Quantification of RNAscope intensity on enteric neurons
Fluorescent signals of RNAscope were colocalized with HuC/D+ enteric
neurons using an automated pipeline in CellProfiler^39. In brief, the Alexa
Fluor 405 channel (HuC/D) was background-corrected using a baseline
subtraction of 10% of the maximum pixel intensity. The background-
subtracted image was then segmented and individual neurons were
detected using the IdentifyPrimaryObjects module and clumped
objects were separated by shape. RNAscope spots (2–30 pixels) were
identified separately for each gene and were processed further only
if colocalized to HuC/D+ neurons. Individual neurons were tracked
through the z-stack by measured overlap. The RNAscope signal inten-
sity of individual neurons was integrated through the image z-stack in
slices in which the cells were identified.
Quantitative PCR
Total RNA was isolated from the colonic muscular layer using Trizol LS
reagent and the PureLink RNA Micro Kit (Invitrogen, 12183016) accord-
ing to the manufacturer’s specifications, and was subjected to reverse
transcription using the High-Capacity cDNA Reverse Transcription Kit
(Applied Biosystems, 4368814). Quantitative PCR was performed with
complementary DNA (cDNA) using Taqman fast universal 2× PCR Mas-
ter Mix (Applied Biosystems) and Taqman probes (Applied Biosystems)
for Actb (Mm02619580_g1) and Cyp1a1 (Mm00487218_m1). Ct values
obtained were normalized to Actb.
Purification of neuronal nuclei from the myenteric plexus
For the isolation of neuronal nuclei from the myenteric plexus, mice
were injected intravenously with AAV9-CaMKII-eGFP-KASH. Five weeks
after intravenous injection of the AAV, the longitudinal smooth muscle
layer and associated myenteric plexus were peeled off the wall of the
small intestine and colon and subjected to Dounce homogenization
in lysis buffer (250 mM sucrose, 25 mM KCl, 5 mM MgCl 2 , 10 mM Tris
buffer with pH 8.0, 1 mM DTT) containing 0.1% Triton X-100, cOmplete
EDTA-free protease inhibitor (Sigma-Aldrich) and DAPI. After filtering
the homogenate to remove large debris, samples were centrifuged
at 1,000g for 10 min at 4 °C to obtain a pellet containing muscularis
externa nuclei. For flow cytometric analysis, doublet discrimination
gating was applied to exclude aggregated nuclei, and intact nuclei were
determined by subsequent gating on the area and height of DAPI inten-
sity. Both eGFP+ and eGFP– nuclear populations were collected directly
into a 1.5-ml tube containing Trizol LS reagent (Invitrogen) using, at the
Francis Crick Institute, the Aria Fusion cell sorter (BD Biosciences) and,
at the University of Bern, the Aria III (BD Biosciences). The obtained
FCS data were further analysed using FlowJo software version 10.5.3.
The list of SPF CUEGs was generated by combining nRNA-seq data
from mice housed in both facilities. Labelling and isolation of neuronal
nuclei in the two facilities was carried out using exactly the same pro-
tocol and reagents, except for the use of two different FACS sorters
(Aria Fusion at the Francis Crick Institute and Aria III in Bern). RNA
extraction and RNA sequencing for all samples was done at the Francis
Crick Institute. The list of germ-free CUEGs was generated with mice
sourced exclusively from the Bern germ-free facility. The identification
of microbiota-dependent CUEGs was done by comparing the transcrip-
tome of colonic neurons from SPF and germ-free mice from the Bern
facility. Finally, the transcriptomic experiment that generated the list
of AHR-induced CUEGs was carried out using exclusively Crick mice.
