Embryo-scale, single-cell spatial transcriptomics

Abstract

Spatial patterns of gene expression manifest at scales ranging from local (e.g., cell-cell interactions) to global (e.g., body axis patterning). However, current spatial transcriptomics methods either average local contexts or are restricted to limited fields of view. Here, we introduce sci-Space, which retains single-cell resolution while resolving spatial heterogeneity at larger scales. Applying sci-Space to developing mouse embryos, we captured approximate spatial coordinates and whole transcriptomes of about 120,000 nuclei. We identify thousands of genes exhibiting anatomically patterned expression, leverage spatial information to annotate cellular subtypes, show that cell types vary substantially in their extent of spatial patterning, and reveal correlations between pseudotime and the migratory patterns of differentiating neurons. Looking forward, we anticipate that sci-Space will facilitate the construction of spatially resolved single-cell atlases of mammalian development.

Figure 1.. sci-Space recovers single cell transcriptomes while recording spatial coordinates.

(A) Arrayed single-stranded oligos are transferred onto permeabilized nuclei in fresh-frozen tissue sections and imaged. Cells from each slide are also labeled with a section-identifying barcode so that multiple sections can be pooled prior to sci-RNA-seq. (B) Joint embedding of E14.0 single cell transcriptomes from this study and published data spanning E9.5 to E13.5 (1). Major trajectories are labeled. (C) UMAP embedding of 121,909 cells from sectioned E14.0 mouse embryos. Cell types are denoted by color and number in legend.

Figure 2.. sci-Space captures spatially and cell type resolved gene expression across the embryo.

(A) Co-registered DAPI stained section image and oligo array, superimposed. SYBR waypoints are highlighted in green. (B) Anatomical regions of Slide 1 (left) and an adjacent immunostained serial section aligned to Slide 1 (right). (C) Highlighted cell types mapping to a single slide. (D) UMAP embedding colored by anatomical regions. (E) Gene expression of dopamine transporter Slc6a3 from sci-Space data (left) and published (27) section/stage matched in situ (right). (F) Smoothed percentage of gene expression for Fgf1, Fgfr1, Fgfr2 and Fgfr3 in cardiomyocytes (top) or endothelial cells (bottom). Color scaled to maximum percentage within each gene. Scale bars in panels (A-C) = 0.5 mm.

Figure 3.. Spatially restricted gene expression in developing neurons.

(A) Number of spatially significant (Moran’s test, FDR < 0.001) autocorrelated genes within each slide (color) and cell type. Only cell types with more than 50 cells per slide were included. (B) Log-log (log10) plot of autocorrelation in UMAP embedding (x-axis) versus autocorrelation in spatial coordinates (y-axis) for each gene. Computed on excitatory neurons from Slide 1. Moran’s I values close to 1 indicate perfect spatial correlation, while a value of 0 indicates a random spatial distribution. Hox genes are highlighted. (C) log10-scale boxplot of Moran’s I statistic for Hox genes displayed in (B) versus all other expression level-matched genes (p-value < 0.001, two sided t-test). (D) Gene expression of HoxC cluster in Slide 1. (E) Similar to (B), log-log (log10) plot of autocorrelation in UMAP embedding (x-axis) versus autocorrelation in spatial coordinates (y-axis) for each gene with genes in different regimes highlighted for Slide 1. (F) Expression patterns across Slide 1 for other spatially restricted genes that are not restricted to a single neuron subcluster. (G) Comparison of sci-Space (Slide 14) and RNA FISH (RNAscope) detected Cyp26b1 patterns of expression (gray) and coexpression with markers (colors as indicated in key) for neuronal and supportive cell types.

Figure 4.. Quantifying the variance in gene expression attributable to spatial position.

(A) Pairwise angular distance (radians) between global transcriptomes of cells of the indicated cell types. Cell pairs are grouped by distance on the physical array (mm) (** p-value < 0.001, *** p-value < 0.0001, Wald linear regression test). (B) Proportion of non-technical variance, explained within cells of each type by spatial position. Point size indicates number of cells and point color indicates the slide of origin. (C) Recovered positions of chondrocytes from Slides 6, 11 and 14 colored by subcluster. Arrows indicate focal concentrations of craniofacial mesenchyme (green) and digit condensate subclusters (red). Insets to the right of each plot 9 show parts of each image with similarly positioned arrows. White text of each inset labels the anatomic structure displayed. Scale bars in panel (C) = 0.25 mm.

Figure 5.. Pseudotemporal spatial trajectories capture migratory patterns in the developing brain.

(A-D) UMAP embedding displaying the neural trajectories colored by (A) cell type, (B) specific gene expression, (C) cortical region, or (D) pseudotime. (E) Neurons and radial glia in the cortex colored by pseudotime or otherwise colored grey. Insets of caudal (above) and rostral (below) brain outlines are shown to the right of each slide (M-Midbrain, P-Pallium, SP-Sub Pallium, V-Ventricle). (F) Scaled and centered gene expression for genes (rows) significantly varying over pseudotime in all three trajectories. Enriched Gene Ontology Biological Processes terms (GO BP) are displayed next to clustered genes.