Rapid and Efficient Live Zebrafish Embryo Genotyping.

Most current methods for genotyping zebrafish embryos require sacrifice or raising the animal to at least 1 month of age for fin amputation. These limitations restrict the use of zebrafish and increase time and costs for experiments. This article introduces an innovative method for genotyping live zebrafish embryos. The method utilizes enzyme to extract a small amount of genetic material from the skin tissue of the embryo. Then, using conventional polymerase chain reaction (PCR) strategy, the embryo is genotyped. This approach was successful >95% of the time while maintaining high viability (>90%) of the embryo. This effective method can facilitate high-throughput screening and other applications in zebrafish.

The transcription factor Foxc1a in zebrafish directly regulates expression of nkx2.5, encoding a transcriptional regulator of cardiac progenitor cells.

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 foxc1a, an ortholog of mammalian Foxc1, 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 foxc1a-null zebrafish embryos. Our results confirmed that foxc1a-null mutants exhibit disrupted cardiac morphology, structure, and function. Performing transcriptome analysis on the foxc1a mutants, we found that the expression of the cardiac progenitor marker gene nkx2.5 was significantly decreased, but the expression of germ layer-patterning genes was unaffected. Dual-fluorescence in situ hybridization assays revealed that foxc1a and nkx2.5 are co-expressed in the anterior lateral plate mesoderm at the somite stage. Chromatin immunoprecipitation and promoter truncation assays disclosed that Foxc1a regulates nkx2.5 expression via direct binding to two noncanonical binding sites in the proximal nkx2.5 promoter. Moreover, functional rescue experiments revealed that developmental stage-specific nkx2.5 overexpression partially rescues the cardiac defects of the foxc1a-null embryos. Taken together, our results indicate that during zebrafish cardiogenesis, Foxc1a is active directly upstream of nkx2.5.