Science - 06.03.2020

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the IF data indicate a distinct neuronal ex-
pression of NECAB1 in subsets of neurons in
various regions of the thalamus and forebrain,
including pyramidal neurons in the cerebral
cortex. The (nor)epinephrine uptake trans-
porter (SLC6A2), also called NET1, is an ex-
ample of an apparent partial discrepancy
between the RNA and the protein location:
The RNA transcript is detected in cell bodies
of the locus coeruleus in the pons with an
expression pattern resembling that of the
well-characterized (nor)epinephrinergic neu-
rons. However, this transporter protein can-
not be detected in the cell bodies with IF. This
is because NET1 is rapidly transported into the
extensive axonal network. Adenylate cyclase–
activating polypeptide 1 (ADCYAP1) is known
to stimulate the generation of cyclic adenosine
monophosphate (cAMP), and all three data-
sets show widespread expression across the
brain regions, with the highest levels in the
hypothalamus and amygdala. In the latter
region, ISH shows that this gene is expressed
by cells located in the cortical amygdaloid
nucleus, whereas protein labeling is found
in nerve terminals in the central amygdaloid
nucleus, which is known to receive an input


from the cortical amygdala. The results sug-
gest that this protein is primarily presynaptic
and support a role in cAMP-mediated synap-
tic plasticity. Expression of the orphan G
protein–coupled receptor 151 (GPR151) can be
visualized both with ISH and IF in neuronal
cell bodies in the habenular nucleus of the
mouse thalamus. However, the protein stain-
ing also shows the projection from the thal-
amus to the interpeduncular nucleus in the
midbrain. This orphan receptor is thus visual-
ized in the neuronal soma, in axons running in
the fasciculus retroflexus, and in the presyn-
aptic terminals. This expression pattern can
also be shown using three-dimensional imag-
ing of solvent-cleared brain (iDISCO) en-
compassing a whole mouse hemisphere (Fig.
3B). In addition, this analysis revealed that
a portion of the axons pass the interpedun-
cular nucleus and innervate the parabrachial-
pericoerulear region. Movie S1 shows the
three-dimensional location of this orphan
receptor. Together, these examples illustrate
how combining three different approaches for
spatial transcriptomics and proteomics results
in insights offering detailed information on
cellular expression and protein location.

Species comparisons of regional brain
expression
To compare the expression profiles in the
three mammalian brains, all genes with one-
to-one orthologs in human, mouse, and pig
were identified, and a total of 12,999 protein-
coding genes were analyzed (fig. S19A). Addi-
tional genes can be included in the analysis
in the future, as additional one-to-one gene
orthologs are identified. A combined hierar-
chical tree including all regions of the three
species based on all regionally elevated genes
across the 10 main regions is shown in Fig. 4A.
The results again support a preserved brain ar-
chitecture, where the hypothalamus and cer-
ebellum of all three species cluster in proximity
to each other. Similarly, the brainstem regions
and the cerebrum regions cluster together.
Neighboring regions from one species cluster
together, but clustering is less tight for cor-
responding regions from different species. The
olfactory bulbs from pig and mouse are clus-
tered tightly together, and the outlier is the
olfactory bulb from humans, which shows
similarities with the cerebrum regions of hu-
mans. This might be due to sampling error, as
discussed above, but could also reflect the

Sjöstedtet al.,Science 367 , eaay5947 (2020) 6 March 2020 4of16


Olfactory bulb Amygdala

Antibody

/

DAPI

Gene ISH

IGFBP5 NECAB1 ADCYAP1

ISH

RNAseq

IF

200 120 100

12 10 10

200 200 200

Cerebral cortex

SLC6A2
10

6

200

Pons & Medulla Thalamus

GPR151
100

10

200

AB

cerebellum

brainstem

cerebral cortex
mhb

mhb

fr

fr

frfr
ip

pb-pc

ca

coa

ca

coa

lc

lc

I
II
III-IV

gra

ml

gl

V
VI

I
II
III-IV
V
VI

mhb

lhb

mhb

lhb

epi

gra

ml

gl

epi

Fig. 3. Comparison of mouse regional data from transcriptomics, in situ
hybridization, and immunofluorescence.(A) Examples of expression
profiles from the HPA Brain Atlas, with mouse expression shown as data from
RNA-seq (this study), in situ hybridization (ISH) data from the Allen Brain
Atlas, and regional staining intensity based on antibody-based immuno-
fluorescence (IF) profiling (this study). The color codes are the same as in
Fig. 2. Below are examples of ISH and IF staining for each gene, from left to
right: insulin-like growth factor binding protein 5 (IGFBP5); N-terminal
EF-hand calcium binding protein 1 (NECAB1); norepinephrine transporter, also


called solute carrier family 6 member 2 (NET1 or SLC6A2); adenylate cyclase–
activating polypeptide 1 (ADCYAP1); and G protein–coupled receptor 151
(GPR151). Glomerular layer, gl; external plexiform layer, epi; mitral cell layer, ml;
granule cell layer, gra; locus coeruleus, lc; central amygdala, ca; cortical
amygdala, coa; lateral habenula, lhb. (B) iDISCO+ volume immunostaining of a
whole mouse brain for GPR151 receptor. The medial habenula (mhb), fasciculus
retroflexus (fr), interpeduncular nucleus (ip), and parabrachial-pericoerulear
region (pb-pc) are strongly stained. The boxed region in the top image is
enlarged in the bottom image. Scale bars, 250mm.

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