Notably,Gpr43mRNA expression in both the
colon and pancreas of GF embryos was sig-
nificantly lower than that in SPF embryos,
althoughGpr41mRNA expression in the sym-
pathetic ganglia of embryos was similar among
thetwogroups(fig.S12,EtoG).Thesefindings
imply that the embryonic metabolic tissues,
such as the sympathetic nervous system, in-
testinal tract, and pancreas, may sense mater-
nal gut microbe–derived SCFAs by expressing
GPR41 and GPR43.
Sympathetic development via GPR41
We further investigated the functions of GPR41
in the sympathetic nervous system and of
GPR43 in the intestine and pancreas during
embryonic development. We found that sym-
pathetic nerve projections to the heart were
significantly reduced inGpr41−/−C57BL/6J em-
bryos compared with wild-type (WT) embryos,
and such an abnormality was also apparent in
GF embryos with the same background (Fig.
3A). Moreover, sympathetic nerve projections
to the heart were significantly reduced in
Gpr41−/−pups on postnatal day 1 (P1), even
though these pups were delivered from mothers
maintained under SPF conditions (Fig. 3B). Gut
microbial compositions were similar for WT,
Gpr41−/−, andGpr43−/−pregnant C57BL/6J
mice(fig.S13,AandB).PlasmaSCFAlevels
were also comparable among the three groups
(fig. S13C).
We further sought to investigate the con-
tribution of GPR41 signaling to sympathetic
neuronal differentiation. In a primary culture
of embryonic superior cervical ganglion (SCG)–
derived neural cells, each of the tested SCFAs,
especially propionate, significantly promoted
sympathetic neuronal differentiation (fig. S14A).
This finding is consistent with the notion thatthe most potent agonist of GPR41 is propi-
onate, followed by butyrate and acetate, with
median effective concentration (EC 50 ) values
of approximately 10, 40, and 2000mM, re-
spectively ( 30 ). Propionate-induced sympathetic
neuronal differentiation was abolished in sym-
pathetic neural cells fromGpr41−/−embryos
(Fig. 3C). Furthermore, Gi/o-mediated (but not
Gi/oa-mediated) GPR41 propionate signaling
increased sympathetic neurite length via Gbg-
mediated mitogen-activated protein kinase
activation (fig. S14, B and C). These findings
strongly suggest that propionate-mediated
GPR41 activation promotes sympathetic neu-
ronal differentiation. To test this notion, we
treated WT pregnant mice with a cocktail of
antibiotics to eliminate intestinal microbiota.
Sympathetic nerve projections to the heart were
significantly attenuated in pups from antibiotic-
treated mice on P1. Notably, this abnormalityKimuraet al.,Science 367 , eaaw8429 (2020) 28 February 2020 4of12
WTGpr41-/-(-) (+)Propionate100 μmABDEF800700600500
Heart rate (beats/min) 0
Abx.
Propionate Propionate++
+++
+
WT Gpr41-/-** ** NS
40383634
Body temperature ( 0)Abx. -++
+++
+
WT Gpr41-/-** ** NS01020304050TH-positive cells(% of DAPI)Propionate - + -+
WT Gpr41-/-** NS00.511.5++
+++
+Abx.
PropionateRelative protein expression-actin)WT Gpr41-/-** ** NSNSTH
-actinTyramine
treatment0102030Energy expenditure(kcal/kg/hr)**NS**WT
WT + Abx.
WT + Abx. + propionate
Gpr41-/-+ Abx.
Gpr41-/-+ Abx. + propionateCWT Gpr41-/-100 μm0
WTGpr41-/-0.40.81.21.6Relative TH+
nerve area*00.511.5Relative protein expression-actin)** **TH
-actinFig. 3. Propionate promotes sympathetic neu-
ronal differentiation via embryonic GPR41.
(A)THexpressioninE18.5wholeheart(n=7or
8 tissues of embryos from three or four litters
per group) and representative Western blots.
(B) Sympathetic nerve projection in P1 whole heart
(upper panels) (TH immunostaining) and ventricle
(lower panels) (TH, green; DAPI, blue). TH density was
quantified (n= 9 or 10 tissues of pups from four litters
per group). (C) Effects of propionate (1 mM) on
sympathetic neuronal differentiation inGpr41−/−mice
(TH, red; DAPI, blue;n= 10 independent experiments
from three biological replicates per condition).
(D) Development of nerve projections in the P1 ventricle
quantified by Western blotting (n= 8 to 11 tissues of
pups from three litters per group) and representative
Western blots. Abx., antibiotic treatment. (E) Heart rate
(left) (n= 7 to 11 animals from three to five litters per
group) and body temperature (right) (n= 8 animals
from three or four litters per group) in 4-week-old
offspring. (F) Energy expenditure in 4-week-old
offspring (n= 8 animals from three litters per group).
Student’sttest [(A) and (B)] and Tukey–Kramer’s
test [(C) to (F)]; **P<0.01and*P<0.05.NS,not
significant. Data are presented as means ± SEM.RESEARCH | RESEARCH ARTICLE