Combining Spatial Transcriptomics And Single-Cell Transcriptomics Analysis To Explore The Regulatory Mechanisms Of Zebrafish

During embryogenesis,a totipotent cell produces cell types with different functions,morphology,and spatial locations.This process is mainly regulated at the level of gene transcription.Therefore,recognizing the transcriptional states of different cell fate decisions is of great significance for understanding and manipulating cell development processes.Previous studies have systematically revealed the molecular regulatory mechanisms of zebrafish cell fate determination using single-cell transcriptomics,but how these regulatory factors interact spatially to induce complex vertebrate embryo development in a precisely controlled manner is one of the fundamental questions of embryogenesis and needs further study.In this study,we analyzed zebrafish embryos at 3.3h,5.25 h,10h,12 h,18h and 24 h after fertilization using transcriptomics and spatiotemporal omics to construct the spatiotemporal transcriptome map of early embryonic development of zebrafish.Then SPOTlight was used to integrate the data from single cell RNA sequencing(sc RNA-seq)and spatial enhanced resolution omics sequencing(Stereo-seq)to confirm the accuracy of both data.Finally,the developmental heterogeneity of zebrafish embryos at 3.3 h and 24 h after fertilization was analyzed by pseudotime and gene differential expression.The main results are as follows:1.In this study,a total of 139,391 sites were captured in zebrafish embryos at 3.3 h,5.25 h,10 h,12 h,18 h and 24 h post fertilization.The number of Unique Molecular identifiers(UMIs)at each time point is: 759(3.3 h),512(5.25 h),552(10 h),512(12 h),1569(18 h),2576(24 h).On this basis,the temporal and spatial transcriptome map of early embryogenesis of zebrafish was constructed.2.A total of 86,307 cells of zebrafish embryos were captured by transcriptome analysis at3.3 h,5.25 h,10 h,12 h,18 h and 24 h post fertilization and 71 cell types were identified.Cells at different developmental stages were visualized by Uniform Manifold Approximation and Projection(UMAP).3.The results of spatial sequencing showed that zebrafish embryos at 3.3 h after fertilization had three different cell subtypes: marginal blastomere,superficial blastomere and deep blastomere.Pseudotime analysis showed that the differentiation sequence was from marginal blastomere to deep blastomere and superficial blastomere.In situ hybridization staining results of cell type specific marker genes grhl3 and thy1 were consistent with sequencing data.4.SPOTlight integration analysis showed that in 3.3 hpf embryos,the early blastocyst was distributed in the ventral side of the embryo,while the late blastocyst disk was mainly distributed in the dorsal side,which was highly consistent with the spatial location of Stereo-seq data.5.The precise spatial transcriptome map of nervous system was obtained by unsupervised cluster analysis of 24 hpf embryos.The pseudotime and gene expression differences of posterior neural stem cells and neural progenitor cells were analyzed,and combined with their spatial location information,the spatial distribution of differentiation and heterogeneity of posterior neural stem cells was revealed.6.In situ hybridization staining was used to verify the expressions of key regulatory genes ntl dependent gene 5(ntd5),E2 F transcription factor 3(e2f3),replicative protein factor A3(rpa3)and apoptosis-inducing factor(siva1)in early embryonic development of zebrafish,and the staining results were consistent with the results of spatial group data analysis.In conclusion,this study constructed a spatio-temporal transcriptome map of early embryonic development of zebrafish,revealed the spatial heterogeneity of neural stem cell differentiation in the posterior brain,and verified the key genes regulating early development,thus will provide a theoretical reference for further understanding of vertebrate embryonic development.