Construction Of TTF-2 Transgenic Mice And Effect Of TTF-2 On Fusion Of Mouse Palate

The formation of the secondary palate in mammals involves the orchestration of several processes to produce the correct separation of the oral and nasal cavities. The first morphological sign of the secondary palate is the presence of two bilateral outgrowths that arise from the medial aspect of the maxillary process of the first branchial arch. They are already visible in the mouse at embryonic day 12 (E12) and they grow down both sides of the tongue until E14, before heading upward to approach one another around E15. Once they meet, the epithelia covering the tip of each palatal shelf (medial edge epithelium, MEE) strongly adhere to form a thick midline epithelial seam (MES). The cells disappear through the combination of programmed cell death , epithelial mesenchymal transformation (EMT), and migration to the oral and nasal palatal epithelia. Finally, the epithelia covering the oral and nasal sides of the palate differentiate into pseudostratified ciliated columnar cells (nasal) or stratifiedsquamous keratinized epithelium (oral). Two areas of mesenchymal condensation are observed both sides of the midline as early as E14.5 in the two anterior thirds of the palate. These areas of condensation undergo membranous ossification at about E16. 5 and ultimately give rise to the hard palate. Alteration in any of these processes results in isolated cleft palate. Several loci have been associated with this congenital malformation.Variants of the TTF-2 gene have been identified in human-isolated cleft palate, suggesting its involvement in the pathogenesis of this disease. Furthermore, a mouse model with a disrupted titf-2 gene exhibited cleft palate and either a sublingual or completely absent thyroid gland. The forkhead domain-containing transcription factor titf-2, which encodes thyroid transcription factor-2(TTF-2), plays a key role in the development of the thyroid. Zannini et al. (1997) cloned the rat cDNA encoding TTF-2 and demonstrated that TTF-2 is a 42-kDa forkhead-containing protein highly enriched in thyroid follicular cells.The forkhead domain-containing proteins are involved in embryonic pattern formation and regional specification. The cleft palate phenotype displayed by human patients led us to analyze in better detail the expression of TTF-2 during palatogenesis. To this end, firstly, we amplify and clone the C57BL/6J mouse titf-2 gene and construct the pBROAD3-titf-2 expression vector,and detect expression of its protein in the mouse marrow mesenchymal cell, which show that it can be used as a tool of transgenic mice. Secondary, we utilize a highly specific anti - TTF-2 polyclonal antibody, to investigate the presence of the TTF-2 protein at several stages during palate development in transgenic mice. We demonstrate the high expression ofTTF-2 protein in the transgenic mouse from embryonic day (E) 12to 15, and its presence in palatal shelf. In addition, we demonstrate that the high expression of TTF-2 inhibits expression of TGF^ 3, similar to TGFP3-/- mouse.