Nature - USA (2020-02-13)

Nature | Vol 578 | 13 February 2020 | 287

(Ahr+/+) mice carrying the lineage reporter Rosa26eYFP, which enabled
us to monitor the efficiency and cell-type specificity of Ahr deletion
by GFP immunostaining (Extended Data Fig. 5n, o). Administration of
AAV9-CaMKII-Cre to Ahr fl/fl;Rosa26eYFP mice resulted in eYFP expression
and ablation of Ahr from the majority of enteric neurons (Extended Data
Fig. 5o, p). eYFP-expressing neurons—which, as expected, lack an AHR
RNAscope signal (Fig. 2o, p)—showed significantly lower levels of Kcnj12
transcripts relative to eYFP-negative neurons (Fig. 2o–s). Together,
our experiments indicate that microbiota-dependent Ahr induction
in enteric neurons enables the cell-autonomous activation of genes
that encode previously known feedback regulators of AHR signalling
as well as newly identified regulators of enteric neuronal excitability.
To determine whether AHR signalling in enteric neurons regulates
intestinal physiology, we analysed the gut motility of AhrEN-KO mice. The

identification of AHR-deficient neurons for at least four weeks after AAV

administration—along with the similar number, morphology and neu-

rochemical properties of colonic neurons in control and AhrEN-KO mice

(Extended Data Fig. 6a)—indicated that Ahr is dispensable for neuronal

survival. Furthermore, no apparent deficit in the organization and cel-

lular composition of the ENS was observed in constitutive Ahr mutant

mice (Ahr−/−)^23 (Extended Data Fig. 6b). Despite the lack of discernible

effects of Ahr deletion on the cellular organization of the ENS, the total

ITT of AhrEN-KO mice was increased relative to two sets of control mice:

AAV-injected wild-type mice (WT + AAV, which act as controls for the

potential effects of AAV on ITT) and Ahr fl/fl mice (cohoused with AhrEN-KO

mice to minimize potential effects of microbiota) (Fig. 3a). WT + AAV

and Ahr fl/fl mice were indistinguishable from one another in terms of

colon histology and motility (Extended Data Fig. 7a, b), but the increase

cde

l m nopq r s

a b

fgh ij k

SPF GF exGF

PERI

HuC/

D

AH

R

HuC/

D

AH

R

PERI PERI

HuC/

D

AH

R

0

100

200

300

400

GF

Kcnj12

SPF

Merge eYFP Ahr Kcnj12

AhrEN-KO

*

*

* *

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*

Intensity

per cell

(^0) SPF GF
200
400
600
Intensit
yp
er
cell
Intensity
per
cell
eYFP

• eYFP

• eYF

P+

eYFP

(^0) –
100
200
300
400
P < 2.2 × 10 –16^500 P = 0.0058 P = 0.0482
AHR HuC/D Merge AHR HuC/D Merge
Colon Duodenum
0
–2 –1 0 123
1
2
3
Relativ
ee
xpression
Control AHR agonist
Cyp1a 1
AHR agonist
Cyp1a1 Kcnj12 log
10 (Cyp1a1)
P = 0.0007 P < 2.2 × 10 –16
Corn oil3MC 14 h
0
0.2
0.4
0.6
log
10
(Kcnj12
)
Fig. 2 | Microbiota- and ligand-dependent activation of AHR signalling.
a, b, AHR (red) and HuC/D (blue) immunostaining of colon (a) and duodenum
(b) muscularis externa (12-week-old SPF mice). Note neurons with cytoplasmic
(arrowhead) or nuclear (arrow) signals. n = 12 mice, 3 experiments.
c–e, Immunostaining of SPF (c), germ-free (d) and conventionalized adult
germ-free mice (exGF) (e) colonic myenteric ganglia with the pan-neuronal
markers peripherin (PERI) (green), HuC/D (blue) and AHR (red). Small panels
show AHR (top) or HuC/D (bottom). n = 6 mice for each condition,
2 experiments. f, g, Colonic myenteric ganglia from control (f) and 3MC-
treated (g) mice hybridized with the Cyp1a1 probe (green). Dotted line, borders
of myenteric ganglia; arrows, positive neurons. n = 4 mice, 2 experiments.
h, Quantitative PCR for Cyp1a1 transcripts (mean ± s.d.) in colonic muscularis
externa from control and 3MC-treated mice. n = 6 control and 8 3MC-treated
mice (two-sided non-parametric Mann–Whitney U-test). i, j, Colonic myenteric
ganglia from 3MC-treated mice hybridized with Cyp1a1 ( green) (i) and Kcnj1 2
(blue) (j) probes. Arrows, neurons co-expressing Cyp1a1 and Kcnj1 2. n = 4 mice,
2 experiments. k, Positive correlation in Cyp1a1 and Kcnj1 2 transcript level in
myenteric neurons (F-test). n = 827 neurons, 4 mice. l, m, Colonic myenteric
ganglia from SPF (l) and germ-free (m) mice hybridized with the Kcnj1 2 probe.
Arrows, positive neurons. n = 6 SPF mice and 6 germ-free mice, 2 experiments.
n, Quantification of signal (mean ± s.d.) in l and m (two-sided non-parametric
Mann–Whitney U-test). n = 510 neurons from SPF mice and 298 neurons from
germ-free mice. o–q, Myenteric ganglia of AhrEN-KO mice immunostained for
eYFP (green) (o) and hybridized with the Ahr (red) (p) and Kcnj1 2 (blue) (q)
probes. o, Merge of signals in p and q. n = 4 mice, 2 experiment s.
r, s, RNAscope signal intensity (mean ± s.d.) for Ahr (r) and Kcnj1 2 (s) in eYFP−
(AHR+) and eYFP+ (AHR−) neurons (two-sided non-parametric Mann–Whitney
U-test). n = 100 eY FP+ and 223 eYFP− neurons, 4 mice. Scale bars, 30 μm.