Nature | Vol 578 | 13 February 2020 | 285neurons from the colon and small intestine of conventionally raised
specific-pathogen-free (SPF) mice. To minimize potential variation in
gene expression due to diet or other environmental factors, we used
mice from two independent animal facilities (the Francis Crick Institute
and University of Bern) to produce two datasets (the Crick and Bern
datasets, respectively). Transcriptional profiles of neuronal nuclei
clustered exclusively according to the intestinal segment of origin
(Extended Data Fig. 1m), indicating that neurons derived from the
small intestine or colon express distinct transcriptional programs.
Differential gene-expression analysis carried out on the combined
Crick and Bern datasets identified 254 genes that were upregulated
in enteric neurons of the colon (termed colon-upregulated ENS genes
(CUEGs)) of SPF mice (hereafter, SPF CUEGs) (Fig. 1b, Supplementary
Table 1). Among the top differentially expressed SPF CUEGs were genes
that are implicated in neuronal development and function, such as
Pou3f3 (which encodes a transcription factor), Ano5 (which encodes
a chloride channel), Pde1c (which encodes a regulator of intracellular
second messengers), Unc5d (which encodes a netrin receptor), Col25a1012(^10)
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–6.0 –4.0–2.0–1.5 02 1.5.0 4. 06 .0
AAV injection(i.v.)
FACS
Tissuehomogenization
pAAV-CaMKII-eGFP-KASH
d
b
Neuronal
nuclei
Neuronal
nuclei
GF SPF
log 2 (foldchange SI versuscolon(SPF))
log 2 (fold change GF versus SPF (colon))
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value)
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( 10
P value)
nRNA-seq
Pou3f3
Pde1c
Pantr2
Col25a1
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SI Colon
Microbiotadiversity/density
SPF condition
SPF conditio
n
log 2 (foldchange SI versuscolon(GF))
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(P
value)
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Microbiota
Ano5
Unc5d
Ahr Prr5
Fam20c
Pou3f3
Pde1c
Pantr2
Ano5
Col25a1
Isolationof myeteric
plexuslayer
ITR CaMKII eGFP KASH WPRE hGH pA ITR
a
GF condition
–8.0 –6.0 –4.0 –2. 00 –1. 51 .52.0 4.0 6.0 –6.0–2.0–1.501.52.06.0
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Fig. 1 | Programming of the enteric neuron transcriptome by microbiota.
a, Experimental design for AAV-mediated expression of nuclear-localized
enhanced green f luorescent protein (eGFP) fused to the K ASH nuclear
membrane retention domain (eGFP–K ASH)^30 in myenteric neurons and RNA
sequencing of neuronal nuclei purified by f luorescence-activated cell sorting
(FACS). The plasmid used to generate the A AV9-CaMKII-eGFP-K ASH vector is
shown at the top. i.v., intravenous. b, c, Volcano plots showing mean log 2 -
transformed fold change (x axis) and significance (−log 10 (adjusted P value)) of
differentially expressed genes between myenteric neurons of the small
intestine and the colon of SPF (b) and germ-free (GF) (c) mice. Differential gene-
expression analysis was carried out using the DESeq2 R package. The default
DESeq2 Wald test (two-sided) was used to determine P values for differential
gene expression. Adjusted P values for differential gene expression were
calculated with the DESeq2 default Benjamini–Hochberg method. log-
transformed fold-change shrinkage was applied by having the betaPrior
parameter set to TRUE. Schematic of the small intestine and colon shown at the
top. SPF CUEGs and germ-free CUEGs are within the area demarcated by red
lines (log 2 -transformed fold change = 1.5 < maximum; P < 0.05) in the plots of
b and c, respectively. n = 4 SPF (Crick), 4 SPF (Bern) and 3 germ-free (Bern)
independent nuclear isolates, each representing 3 mice. d, Volcano plot
showing mean log 2 -transformed fold change (x axis) and significance
(−l o g 10 (adjusted P value)) of differentially expressed genes between colonic
myenteric neurons from SPF and germ-free mice (Bern). Schematics of the
colon from germ-free and SPF mice are shown at the top. Microbiota-
dependent CUEGs are within the area demarcated by red lines (log 2 -
transformed fold change = 1.5 < maximum; P < 0.05). e, Representative images
of myenteric ganglia (outlined by dotted line based on HuC/D immunostaining)
from germ-free (left) and SPF (right) mice hybridized with RNAscope probes
for Ahr, Prr5 and Fam 20c. Scale bars, 30 μm. Data represent two independent
experiments.