Spatially resolved single-cell transcriptomics census of developing embryonic chicken hearts This analysis identifies transcriptional differences between epithelial and mesenchymal cells, further clarifies the differentiation transitions within the epicardial lineage, and points to the utility of spatiotemporal single-cell RNA sequencing to study cardiogenesis. We furthermore construct a similarity map between single-cell and spatial transcriptomes, which enables us to spatially map lineage-associated differentiation trajectories within the tissue. We use these cell-type predictions to construct proximity maps identifying changes in local cellular environments and uncovered spatially restricted regulatory programs. We integrate scRNA-seq and spatial RNA-seq data using an anchor-based method to predict cell type annotations for spatially resolved transcriptomes. We characterize and spatially resolve progenitor and differentiated cell types, identify stage-specific transcriptional programs and cellular interactions, reconstruct differentiation lineages, and delineate important regulatory programs in cardiac development. In this study, the combination of single-cell and spatial transcriptomics uniquely enables us to unravel cellular interactions that drive cardiogenesis and reconstruct a high-resolution, spatially resolved gene expression census of developmental cardiac lineages. In addition, we performed spatially resolved RNA-seq on a total of 12 heart tissue sections at the same four stages. The data encompasses common and rare cell types, including progenitor and mature cell types from multiple lineages. We generated over 22,000 single-cell transcriptomes across four key Hamburger-Hamilton ventricular development stages (HH21-HH24, HH30-HH31, HH35-HH36, and HH40). Current spatial transcriptomics approaches lack single-cell resolution, which we addressed here using approaches to integrate high-throughput spatial and single-cell transcriptomic data.Ĭhicken embryos were used as a cardiogenesis model system since the ex-utero development in an egg allows unique access to early cardiogenesis stages and the chicken heart anatomy resembles many aspects of human heart anatomy 3. Here, we combined spatially resolved RNA-seq with high-throughput scRNA-seq to study the spatiotemporal interactions and regulatory programs that drive embryonic chicken heart development. Single-cell RNA-sequencing (scRNA-seq) has been used to study the cellular mechanisms involved in heart development, but does not preserve spatial information, and does not enable studies of the complex interplay between cellular differentiation and morphogenesis. The heterogeneity of cell types during cardiac development makes it difficult to study cardiac fate decisions using traditional developmental biology techniques. Cardiogenesis involves heterogeneous cell populations from multiple lineages that spatiotemporally interact to drive cardiac fate decisions 2. The heart is the first fully functional organ to develop and is vital for embryogenesis 1. Overall, our study identifies an intricate interplay between cellular differentiation and morphogenesis.
Last, we discover a persistent enrichment of the small, secreted peptide, thymosin beta-4, throughout coronary vascular development. Using spatially resolved expression analysis, we identify anatomically restricted expression programs, including expression of genes implicated in congenital heart disease. Spatial mapping of differentiation transitions in cardiac lineages defines transcriptional differences between epithelial and mesenchymal cells within the epicardial lineage.
We create a census of the diverse cellular lineages in developing hearts, their spatial organization, and their interactions during development. Here, we combine single-cell and spatial transcriptomics with algorithms for data integration to study the development of the chicken heart from the early to late four-chambered heart stage. Single-cell RNA sequencing is a powerful tool to study developmental biology but does not preserve spatial information about tissue morphology and cellular interactions.
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