Associated With The Development Of Gene Cloning And Functional Studies. Zebrafish Blastula

The midblastula transition (MBT) represents a major phase in embryonic development.Prior to the MBT, the early stage of embryo development is mostly regulated by maternal genes and shows a very low level of transcription. Although Cdk7/Cyclin H has also been implicated to be involved in the regulation of Cdk-activating kinase (CAK) and of RNA polymerase, the function of these orthologues in zebrafish MBT has not been fully elucidated. In the current study, two novel cDNAs encoding Cyclin H and Cdk7 were cloned from zebrafish blastula embryos. Sequence analysis data and Phylogenetic parsimony tree analysis suggested zebrafish Cyclin H and Cdk7 cDNA were conservative to the human orthologue respectively. RT-PCR and whole-mount in situ hybridization (ISH) analysis, suggested that Cyclin H and Cdk7 were maternally loaded in embryos and organs of adult fish.Distribution of Cyclin H and Cdk7 transcripts was ubiquitous during early stages, becoming restricted to the anterior neural tube, brain, eyes, procreate tissues, liver and heart by 5 days post-fertilization (dpf). Expression of cdk7 was lower and more concentrated than Cyclin H. Overexpression of Cyclin H affected the rate of epiboly of early embryos and induced malformation of tail; Expression of dominant-negative forms of Cyclin H delayed the onset of zygotic transcription in the early embryo, resulting in apoptosis at 5 hours post-fertilization (hpf) and leading to sever defects in tissues with high Cyclin H expression. These results indicated that Cyclin H played a critical role in the regulation of the transcriptional machinery during MBT, suggesting that it was an essential gene in early zebrafish larval development. In zebrafish, the genetic approach by ENU mutagenesis discovered some genes related to MBTdevelopment,however,many genes with critical roles during the MBT remain undemonstrated. Present reseaarch analyzed those differentially expressed genes in zebrafish MBT using GeneChip? Zebrafish Genome Array of Affymetrix. Total RNA of 2.5hpf and 4hpf were subjected to GeneChip? Zebrafish Genome Array. We detected 360 up-regulated and 119 down-regulated genes in 4hpf compared to 2.5hpf. There were two criterions for selecting genes for further study: one was significantly different expression level between 2.5 hpf and 4 hpf; the other was genes related to cell cycle and regulation of cellcycle.PCR was used to confirm the reliability of genechip results.Then, the whole-mount in situ hybridization was also applied to investigate the spatial specificity of the expression of the genes. Subsequently, based on above information and background of these genes, more could be done to study the functions of the genes during MBT.