RNA sequencing and bioinformatic analysis
Extraction of nuclear RNA and nuclear RNA sequencing were carried out
at the Francis Crick Institute. Nuclear RNA was isolated using PureLink
RNA Micro Kit (Invitrogen, 12183016) according to the manufacturer’s
instructions. Double-stranded full-length cDNA was generated using
the Ovation RNA-Seq System V2 (NuGen Technologies). Following
quantification on a Qubit 3.0 fluorometer (Thermo Fisher Scientific),
cDNA was fragmented to 200 bp by acoustic shearing using Covaris
E220 instrument (Covaris) at standard settings. The fragmented cDNA
was then normalized to 100 ng, which was used for sequencing library
preparation using the Ovation Ultralow System V2 1-96 protocol (NuGen
Technologies). A total of 7 PCR cycles was used for library amplifica-
tion. The quality and quantity of the final libraries were assessed with
TapeStation D1000 Assay (Agilent Technologies). The libraries were
then normalized to 2.5 nM, pooled and loaded onto a HiSeq 4000
(Illumina) to generate 75-bp single-end reads. For the transcriptomic
comparison of colon to small-intestine myenteric neurons from SPF
mice (Fig. 1b) we used eight nuclear isolates (four from Crick, and four
from Bern, mice), each representing three mice. All mice in the Crick
samples were male. Two of the Bern samples were generated from male
and two from female mice. Male and female samples were indistinguish-
able by principal component analysis. The transcriptomic comparison
of colon to small-intestine myenteric neurons from germ-free mice
(Fig. 1c) were generated using three nuclear isolates (all Bern mice),
each representing three mice.
For the bioinformatics analysis, the ‘Trim Galore!’ utility version
0.4.2 was used to remove sequencing adaptors and to quality trim
individual reads with the q-parameter set to 20. The sequencing reads
were then aligned to the mouse genome and transcriptome (Ensembl
GRCm38 release-86) using RSEM version 1.3.0 in conjunction with the
STAR aligner version 2.5.2. Sequencing quality of individual samples
was assessed using FASTQC version 0.11.5 and RNA-SeQC version 1.1.8.
Differential gene expression was determined using the R Bioconductor
package DESeq2 version 1.14.1.ITT assay
The total ITT was measured as previously described^38. Mice were placed
individually in bedding-free cages with the diet and HydroGel (Clear
H 2 O). For all experiments, 250 μl of 6% (w/v) carmine red dye (Sigma-
Aldrich) in 0.5% (w/v) methylcellulose (Sigma-Aldrich) was orally admin-
istered to each mouse at 09:00. The time period from gavage until
the emergence of the first red-colour pellet was recorded as total ITT.Live video imaging and spatiotemporal mapping of colonic
motility
Ex vivo video imaging and analysis of colonic motility was performed as
previously described^38. In brief, the entire colon was carefully isolated
and loosely pinned in an organ bath chamber continuously superfused
(flow rate of 4 ml/min) with oxygenated (95% O 2 and 5% CO 2 ) Krebs
solution (in mM, 120.9 NaCl, 5.9 KCl, 1.2 MgCl 2 , 2.5 CaCl 2 , 1.2 NaH 2 PO 4 ,
14.4 NaHCO 3 , 11.5 glucose) kept at 37 °C. Following equilibration of
the colon for 30 min, movies of colonic motility were captured (2.5-Hz
frame rate) with a QICAM-Fast camera using QCapture Pro 6.0 software
(Q-Imaging). Images were read into an Igor Pro (WaveMetrics) and ana-
lysed using custom-written algorithms. The edges of the bowel were
determined, and the width computed and mapped over time. From
the generated spatiotemporal maps, the frequency of propagating
contractions was determined.Ca2+ imaging of colonic myenteric plexus
For ex vivo Ca2+ imaging experiments, the large intestine of adult
Wnt1::cre;Rosa26-GCaMP6f mice^33 ,^34 was isolated and pinned flat in a
Sylgard-lined dish filled with Krebs solution, bubbled with 95% O 2 and 5%
CO 2 at room temperature. The mucosal, submucosal and longitudinal
muscle layers were carefully removed to obtain a circular muscle with
adherent myenteric-plexus preparation, which was mounted over a
small inox ring, immobilized by a matched rubber O-ring^40. Myenteric-
plexus preparations were placed in a recording chamber mounted
on an upright Zeiss Axio Examiner.Z1 microscope equipped with a
Poly V xenon monochromator (TILL Photonics) and water dipping
lens (20×, NA 1.0, Zeiss). GCaMP6f was excited at 475 nm and images
were recorded at 525/50 nm (at 2 Hz) on a Sensicam-QE CCD camera