By contrast, subcluster 3 cells were in the
spinal cord parenchyma, whereas subclus-
ter 1 cells were mainly outside the CNS, and
thus might represent neural crest progenitors
(e.g., activeSox10) (Fig. 2I). Additionally, two
subclusters were found in the chondrocytes
and osteoblasts, and the genes related to de-
veloping teeth (e.g.,Barx1) had higher GAS in
subcluster 2 (fig. S12B).
The data were further used to examine the
developmental process from radial glia to
excitatory neurons through postmitotic pre-
mature neurons as the immediate state and
the cells were ordered in pseudotime. Spatial
projection of each pixel’s pseudotime revealed
the spatially organized developmental tra-
jectory (fig. S13). Cells early in differentiation
were enriched around the ventricles in the
developing brainstem, whereas those farther
away exhibited a more differentiated pheno-
type ( 21 ) (fig. S13B). We then identified changes
in gene activity based on H3K4me3 across this
developmental process, and many of the genes
recovered are important in neuron develop-
ment, includingPou4f1andCar10( 19 , 22 ) (fig.
S13, C and D).
We next combined spatial-CUT&Tag with
immunofluorescence staining in the same
tissue section (Fig. 3). We stained an olfactory
bulb with 4′,6-diamidino-2-phenylindole (DAPI)
for nuclear DNA (Fig. 3B) and next performed
spatial-CUT&Tag against H3K27me3 with
20-mm pixel size, which distinguished the
major cell types, including the glomerular
(C1) and granular (C2) layer (Fig. 3). Spatial
patterns of H3K27me3 modification were
validated by in situ hybridization (Fig. 3D).
With DAPI staining for the nucleus, we could
select the pixels of interest such as those
containing a single nucleus or those showing
specific histone modifications, allowing for
extracting single-cell epigenome data without
tissue dissociation (Fig. 3, E to I).
We also conducted spatial-CUT&Tag with
20-mm pixel size to analyze the brain region of
an E11 mouse embryo (fig. S14) and observed
distinct spatial patterns. H3K27me3 yielded
the most spatial clusters (fig. S14, A to C). The
clusters identified agreed with the projection
of ENCODE organ-specific bulk ChIP-seq data
in the UMAP embedding (fig. S14, C and D).
We further surveyed H3K27me3 modifications
and observed distinct modification patterns
across clusters (figs. S14E and S15A).Cfap77
was repressed extensively except in a portion
of the forebrain.Six1, which is involved in
limb development, had low CSS in cluster 5.
AlthoughSfta3-psandRhcglack H3K27me3
enrichment in the forebrain, they had distinct
spatial patterns. Pathway analysis of marker
genes revealed that cluster 1 was involved in
the forebrain development, cluster 2 corre-
sponded to anterior/posterior pattern spec-
ification, and cluster 4 was associated with684 11 FEBRUARY 2022•VOL 375 ISSUE 6581 science.orgSCIENCE
Fig. 3. Spatial mapping of an immunofluorescence-stained mouse olfactory bulb tissue section.
(A) H&E-stained image from an adjacent tissue section and a region of interest for spatial mapping.
(B) Fluorescent image of nuclear staining with DAPI. (C) Unsupervised clustering analysis and spatial
distribution of each cluster (20-mm pixel size). (D) Spatial mapping (left) of H3K27me3 modification for
selected marker genes. In situ hybridization (right) and expression images (middle) of corresponding
genes are from the Allen Mouse Brain Atlas. (E) Fluorescent images of selected pixels containing a single
nucleus (DAPI). (F) Heatmap of chromatin silencing score of selected pixels. (G) Comparison of unique
fragments in pixels with more than one nucleus and those with a single nucleus. (HandI) Unsupervised
clustering of selected pixels containing single nuclei (H) and colored by CSS for selected genes (I).
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