Function of human TBX22 during chicken facial development

Objectives: The T-box transcription factor TBX22 is a causative gene for X-linked cleft palate and ankyloglossia (CPX, OMIM 303400). In this study, we tested the function of human TBX22 during facial development by overexpressing the gene in avian embryos. The phenotype and several downstream targets of the TBX gene were characterized.

Methods: The full-length human TBX22 open reading frame was cloned into RCAS (an avian retrovirus). Live virus was injected adjacent to the optic stalks of stage 10 embryos. Specimens were collected 4 and 12 days after for gene expression and skeletal analysis. DIG-labeled chicken TBX22 and DLX5, and FLU-labeled human TBX22 riboprobes were used to simultaneously detect gene expression changes and to localize the virus in the same embryo.

Results: Striking clefts (10/23) and tongue deformities (11/23) were found in chicken embryos infected with hTBX22. There were reductions in size of the premaxillary, maxillary and palatine bones. In addition the palatine bones were more widely separated than usual.  The tip of the tongue cartilage, the entoglossum, was usually missing. In gene expression analysis, we found endogenous chicken gTBX22  was repressed while and gDLX5 was induced by hTBX22.

Conclusions: There was a close fit between the regions affected in viral-infected chicken embryos and those affected in human CPX syndrome. Since the hTBX22 gene is a repressor and is capable of self-regulation, we hypothesize that phenotypes caused by the virus are due to repression gTBX22 and possibly other Tbox genes. The activation of gDlx5 suggests that it is also a target of TBX22 however its regulation is likely to be via an intermediary transcription factor.

Aknowledgements: This work was funded by Alpha Omega, and CIHR grants to JMR. NH is supported by a fellowship from the Canadian Government (CPDRF). JMR is a MSFHR Distinguished Scholar