Forebrain Midline Development Defects In Chd7 Mutant Mice

CHARGE syndrome is a recognizable genetic pattern of birth defects, including ocular coloboma, heart defects of any type, atresia of the choanae, retarded growth and development, genital hypoplasia, ear abnormalities, CNS anomalies and other defects. The 3C triad (Coloboma-Choanall atresia-abnormal semicircular Canals), arhinencephaly and rhombencephalic dysfunctions are regarded the most important and constant clues to the diagnosis. But the etiology of CHARGE syndrome remains to be elucidated.60%-80% individuals with CHARGE syndrome have been identified with Chd7 mutations. CHD7 protein belongs to the CHD family, which utilizes the energy from ATP hydrolysis to alter the histone-DNA contacts within the nucleosome. And CHD7 protein is predicted to be involved in chromatin remodeling and gene transcriptional regulation. However, the mechanisms whereby CHD7 participate in the gene transcription regulation and cause CHARGE syndrome remains poorly understood.In this study, we described a novel Chd7 mutation mouse strain derived from ENU mutagenesis, as a mouse model for CHARGE syndrome. The mutation COA1 was mapped to the proximal region of chromosome 4. Sequence analysis revealed an A-to-T transition, which resulted in a premature stop codon in the exon4 of Chd7 gene. Homozygous mutant died at around E10.5, displayed delayed turning and hypoplasia of neuroepithelium. The Chd7COA1/+ mice displayed coloboma, retarded development, edema, haemorrhaging, cardiovascular defects, angiogenesis, choanal atresia, skeletal abnormalities and CNS anomalies, that recapitulated the phenotype of CHARGE syndrome. Thus the Chd7COA1/+ mice are a faithful animal model for CHARGE syndrome.55-85% of CHARGE syndrome patients have definite CNS anomolies, among which the forebrain development defects including arhinencephaly, holoprosencephaly and hypoplastic olfactory bulbs are the most common. However, the pathology and etiology of the forebrain defects are poorly understood. Interestingly, we found Chd7 is highly expressed in CNS in early embryonic days, suggesting a role of CHD7 in forebrain development. Subsequent histological analysis revealed that Chd7COA1/+ embryos displayed telencephalic midline developmental defects, including dilated third and lateral ventricles, reduced cerebral cortex, the corpus collasum crossing failure, the choroid plexus morphogenesis disruption.In order to address the pathogenesis of the CNS defects of CHARGE syndrome, we next examined midline apoptosis and neuroectoderm proliferation, the most crucial process in the midline induction and forebrain development. In the Chd7COA1/+ embryos, the midline apoptosis was attenuated, while the neuroectoderm proliferation was roughly reserved. Importantly, we found that Bmp4, a major morphogen regulating development of telencephalic midline, was down-regulated in midline of Chd7COA1/+ embryos and its expression pattern was also altered. The expression of Bmp4 targets, Msx1 and Msx2, and the expression of Foxg1(Bf1), whose expression is complementary to Bmp4, were altered consistently.Seeking to understand the mechanism whereby CHD7 regulates Bmp4 expression, we found a highly conserved fragment located downstream of Bmp4 bound with CHD7. We further studied the function of this fragment with luciferase report assay. This fragment showed repressive activity on the SV40 promoter and Bmp4 promoter, and the repressive activity was antagonized by CHD7.Altogether, our results presented first characterization of the forebrain defects in the Chd7 mutant mice, thus providing new (or first) insight into the pathogenesis of CHARGE syndrome. And our data suggested that CHD7 regulates the forebrain midline development via modulating Bmp4 expression, and this modulation was through antagonizing the inhibitory activity of the repressive element.