Integration of spatial and single-cell transcriptomic data elucidates mouse organogenesis

Abstract

Molecular profiling of single cells has advanced our knowledge of the molecular basis of development. However, current approaches mostly rely on dissociating cells from tissues, thereby losing the crucial spatial context of regulatory processes. Here, we apply an image-based single-cell transcriptomics method, sequential fluorescence in situ hybridization (seqFISH), to detect mRNAs for 387 target genes in tissue sections of mouse embryos at the 8–12 somite stage. By integrating spatial context and multiplexed transcriptional measurements with two single-cell transcriptome atlases, we characterize cell types across the embryo and demonstrate that spatially resolved expression of genes not profiled by seqFISH can be imputed. We use this high-resolution spatial map to characterize fundamental steps in the patterning of the midbrain–hindbrain boundary (MHB) and the developing gut tube. We uncover axes of cell differentiation that are not apparent from single-cell RNA-sequencing (scRNA-seq) data, such as early dorsal–ventral separation of esophageal and tracheal progenitor populations in the gut tube. Our method provides an approach for studying cell fate decisions in complex tissues and development.

Squidpy: a scalable framework for spatial omics analysis

“…Clusters of enriched cell types (such as 'Lateral plate mesoderm' with ' Allantois' and 'Intermediate mesoderm' clusters, 'Endothelium' with 'Hematoendothelial progenitors'; Fig. 2b) are consistent with the original publication 33 and the spatial proximity can be visualized in Fig. 2c.…”

“…Squidpy provides several tools that leverage the spatial graph to address such questions. On a recently published seqFISH 33 dataset we built a spatial nearest-neighbor graph based on Delaunay triangulation and computed a permutation-based neighborhood enrichment across cell-type annotations (Methods). Clusters of enriched cell types (such as 'Lateral plate mesoderm' with ' Allantois' and 'Intermediate mesoderm' clusters, 'Endothelium' with 'Hematoendothelial progenitors'; Fig.…”

Tbx5 drives Aldh1a2 expression to regulate a RA-Hedgehog-Wnt gene regulatory network coordinating cardiopulmonary development

“…Immunostaining of transgenic Shh:GFP embryos confirmed previous reports that Aldh1a2 is co-expressed with Tbx5 in a subset of pSHF cells adjacent to the foregut Shh/Nkx2-1-expressing pulmonary endoderm ( Figure 1D–F” and Figure 1—figure supplement 1D-E'' Hochgreb et al, 2003 ; Ryckebusch et al, 2008 ; De Bono et al, 2018 ; de Soysa et al, 2019 ). Co-expression of Tbx5 and Aldh1a2 transcripts in the pSHF adjacent to Tbx1 + aSHF and Shh + endoderm were also evident in an online spatial atlas of single-cell gene expression ( Figure 1—figure supplement 1F ; Lohoff et al, 2021 ). To further define the spatial distinct aSHF-FGF and pSHF-RA signaling domains, we generated 3D reconstructions using serial sections of Aldh1a2 , Fgf8, Fgf10, and Shh in-situ hybridizations from the WT mouse E9.5 ( Figure 1—figure supplement 1G-H ).…”

“…( D, E ) Immunostaining of E8.5 ( D ) or E9 ( E ) mouse embryo foregut region shows co-expression of Tbx5 (green) and Aldh1a2 (red) protein in the foregut lateral plate mesoderm (fg lpm)/posterior second heart field (pSHF). ( F ) Digital in-situ hybridization using as online Spatial Mouse Atlas of single cell gene expression ( Lohoff et al, 2021 ). E9.5 digital in-situ hybridizations show the domains of Aldh1a2, Tbx5, Tbx1 , and Shh expression; yellow boxed region indicates Tbx5/Aldh1a2 co-expression in the pSHF lpm.…”

“…Digital in-situ hybridization for Aldh1a2 , Tbx1 , Tbx5 , and Shh was generated using the single cells Spatial Mouse Atlas ( Lohoff et al, 2021 ; https://crukci.shinyapps.io/SpatialMouseAtlas/ ). Images are centered around the cardiothoracic region with cardiomyocytes highlighted.…”