S7,AtoH).Furthermore,bothmaleandfe-
male GF offspring from C57BL/6J mothers
developed severe obesity and metabolic dis-
orders upon HFD feeding (figs. S7, A to H,
and S8, A to H). Thus, to a greater or lesser
extent, metabolic disorders in GF offspring
were commonly observed regardless of strain
and sex.
Sensing maternal SCFAs in the embryo
We could not detect bacteria in the amniotic
fluidofpregnantSPFICRmiceatouranimal
facility (fig. S9, A and B). We hypothesized that
metabolite signals ( 23 – 25 ), derived from the
maternal gut microbiota, may translocate to
the fetus and influence the development of the
metabolic system. Thus, we profiled the plasma
levels of hydrophilic and lipophilic metabolites
in SPF and GF ICR mothers and their embryos
during pregnancy. The levels of 5 metabolites
in the mothers and 12 metabolites in the em-
bryos were significantly different between the
SPF and GF groups (fig. S10); among them,
only 5 metabolites showed similar changes in
the mothers and embryos in response to breed-
ing conditions (Fig. 2A). In particular, the plasma
levels of SCFAs (acetate,propionate, and butyr-
ate) were significantly lower in GF mothers
and embryos than in their SPF counterparts
(Fig. 2B). Given that plasma SCFA levels were
constant in both mothers and embryos of the
SPF group during pregnancy (fig. S11), the ma-
ternal gut microbiota appears to constitutively
supply SCFAs to embryos via the bloodstream.
Regulation of host energy metabolism through
activation of GPR41 and GPR43 by gut microbial
SCFAs has been documented ( 15 , 16 , 26 , 27 ).
We detectedGpr41mRNA in the sympathetic
ganglia of embryos (Fig. 2C and fig. S12, A and
B) with biphasic expression in the embryonic
and adult stages (Fig. 2C). Such an expression
pattern was not observed for nestin (Nes;an
undifferentiated neural marker), tyrosine hy-
droxylase (Th; a sympathetic neuronal marker),or glial fibrillary acidic protein (Gfap; a glial
marker) (Fig. 2C). Meanwhile,Gpr43mRNA
was detected in the intestinal tract from em-
bryonic day 15.5 (E15.5) onward (fig. S12C),
although its expression was restricted to en-
teroendocrine cells in the adult stage (fig. S12D),
as previously reported ( 28 ). Biphasic increases
inGpr43expression were observed in the colon
in the embryonic and adult stages, with ex-
pression peaking later than that ofPax4and
Pax6(regulators of intestinal enteroendocrine
cell differentiation) and at a similar time point
to that ofGcg(intestinal enteroendocrine cell
maker, GLP-1) (Fig. 2D). These SCFA receptors
are also expressed in the pancreas and regu-
late insulin secretion in adult mice ( 27 , 29 ).
Levels of pancreaticGpr43mRNA, but notGpr41,
transiently increased during the perinatal-
postnatal period, with expression peaking later
than that ofNkx6.1(earlybcell differentiation–
related factor) and at a similar time point to
that ofIns2(pancreaticbcell maker) (Fig. 2E).Kimuraet al.,Science 367 , eaaw8429 (2020) 28 February 2020 3of12
ABECD012310 -3
Th023410 -6
Gfap1
046810 -5
Nes2024810 -6
Gpr41
6mRNA expressionSCG00.61.21.810 -4ColonmRNA expressionGpr43
Gpr4100.61.21.810 -4
Gcg00.511.510 -7
Pax4024610 -6
Pax6023410 -6PancreasmRNA expressionGpr43
Gpr41
1
02310 -3
Ins21023410 -6
Nkx6.11SPFGFSPFGF
Mo E18.5500
400
300
200
100
0Acetate (μM)** **SPFGFSPFGF
Mo E18.550
40
30
20
10
0Propionate (μM)** **SPFGFSPFGF
Mo E18.52015100n-Butyrate (μM)** **5Mothers
4310
Significance (-log10q
value)-6 -4 -2 0 2 4 6
SPF / GF (log 2 fold change)acetate
n-butyrate(^1) propionate
Embryos (E18.5)
acetate
n-butyrate
propionate
1
2
2
4
3
1
0
-6 -4 -2 0 2 4 6
SPF / GF (log 2 fold change)
2
2
Fig. 2. Embryonic SCFAs depend on
maternal gut microbiota, with receptors
already expressed at the embryonic
stage.(A) Volcano plot showing the
significance and magnitude of differences
in the relative abundances of plasma
metabolites in mothers or embryos from
ICR SPF and GF mothers (n= 5 plasma
samples of mothers andn= 5 plasma
samples of embryos from five litters per
group). 1, 1,5-anhydro-D-glucitol; 2, 13-hy-
6c,9c-18:2. (B) Levels of plasma SCFAs,
as determined by GC-MS [n= 8 plasma
samples of mothers (Mo) andn= 8 plasma
samples of embryos from eight litters per
group]. (C) Expression ofGpr41,Th,Nes,
andGfapduring growth in the SCG (n=
8 tissues from four litters per group).
(D)ExpressionofcolonicGpr41,Gpr43,Gcg,
Pax4,andPax6during growth (n=7or
8 tissues from four litters per group).
(E) Expression of pancreaticGpr41,Gpr43,
Ins2,andNkx6.1during growth (n=7or
8 tissues from four litters per group). Values are
shown relative to 18SrRNA gene expression
[(C) to (E)]. Student’sttest (B); **P<0.01.
Data are presented as means ± SEM [(B) to (E)].
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