silencing score (CSS) was calculated to pre-
dict the gene expression ( 9 ), and active genes
should have a low CSS because of the lack of
an H3K27me3-repressive mark (Fig. 2A and
fig. S7A). For example,Hand2, which is re-
quired for vascular development and plays an
essential role in cardiac morphogenesis ( 19 ),
showed a lack of H3K27me3 enrichment in
the heart (C1). For H3K4me3 and H3K27ac,
gene activity score (GAS) was used because
they are related to active genes (Fig. 2B and
figs.S8AandS10A).Forexample,Nfe2, which
is essential for regulating erythroid and hema-
topoietic cell maturation and differentiation
( 19 ), was active in liver and to some extent in
the heart (C2 and C6 for H3K4me3). We further
analyzed Gene Ontology (GO) pathways, and
the results agreed with the anatomical an-
notation (figs. S7B, S8B, and S10B). To un-
derstand which regulatory factors are most
active across clusters, we calculated tran-
scription factor (TF) motif enrichments in
H3K4me3 and H3K27ac modification loci
(figs. S9 and S11). As expected, the most en-
riched motifs in liver correspond to GATA
transcription factors includingGata2, where-
asMef2awas enriched in the heart region. To
predict the gene-regulatory interactions of en-
hancers and their target genes across clusters,
we correlated gene expression from single-cell
RNA-sequencing (scRNA-seq) ( 5 ) and H3K27ac
modifications at the candidate enhancers
(Fig. 2C). The correlation-based map success-
fully predicted some enhancer-gene interac-
tions that have been experimentally validated.
For example, the predicted enhancers ofAscl1
andKcnq3were enriched in the CNS (C1, C2,
C3,andC6),whichisinagreementwiththe
VISTA validated elements ( 20 ).
We then integrated scRNA-seq data ( 5 ) with
spatial-CUT&Tag data to identify cell types
(Fig.2,DtoH,andfig.S12).Spatialtissue
pixels were found to conform well into the
clusters of single-cell transcriptomes. Sev-eral organ-specific cell types were detected
(Fig. 2, E and G). For example, the definitive
erythroid lineage cells were exclusively en-
riched in the liver, which is the major hema-
topoietic organ at day 11.5 ( 19 ). Cardiac muscle
cell types were observed only in the heart re-
gion in agreement with the anatomical an-
notation. Chondrocytes and osteoblasts were
observed widely in the embryonic facial prom-
inence. Inhibitory interneurons were highly
enriched in the brain stem. Postmitotic pre-
mature neurons were observed extensively
in the spinal cord region. A high-resolution
clustering further identified subpopulations
of developing neurons with distinct spatial
distributions and chromatin states (Fig. 2I and
fig. S12B). For instance, the H3K27ac radial
glia could be further divided to three clusters.
Genes related to stem cell maintenance in the
CNS (e.g.,Sox1) had higher GAS in subcluster 2,
which was enriched along the ventricles in
the developing brain stem and spinal cord.SCIENCEscience.org 11 FEBRUARY 2022¥VOL 375 ISSUE 6581 683
Fig. 2. Spatial mapping and integrative analysis of mouse embryos.(Aand
B) Genome browser tracks (left) and spatial mapping (right) of gene silencing
by H3K27me3 and gene activity by H3K4me3 modification. (C) Predicted
enhancers ofAscl1(chr10: 87,463,659 to 87,513,660; mm10) (left) andKcnq3
(chr15: 66,231,223 to 66,331,224; mm10) (right) from H3K27ac profiling. Cluster
of each track corresponds to Fig. 1F. Enhancers validated by in vivo reporter assays
are shown between main panels. (DandF) Integration of scRNA-seq ( 5 ) and
spatial-CUT&Tag data. (EandG) Spatial mapping of selected cell types identified
by label transferring. (H) List of cell types in scRNA-seq. (I) Refined clustering of
radial glial enabled identification of subpopulations. Scale bar, 1 mm.RESEARCH | REPORTS