Science 6.03.2020

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

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