Science - 06.03.2020

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are essential for differentiation and mainte-
nance of cell identities in the postmitotic brain
( 29 ); (ii) cell identity genes, including neuro-
peptides, proteins, and enzymes responsible
for the production, transport, and clearance of
neurotransmitters (n=63);and(iii)allknown
neurotransmitter and neuropeptide receptors
and receptor subunits (n= 118). Comparing
the correlation values between species for
these protein classes reveals a higher correla-
tion for transcription factors (P< 0.001), cell
identity genes (P< 0.001), and receptors (P<
0.01) relative to the gene expression of all other
11,765 genes with their one-to-one ortholog
(Fig. 5A).
We found that expression of some transcrip-
tion factors is highly conserved across the


three species, while other transcription factors
have a less-maintained expression profile, thus
affecting the overall correlation (fig. S28). Ex-
amples of some of the transcription factors
with conserved distribution across the brain
regions are illustrated in Fig. 5B. Homeobox
protein 1 (EMX1), known to be expressed by
most of the neurons in the cerebral cortex
and hippocampus ( 30 ), has a similar expres-
sion pattern and expression levels in all three
species. Class E basic helix-loop-helix protein
22 (BHLHE22, or Bhlhb5), which regulates
postmitotic differentiation of cortical neurons
( 31 ), has a highly similar expression pattern in
the cerebrum regions. In contrast, expression
of the transcription factor SIX6 is restricted to
the hypothalamus of all three species, which

is in line with earlier reports ( 32 ). Pituitary
homeobox 3 (PITX3) is mainly expressed in
the midbrain in all three species, with the
highest expression in the substantia nigra in
pig, also supporting earlier observations that
PITX3 plays a role in the development of do-
paminergic neurons of the substantia nigra in
mice ( 33 ). Homeobox protein Hox-B5 (HOXB5)
is specifically expressed in the pons–medulla
oblongata region, supporting the observation
that Hox genes are expressed in the hindbrain
and known to be important for the segmental
patterning of this part of the brain. Neurogenic
differentiation factor 1 (NEUROD1) is essential
for the development of the cerebellum in rats
( 34 ). We find that this transcription factor is
restricted to the cerebellum in all three species

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


Fig. 5. Expression profiles of cell identity genes in
the mammalian brain.(A) Overall Pearson correlation
between species for transcription factors (red),
genes involved in the production and processing of
neurotransmitters and neuropeptides (blue), and
metabotropic and ionotropic neurotransmitter and
neuropeptide receptors (green) in contrast to all other
genes (dark blue). (B) Examples showing the relative
expression in the 10 regions of human, pig, and mouse
brains display elevated expression in developmental
and anatomical defined regions of the brain.
(C) Comparing the relative distribution of the mono-
aminergic systems and (D) selected neuropeptide
genes reveals a conserved pattern of expression,
especially of the enzymes responsible for the produc-
tion of dopamine (TH, DDC), noradrenaline (+DBH),
adrenaline (+PNMT), and serotonin (TPH2) as well as
the opioid peptide proenkephalin (PENK). Although
many neurotransmitter receptors show a similar
distribution profile [DDRs, ADRs, HTR1A, and 5-
hydroxytryptamine receptor 6 (HTR6)], several excep-
tions with clear on/off differences between species
could be observed, especially in the cerebellum
(HTR5A, OPRM1, OPRD1, CCKBR, and NPFFR1).
(E) Relative expression of all GABAAreceptor subunits
in the cerebral cortex and cerebellum and (F) nicotinic
receptor subunits in all three species suggests
alternative nicotinic subunit composition in different
species, especially the pig. (G) Four brain-elevated
orphan GPCRs (GPR6, GPR52, GPR88, and GPR149)
are elevated in the caudate and putamen,
whereas GPR150 is group-enriched in the forebrain
regions. The nonvisual photoreceptor melanopsin
(OPN4) is only expressed in the human basal
ganglia. Missing values (gray bars) are due to missing
one-to-one orthologs or genes not included in
all used datasets. (H) The relative distribution
of brain-elevated GPCRs with unknown function
reveals widespread expression of the orphan
GPR37L1 and GPR162, including the cerebellum,
whereas GPRC5C expression is elevated in cerebellum.
Scale bars, 40 um. Full overviews with NX for all
transcription factors, neurotransmitters, and GPCRs
are available in figs. S28 to S30.


human vs pig human vs mouse pig vs mouse

A

B

serotonin

dopamine

(nor)epinephrine

HTR6

ADRB3

ADRA2A

ADRA2C

SLC6A4

DRD2

SLC6A3
SLC6A2
TPH2

TH
DDC
DBH
PNMT

C

D

G

H

E

47%

9%

9%
9%

6%

9%

44%

7%

37%

7%

9%

6%

36%

ptpm fraction

CHRNA2
CHRNA3
CHRNA4
CHRNA6
CHRNA7
CHRNA9
CHRNB2
CHRNB3
CHRNB4CHRND
CHRNE
CHRNG

Cerebral cortex Basal ganglia

DRD3

ADRA2B

ADRB2

ob ctx hf am bg hy th mb pm cb

BHLHE22

EOMES

SIX6
PITX3

PAX2

PENK

CCKBR

obctx hf am bghythmbpmcb

OPN4

GPR6

Gene name obctx hf am bghythmbpmcb

F

0% 50% 100%
human pig mouse

human pig mouse
GABRA2
GABRA3
GABRA4
GABRA6
GABRB2
GABRB3
GABRG2
GABRG3
GABRD
GABRE
GABRP
GABRQ

ptpm fraction

human

pig
mousehuman
pig
mouse

Cerebral cortex Cerebellum
27% 6%

7%
6%
9%

7% 9% 9%

7%

7%

human pig mouse

Transcription factor
Neurotransmitter

Receptor
Category Other

Pearson correlation

obctx hf am bghythmbpmcb

human

pig
mousehuman

pig
mouse

relative expression

relative expression

relative expression

relative expression

Gene name

Gene name

Gene name

Transcription factor

Neuropeptides

Orphan GPCRs
Monoaminergic systems

GPR37L1 GPR162 GPRC5C

relative expression levels

RESEARCH | RESEARCH ARTICLE

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