The Role And Mechanism Of Maternal Factors During Zygotic Genome Activation In Zebrafish

Zygotic genome activation(ZGA),as the core transcriptional regulation event in the early embryonic development of metazoans,has become a hot spot in the research of embryogenesis in recent years.However,there are still many technical challenges in the research on the role of maternal factors in early embryonic development and the mechanism of ZGA,due to the small amount of cells,the difficulty of operation and the short response time to drugs in early embryonic development.The first is the research method of gene function.The emergence of CRISPR/Cas9 as gene editing technology provides the foundation for simple,accurate and effective gene mutations.However,multiple generations of crossing and screening are still required to obtain homozygous mutant lines due to the low gene knockout efficiency.Therefore,systematic and large-scale research on gene function requires a lot of manpower and material resources and is difficult to achieve.The second is high-throughput omics technology,especially the method for detecting genome-wide binding sites of transcription factors(TFs).The common method for TF profiling is Ch IP-seq,but which is difficult to be applied to the research of early embryonic development due to its requirement for large number of cells and dependence on high-quality specific antibodies.Therefore,due to the lack of these necessary technologies,researchers still know little about the regulatory mechanism of early embryo development.Zebrafish has been widely used as a model organism in the study of vertebrate development,human genetic diseases and drug screening.Therefore,in this study,zebrafish was used as a model to establish the F0 homozygous mutation technology and develop an effective method for detecting the genome-wide binding site of TFs during early embryogenesis on the basis of existing technologies.Using these techniques,the functions and possible regulatory mechanisms of oocyte-specific genes and known pioneer TFs in early embryonic development were systematically studied.Previous studies have shown that oocyte-specific genes play an important role in early embryonic development,but there is still a lack of systematic research.Hence,in the first part of this study,based on the existing CRISPR/Cas9 technology,the effects of different concentrations of Cas9 m RNA and Cas9 protein,a single guide RNA(g RNA)and multiple g RNAs on the mutation efficiency were compared,and it was found that by co-injecting Cas9 protein and 4 g RNAs,nearly 100%gene knockout efficiency could be achieved and the F0 homozygous mutation technology could be constructed.At the same time,bioinformatics analysis was used to standardize the gene expression data of oocyte and other adult tissues or development processes,The genes and transcripts specifically expressed in oocyte were screened out,and the genes related to DNA binding,methylation and chromatin structure were found out by analyzing their conservative functional domains,and then the oocyte-specific genes to be further studied was also confirmed by RT-PCR experiment.Then,the F0 homozygous mutation technology was used to knock out these oocyte-specific genes one by one,and the developmental phenotypes of F1 embryos after gene mutation were observed.It was found that after the maternal deletion of 4genes(mcm3l,mcm6l,npm2a and orc1),the F1 embryos stopped developing before the completion of ZGA,indicating that these 4 genes are very important for the normal development of zebrafish early embryos.In addition,by comparing and analyzing gene expression data(m RNA-seq)and open chromatin capture data(ATAC-seq)in wild-type and mutant embryos,it was found that mutations of these 4genes affected the transcription activation of some genes and the establishment of open chromatin during ZGA;Moreover,through genome quantitative analysis,immunofluorescence staining and flow cytometry analysis,it was found that gene mutation also affected the structure of nucleus,normal replication of genome and equal division of cells.These results indicate that the maternal deletions of these 4oocyte-specific genes affect the rapid replication of DNA and normal cell division in zebrafish early embryos,which makes some genes unable to be activated during ZGA,and embryonic development arrests before the completion of ZGA.In addition,researchers believe that maternal TFs are the core transcription activators involved in regulating the occurrence of ZGA.Therefore,in the second part of this study,based on the existing―cleavage under targets and release using nuclease‖(CUT&RUN)technology,by fusing and expressing the Fc fragment of rabbit immunoglobulin G with the target TF,a method was developed to capture genome-wide binding site of TFs without the need for antibodies,called FitCUT&RUN(Fc fragment of immunoglobulin G tagging followed by CUT&RUN).Firstly,to test the effectiveness of FitCUT&RUN method,the FitCUT&RUN data of ELF1 and ATF1 were generated in 1x10~5 K562 cells,and the authenticity of FitCUT&RUN data was verified from multiple angles by comparing with the public Ch IP-seq data;Moreover,when the number of cells drops to 5x10~3,the TF profiling was consistent with that in a large number of cells by FitCUT&RUN,which indicates that this technology is still reliable in a small number of cells.Then,the genome-wide profilings of known pioneer transcription factors(Nanog,Pou5f3 and Sox19b)were generated in zebrafish early embryos by FitCUT&RUN technology.Compared with the limited public Ch IP-seq data,it was found that FitCUT&RUN technology could detect more real TF binding sites in a small number of embryos(50 embryos at dome stage).Finally,by analyzing the time-series FitCUT&RUN data of Nanog during the whole process of zebrafish ZGA(256-cell,1k-cell and dome stages),it was found that the occupancy of Nanog in the genome showed a gradual increasing trend with the development of embryos,and its occupancy at 256-cell stage was crucial to the activation of significant proportion of early transcribed genes.In addition,combining with genome replication timing data and motif analysis,regions occupied by Nanog earlier showed earlier replication timing and stonger motif strength,so it can be inferred that the sequential binding of Nanog may be regulated by replication timing and motif strength.To sum up,in this study F0 homozygous mutation technology was constructed in zebrafish and systematically studied the function of oocyte-specific genes in early embryo development by using this method,which is helpful to understand the underlying mechanisms of maternal factors regulating early embryo development;Meanwhile,a method for detecting TF binding sites in early embryos was developed and called as FitCUT&RUN.Using this technology,the genome-wide profiling of known pioneer transcription factors was obtained around zebrafish ZGA,which provided strong support for further study of the functions and regulation mechanisms of transcription activators during ZGA.