Bioinformatics Study Of The Spatiotemporal Transcriptional Regulation Mechanism Of Human Embryonic Retinal Development

The retina is part of the central nervous system and consists of various types of neurons(photoreceptor cells,horizontal cells,bipolar cells,amacrine cells,and retinal ganglion cells)and glial cells,responsible for processing visual information.Deciphering the molecular mechanisms of human embryonic retinal development is important for retinal disease prevention and treatment.Most of the current research on retinal development is based on model animals,yet little is known about the molecular mechanisms of human embryonic retinogenesis.The development of high-throughput sequencing technology in recent years has made it possible for us to systematically and comprehensively study the process of organ differentiation and development.Existing omics studies have revealed gene expression and histone epigenetic maps in human retinal development,while the transcriptional regulatory mechanisms during human embryonic retinal development remain poorly understood.In addition,retinal organoids formed by human induced pluripotent stem cells in vitro provide a new model for studying human embryonic retinas.However,the similarities and differences between these two differentiation processes at the transcriptional and chromatin levels remain unclear.In addition,gene expression is spatiotemporally specific,and the construction of spatiotemporal expression profiles for human retina is crucial for us to understand the human embryonic retinal development.Therefore,we used ATAC-seq,scATACseq,Spatial Transcriptomics,and other multi-omics technologies to integrate epigenome and transcriptomics data to decipher the transcriptional regulation network in human embryonic retinal development.The content of this thesis is mainly divided into two parts.The first part of the work is to use ATAC-seq technology to study the transcriptional regulation mechanism of human embryonic retinal development.To systematically reveal the landscape of chromatin accessibility and gene expression in developing human retina and retinal organoids,we collected ATAC-seq and RNA-seq data of human embryonic retina and retinal organoids samples at different developmental time.Our results showed that the chromatin accessibility and transcriptional state of the retinal organoid differentiation process were similar to those of human embryonic retinal development,suggesting that retinal organoids would be a good model for studying the molecular mechanisms of human embryonic retina.Moreover,we found that the region related to neural differentiation in the middle stage of human embryonic retinal development was in a bivalently modified state(H3K27me3 and H3K4me3),and this region had a phenomenon of delayed opening in retinal organoids differentiation.Through integrated analysis of RNA-seq data and ATAC-seq data,we identified transcription factors NFIB and THRA as necessary regulators for human retinal development,and validated by retinal organoids.Finally,we reconstructed the transcriptional regulatory network at different stages of retinal development.Further,we collected published single-cell ATAC-seq data during human embryonic retinal development to identified the cell-type-specific chromatin open regions and transcriptional regulators of human embryonic retina at different developmental stages.In addition,human retina has a complex spatial structure,with cells spatially arranged to form three nuclear layers and two synaptic layers.To study the spatial development map of retinal cells,in the second part of the work,we used spatial transcriptomic technology to study the spatiotemporal expression profiles during retinal development.Yet,the current mainstream spatial transcriptome technologies have shortcomings such as low gene throughput(only a small number of RNA molecules can be detected)or low resolution(each spot contains multiple cells),respectively,making it difficult for researchers to obtain the complete transcriptome information of a single cell.To overcome the limitations of these spatial transcriptome technologies,researchers have developed many intergration methods that combing spatial transcriptome and single-cell transcriptome data to predict the spatial distribution patterns of genes or cellular subtypes.To choose appropriate integration methods for spatial data analysis,we designed a standard pipeline to compare the performance of 16 integrated algorithms in predicting the spatial distribution of genes or cells with 45 pairs of real spatial transcriptome and single-cell transcriptome datasets,and 32 pairs of simulated datasets.Afterwards,we obtained a spatiotemporal overview of human embryonic retina at gestation weeks 11 and 14,and performed an integrative analysis of spatial transcriptome data using Tangram and Cell2location.Our research revealed the spatial distribution of genes and retinal cell types,and systematically studied the functions of different spatial domains in the human retina by identifying functional spatial regions.Finally,we constructed the spatial differentiation trajectories of retinal cells by pseudotime analysis.In summary,this study used bioinformatics methods to comprehensively demonstrate the dynamic changes of chromatin accessibility during human embryonic retinal development and retinal organoids differentiation;draw the map of regulatory element and transcription factor binding during human embryonic retinal development;identified transcription factors NFIB and THRA as necessary regulators for human retinal development,and validated by retinal organoids;constructe the transcriptional regulatory network for human embryonic retinal development;identificatr the celltype-specific chromatin open regions and transcription factors;developed an unbiased,systematic evaluation pipeline for evaluating the performance of intergration methods for spatial transcriptome and single-cell transcriptome data;and reveal spatial distribution patterns of key genes and cell types for human embryonic retinal development.It fills the gap in the research on epigenetic regulation of human retinal development in the field,and provides an important research basis and valuable data resources for future research on human embryonic retinal development and improvement of retinal organoids differentiation systems.