, a member of the T-box family of transcription factors, has been identified as a major candidate gene for 22q11.2 deletion syndrome (Velo-cardio-facial/DiGeorge syndrome). 22q11.2 deletion syndrome is a common congenital disorder affecting approximately 1 in 4000 live births, characterized by craniofacial defects, thymic hypoplasia, cardiovascular anomalies, velopharyngeal insufficiency and skeletal muscle hypotonia (1
). The craniofacial malformations occur in ~60% of 22q11.2 deletion syndrome patients (5
). The most frequent features include micrognathia, ear abnormalities, blunted nose, hypertelorism, complete cleft palate, submucosal and soft palate cleft (5
) and single central incisor (6
). In humans, Tbx1
mutation is responsible for the major phenotypes of 22q11.2 deletion syndrome as well as non-syndromic submucous cleft palate (7
), suggesting that Tbx1 is a regulator of palatogenesis. It is reported that homozygous-null mice of Tbx1
have most features of 22q11.2 deletion syndrome, including cleft palate, micrognathia and ear abnormalities (1
); however, the etiology of cleft palate in Tbx1−/−
mice and biological roles of Tbx1 in palatal epithelium have not been determined.
Cleft palate is the most frequent congenital craniofacial birth defect in the human population, occurring in 1 in 500 to 1 in 1000 births worldwide (10
). Higher incidences of cleft palate are reported in offspring of individuals with cleft palate than in those without a family history (11
), suggesting a multifactorial etiology, involving both genetic and environmental factors. Cleft palate as part of a syndrome accounts for 55% of reported cases (12
) and several cleft-causing mutations have been identified in the IRF6
). In humans, cleft palate manifests as cleft lip and/or palate. In addition to isolated cleft palate, a large number of cleft palate cases include incomplete cleft palate, soft palate cleft and submucosal cleft palate, and the underlying mechanism for each cleft appears to be distinct. As initial palatal closure occurs in the anterior third of the palatal shelves, the mildest form of cleft palate in humans is bifid uvula or soft palate cleft (20
). Cleft palate occurs in a number of mouse models carrying mutations in genes encoding transcription factors, signaling proteins and their receptors and extracellular matrix components (reviewed by 21
). However, most of the studies are focused on complete cleft palate, and the etiology of submucosal cleft palate and soft palate cleft is not well understood.
The mammalian palate is formed by the union of a primary palate and two secondary palatal shelves. The primary palate is derived from the frontonasal process, whereas the secondary palatal shelves extend bilaterally from the internal prominences of the maxillary processes. In the mouse embryo, the secondary palatal shelves start to develop at embryonic day (E) 11.5. They grow downward vertically beside the tongue (E12.5–E13.5), and then grow and elevate into a horizontal position above the dorsum of the tongue (E14.0). With continuous growth, the bilateral palatal shelves appose at the midline (E14.5) and fuse (E15.5). Palatal shelves also extend anteriorly to fuse with the primary palate and nasal septum, thereby forming a barrier between the oral and nasal cavities to allow simultaneous breathing and feeding (23
). Ossification subsequently occurs in the anterior two-thirds of the palate to form the hard palate. The posterior third forms the soft palate is bone-free and involved in velopharyngeal sealing (24
). Although the palatal epithelium transiently contacts with the mandibular and tongue epithelium, oral fusions or adhesions between the palatal shelves and the mandible or the tongue are rare (26
). Disruption at any stage of growth, elevation or fusion of the palatal shelves, genetically or environmentally, results in cleft palate (27
Here we show that Tbx1−/− mice exhibited abnormal epithelial adhesions between the palatal shelves and the mandible. The epithelium of Tbx1−/− mice was hyperproliferative and failed to undergo a normal differentiation program. Furthermore, ectopic Tbx1 expression in MCF7 human epithelial cells resulted in altered cell cycle with an increase in G1 phase. Taken together, we show that Tbx1 is a novel mediator of cellular proliferation and differentiation in oral epithelial cells, leading to various cleft palate phenotypes including submucosal cleft palate and soft palate cleft.