Transcription factors play crucial roles in patterning posterior neuroectoderm. Previously, zinc finger transcription factor was reported to be expressed in the posterior neuroectoderm of zebrafish embryos. However, its roles remain unknown. Here, we report that there are 13 copies of in the zebrafish genome, and all the paralogues share highly identical protein sequences and cDNA sequences. When are knocked down using a morpholino to inhibit their translation or dCas9-Eve to inhibit their transcription, the zebrafish gastrula displays reduced expression of, the marker gene for the posterior neuroectoderm. Further analyses reveal that diminishing produces the decreased expressions of, whereas overexpression of effectively rescues the reduced expression of in the posterior neuroectoderm. Additionally, knocking down causes the reduced expression of, a direct regulator of , in the posterior neuroectoderm, and overexpression of rescues the expression of in the knockdown embryos. In contrast, knocking down either or does not affect the expressions of Taken together, our results demonstrate that zebrafish control the expression of in the posterior neuroectoderm by acting upstream of and .

Cardiogenesis is a tightly controlled biological process required for formation of a functional heart. The transcription factor Foxc1 not only plays a crucial role in outflow tract development in mice, but is also involved in cardiac structure formation and normal function in humans. However, the molecular mechanisms by which Foxc1 controls cardiac development remain poorly understood. Previously, we reported that zebrafish embryos deficient in , an ortholog of mammalian, display pericardial edemas and die 9-10 days postfertilization. To further investigate Foxc1a's role in zebrafish cardiogenesis and identify its downstream target genes during early heart development, we comprehensively analyzed the cardiovascular phenotype of -null zebrafish embryos. Our results confirmed that-null mutants exhibit disrupted cardiac morphology, structure, and function. Performing transcriptome analysis on the mutants, we found that the expression of the cardiac progenitor marker gene was significantly decreased, but the expression of germ layer-patterning genes was unaffected. Dual-fluorescence hybridization assays revealed that and are co-expressed in the anterior lateral plate mesoderm at the somite stage. Chromatin immunoprecipitation and promoter truncation assays disclosed that Foxc1a regulates expression via direct binding to two noncanonical binding sites in the proximal promoter. Moreover, functional rescue experiments revealed that developmental stage-specific overexpression partially rescues the cardiac defects of the -null embryos. Taken together, our results indicate that during zebrafish cardiogenesis, Foxc1a is active directly upstream of.

FOXC1 is a member of the forkhead family of transcription factors, and whose function is poorly understood. A variety of FOXC1 mutants have been identified in patients diagnosed with the autosomal dominant disease Axenfeld-Rieger syndrome, which is mainly characterized by abnormal development of the eyes, particularly those who also have accompanying congenital heart defects (CHD). However, the role of FOXC1 in CHD, and how these mutations might impact FOXC1 function, remains elusive. Our previous work provided one clue to possible function, demonstrating that zebrafish foxc1a, an orthologue of human FOXC1 essential for heart development, directly regulates the expression of nkx2.5, encoding a transcriptional regulator of cardiac progenitor cells. Abnormal expression of Nkx2-5 leads to CHD in mice and is also associated with CHD patients. Whether this link extends to the human system, however, requires investigation. In this study, we demonstrate that FOXC1 does regulate human NKX2-5 expression in a dose-dependent manner via direct binding to its proximal promoter. A comparison of FOXC1 mutant function in the rat cardiac cell line H9c2 and zebrafish embryos suggested that the zebrafish embryos might serve as a more representative model system than the H9c2 cells. Finally, we noted that three of the Axenfeld-Rieger syndrome FOXC1 mutations tested increased whereas a fourth repressed the expression of NKX2-5. These results imply that mutant FOXC1s might play etiological roles in CHD by abnormally regulating NKX2-5 in the patients. And zebrafish embryos can serve as a useful in vivo platform for rapidly evaluating disease-causing roles of mutated genes.