Overexpression of mouse TTF-2 gene causes cleft palate

Abstract

In humans, mutations of the gene encoding for thyroid transcription factor-2 (TTF-2 or FOXE1) result in Bamforth syndrome. Bamforth syndrome is characterized by agenesis, cleft palate, spiky hair and choanal atresia. TTF-2 null mice (TTF-2(-/-) ) also exhibit cleft palate, suggesting its involvement in the palatogenesis. However, the molecular pathology and genetic regulation by TTF2 remain largely unknown. In the present study, the recombinant expression vector pBROAD3-TTF-2 containing the promoter of the mouse ROSA26 gene was created to form the structural gene of mouse TTF-2 and was microinjected into the male pronuclei of fertilized ova. Sequence analysis confirmed that the TTF-2 transgenic mouse model was established successfully. The transgenic mice displayed a phenotype of cleft palate. In addition, we found that TTF-2 was highly expressed in the medial edge epithelium (MEE) from the embryonic day 12.5 (E12.5) to E14.5 in TTF-2 transgenic mice. These observations suggest that overexpression of TTF-2 during palatogenesis may contribute to formation of cleft palate.

Fig 1

Micrographs and Scanning electron micrographs of the palatal shelves region of control mice and TTF-2 transgenic mice. (A) and (C) showed that the palatal shelves were fused on E18 in the control groups. However, the palatal shelves were not fused on E18 in the TTF-2 transgenic mice as shown in (B) and (D).

Fig 2

(A) Results of PCR using genomic DNA extracted from tail biopsies of the transgenic foetus. (B) Results of Southern blots detection of the genome DNA of the positive PCR transgenic mice.

Fig 3

In the transgenic mice group, the 508 bp band was found on E12.5, 13.5, 14.5 and 15.5. In the control group, the 508 bp band was found on the E12.5, 13.5, 14.5 However, there was no 508 bp band found on E15.5 in the control group. 0:Marker;1: Positive control;2: E 12 Trs;3: E 13 Trs;4: E 14 Trs;5: E 15 Trs;6: E 12 Ctr;7: E 13 Ctr;8: E 14 Ctr;9: Ed15 Ctr (E: embryonic day;Trs: transgenic group;Ctr: control group).

Fig 4

(A) Relative TTF-2 mRNA expression in the TTF-2 transgenic mice and the control group. As can be seen from (A), there was no obvious change on TTF-2 mRNA expression in the TTF-2 transgenic mice. However, there was an apparent change in the control group and reached its peak on E13.5 and started to decline later. The TTF-2 mRNA expression was not detected on E15.5. (B) TTF-2 protein expression in the TTF-2 transgenic mice and the control group. The change in tendency of the TTF-2 protein in the control group was basically consistent with the change in TTF-2 mRNA level. The tendency of the TTF-2 protein expression change of the TTF-2 transgenic mice was not obvious. (C) The Western blot results of the TTF-2 expression of the transgenic mice and control group on E12.5, 13.5, 14.5 and 15.5.

Fig 5

Immunohistochemical staining of TTF-2. TTF-2 expression in the palatal shelves on E12.5-15.5 in the control group mice (A–D) and in the transgenic mice (E–H).In the transgenic group, there was positive immunohistochemical staining of MEE cells, but no expression differences were found in the edge epithelium tissue of the palatine process from E 12.5 to E15.5. The positive immunohistochemical staining of MEE cells was increased in transgenic group compared with control group on the corresponding phase. N: nasal epithelium. O: oral epithelium. PS: palatal shelf. T: tongue. The positive immunohistochemical staining of MEE cells is indicated with arrows. Scale bars represent 50 μm.