the hard palate. Lack of palatine bones is the most recognisable structural defect in Tbx22 mutants. We will investigate if the poor intramembranous bone development is a consequence of insufficient cranial neural crest (CNC), an imbalance with non-CNC mesoderm or indeed an inappropriate differentiation towards a muscle lineage. This will be studied using the ROSA26YFP Wnt1-Cre reporter mice crossed to both wild type and Tbx22null animals. Tbx22 expression will then be investigated for co-localisation with the YFP positive cells in the wildtype animals. A similar experiment will also be performed with a mesoderm specific Cre such as Mesp1-Cre (Yoshida et al, 2008). This experiment will then inform a confirmatory experiment to cross the Wnt1-Cre mice or Mesp1-Cre to floxed Tbx22 mice to investigate the phenotype recovered when Tbx22 is only deleted from the respective cell type.
Expression studies indicate that Tbx22null mice undergo an upregulation of muscle development in the palatal shelves prior to the onset of osteogenesis. Therefore, genetic markers of muscle differentiation will be investigated in E10.5 to E14.5 wild type and mutant embryos. The regulation of osteoblastic differentiation into intramembranous bone will also be investigated in E13.5-E17.5 embryos. Analysis will also include expression of Bmp and Fgf signalling pathways, which are likely to act upstream of TBX22 and are known to have critical roles in providing instructive cues for normal palatal intramembranous bone development. Next, differential cell proliferation will be investigated in the rapidly elongating palatal shelves (E13.5-E14.5) and at the time of fusion using immunohistochemistry with anti-phosphohistone H3.
An in vitro palate culture system will be used to compare palate development between wildtype and mutant mice both in the context of specific pathway inhibitors or modifiers and as a model to investigate repair of palatine bone defects using osteogenically differentiated stem cells (6).
1) Braybrook C, Doudney K, Marçano ACB, Arnason A, Bjornsson A, Patton MA, Goodfellow PJ, Moore GE and Stanier P. X-linked cleft palate and ankyloglossia (CPX) is caused by mutations in the T-box transcription factor gene TBX22. Nat Genet (2001) 29:179-183.
2) Marçano ACB, Doudney K, Braybrook C, Squires R, Patton MA, Lees M, Richieri-Costa A, Lideral AC, Murray JC, Moore GE and Stanier P. TBX22 mutations are a frequent cause of cleft palate. J Med Genet (2004) 41: 68-74.
3) Stanier P and Moore GE. Genetic basis for cleft lip and palate: syndromic genes contribute to the incidence of non-syndromic clefts. Hum Mol Genet (2004) 13: R73-R81.
4) Pauws E, Hoshino A, Bentley L, Prajapati S, Keller C, Hammond P, Martinez-Barbera JP, Moore GE, Stanier P. Tbx22null mice have a submucous cleft palate due to reduced palatal bone formation and also display ankyloglossia and choanal atresia phenotypes. Hum Mol Genet (2009) 18: 4171-4179.
5) Yoshida T, Vivatbutsiri P, Morris-Kay G, Saga Y, Iseki S. Cell lineage in mammalian craniofacial mesenchyme. Mech Dev 2008 125: 797-808.
6) Congejero JA, Lee JA, Parrett BM, Terry M, Wear-Maggitti K, Grant RT, Breitbart AS. Repair of palatal bone defects using osteogenically differentiated fat-derived stem cells. Plastic Reconstr Surg 2006 117: 857-863.