Roles Of FOXC1 Invertebrate Cardiogenesis

Transcription factor FOXC1 not only plays crucial role in vertebrate heart development but also involves in cardiac pathology.However,the molecular mechanisms remain poorly understood.In this study,we aim to investigate the role of FOXC1 in vertebrate cardiogenesis,and identify novel downstream target gene of FOXC1 during vertebrate early heart development.We comprehensively analyzed the cardiovascular phenotype of foxc1a,an orthologous of mammalian FOXC1,null zebrafish embryos.The results showed that the cardiac morphology,structure and function were disrupted in the mutants.Performing transcriptome analysis on foxc1a mutants,we demonstrated the expression of cardiac progenitor marker gene nkx2.5 was significantly decreased while the mesoderm formation was not affected.Dual fluorescent in situ hybridization assay showed that foxc1a and nkx2.5 were co-expressed in anterior lateral plate mesoderm at somite stage.Chromatin immunoprecipitation and promoter truncated assays demonstrated that Foxc1a regulated nkx2.5 expression through directly binding to two non-canonical binding sites in nkx2.5 promoter.Moreover,functional rescue experiments revealed that time specific overexpression of nkx2.5 partially rescued the cardiac defects of foxc1a null embryos.Furthermore,both Foxcl knock-down and overexpression assays supported the direct regulation of NKX2-5 by Foxcl is conserved in mammals.Interestingly,human FOXC1 with F112S or L86F mutation enhances the promoter activity of NKX2-5 abnormally,while FOXC1 with Q70Hfs*8,S82T,G149D and R170W mutation inversely repress the transcriptional activation of NKX2-5.On the other hand,the expression of some genes associated with RA signaling,WNT signaling and microRNAs have also been changed in foxcl a mutant zebrafish embryos.Taken together,our results demonstrate that Foxc1 acts direct upstream of Nkx2-5 or indirectly regulates Nkx2-5 by other signaling,such as RA,WNT or some microRNAs to regulate vertebrate cardiogenesis.Our findings will be helpful for us to understand the molecular mechanism of congenital heart diseases and the regulatory network of FOXC1 during vertebrate cardiogenesis.