cells and physically joining barcodes in adjacent
cells connected by synapses. A variation of this
idea but based on injection of barcoded viral
particles [multiplexed analysis of projections by
sequencing (MAPseq)] ( 61) can be used to map
thousands of long-range projections but not link
them to molecular cell identities or to specific tar-
get cells. An imaging-based approach, FISSEQ-
BOINC ( 62), envisaged direct in situ sequencing
of barcodes located in the pre- and postysynaptic
compartments across a synaptic cleft. Although
presently none of these strategies link connectiv-
ity to molecular identity, this could be achieved
in principle through simultaneous detection of
gene expression in the pre- and postsynaptic cells.
However, any mapping of complete circuits would
require imaging very large volumes, increasing
the imaging throughput challenge.
Spatial transcriptomics of brain disease
Although the greatest short-term scientific im-
pact of spatial transcriptomics may be on the
fundamental question of brain architecture, the
greatest long-term societal impact may instead
come from applying integrated spatial transcrip-
tomics to human neurological disease. Most brain
disorders are either developmental or degenera-
tive, and in both cases, the spatial organization of
the diseased brain is of fundamental importance.
With a detailed understanding of brain cell types,
and their localization, comes an opportunity to
develop new molecular pathology to diagnose and
understand brain disease. For example, a type of
disease-associated activated microglia was recently
discovered by using single-cell RNA-seq in a mouse
Alzheimer’s disease model, and the authors used
spatial methods to show that these activated
microglia are specifically located near b-amyloid
plaques (Fig. 1B) ( 63). This immediately suggests
a plausible mechanism for the origin of Alzheimer’s
disease in humans (insufficient microglia activa-
tion) and an avenue for targeted treatment with
Many brain diseases similarly show strong
spatial organization, including multiple sclerosis
(white matter lesions) and Parkinson’s disease
(Lewy bodies). Brain tumors, in particular, are
complex cellular environments, and single-cell
RNA-seq has been used to show how multiple
parallel tumor clones are intermingled with nor-
mal neurons and glia as well as activated immune
and vascular cells ( 64–66). To fully understand
the interplay of cell-cell interactions and com-
plex differentiation in tumors, spatial transcrip-
tomics will be an invaluable contribution.
Transcriptomics has provided a new para-
digm for understanding the nervous system in
terms of its genetically defined cell types. Spa-
tial transcriptomics methods will contribute to
a deep understanding of cell types and neuro-
nal circuitry in developing, adult, and diseased
brains, provided that technical hurdles can be
overcome to make them broadly applicable in
combination with other analytical methods.
The clear definition of specific-use cases and
associated requirements, and standardization
of experimental and analytical methods, will
help this field realize its remarkable potential
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G TG A
Fig. 3. Spatially resolved transcriptomics achieve high levels of multiplexing through multiple
rounds of probing, imaging, and stripping. In additive smFISH, a small number of spectrally
resolved probes is used in each round. seqFISH uses temporal barcodes, in which the combination
of signal across all cycles is specific to each target. MERFISH uses an error-corrected barcode to
reduce the effect of false-positive and -negative signals. In situ sequencing uses padlock probes
to target specific mRNAs, with cDNA synthesis and rolling-circle amplification in situ, followed
by sequencing by ligation. FISSEQ uses a similar principle, but reverse-transcribes RNA in an