Article
(PCO) using TillVisION (TILL Photonics). Myenteric plexus prepara-
tions were constantly superfused with carbogenated Krebs solution
at room temperature containing 1 μM nifedipine (Sigma-Aldrich) via a
local gravity-fed (±1 ml/min) perfusion pipette. Myenteric ganglia were
stimulated electrically using single and a train (20 Hz, 2 s) of pulses
(300 μs) transmitted from a Grass stimulation unit via a focal electrode
(50-μm diameter tungsten wire) placed on an interganglionic connec-
tive leading to the selected myenteric ganglia within the field of view.
Analysis was performed with custom-written routines in Igor Pro
(Wavemetrics)^40. Regions of interest were drawn, after which the
average Ca2+ signal intensity was calculated, normalized to the initial
GCaMP6f values and reported as F/F 0. Cells were considered as respond-
ers when the GCaMP6f signal rose above baseline plus 3× the intrinsic
noise (standard deviation) during the recording. The Ca2+ transient
amplitudes were measured as the maximum increase in [Ca2+]i above
baseline (maximum Fi/F 0 ).
Statistical analysis
Statistical comparisons between samples were performed in GraphPad
Prism software using Student’s t-test. When variances were not homo-
geneous, the data were analysed by the non-parametric Mann–Whitney
U-test. For correlation analysis of RNAscope intensity (Fig. 2k, Extended
Data Fig. 5m), data were filtered to remove pairs for which either obser-
vation was zero and data were then log 10 -transformed. The correlation
coefficient was calculated by fitting a standard linear regression model
to this log 10 -transformed data and quality of the overall model was
assessed with an F-test. Data excluded from the analysis were overlaid
on the scatter plot by log 10 -transforming the non-zero observation
and retaining the zero value. The analysis was performed using R 3.3.1.
Reporting summary
Further information on research design is available in the Nature
Research Reporting Summary linked to this paper.
Data availability
All RNA-seq data are available at Gene Expression Omnibus (GEO) under
accession number GSE140293. Source Data for Figs. 2, 3 and Extended
Data Fig. 1–5, 7 are provided with the paper. All datasets analysed
during the current study are presented in this manuscript, or are
available from the corresponding authors upon reasonable request.
Code availability
The source code and installation instructions for colonic migrating
motor complex evaluation and Ca2+ imaging can be found at https://
doi.org/10.7554/eLife.42914.039 (Ca2+ imaging analysis source code)
and https://doi.org/10.7554/eLife.42914.040 (installation instruc-
tions and user guide). For more information, please contact pieter.
[email protected]. The code related to the RNAscope signal
quantification is available at GitHub (https://github.com/FrancisCrick-
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Acknowledgements We thank the Crick Science Technology Platforms, the University of Bern
FACSLab and the Bern Clean Mouse Facility for expert support; R. Lasrado and S.-H. Chng for
assistance with tissue dissection; M. Shapiro for bioinformatic input; C. Schiering for useful
advice; all members of the Pachnis laboratory for insightful comments on the manuscript and
discussions; and M. D’Amato for insightful comments on the manuscript. Y.O. was supported
by an EMBO long-term fellowship (ALTF 1214-2015), travel grants from Boehringer Ingelheim
Fonds and the Society for Mucosal Immunology (SMI); he is currently supported by an HFSP
postdoctoral fellowship (LT000176/2016). This work was supported by the Medical Research
Council (MRC) and The Francis Crick Institute (which receives funding from the MRC, Cancer
Research UK and the Wellcome Trust). V.P. was also funded by BBSRC (BB/L022974) and the
Wellcome Trust (212300/Z/18/Z).
Author contributions Y.O. and V.P. conceived the study and together with B.S. and A. J.
Macpherson designed the experiments. Á.C., A.C.B.-F., C.F., M.G.d.A., B.Y., M.R.M., W.B. and B.Y.
helped with the experiments. Á.C. performed the RNAscope in situ hybridization experiments;
T.F. helped with the quantification of RNAscope data; M.G.d.A. and B.Y. helped to organize
experiments with germ-free and exGF mice; A.C.B.-F., W.B. and P.V.B. provided help with the
spatiotemporal mapping experiments and the analysis. C.F. carried out and analysed the Ca2+
imaging experiments with help from W.B. and P.V.B. A.H. prepared the cDNA library for the bulk
nRNA-seq. S.B. performed bioinformatics analysis. S.H. performed statistical analysis. R.L.
generated ChAT-TVA-mCherry mice. Y.O. generated the AAV-CaMKII-eGFP-KASH construct
with help from A. J. Murray. V.P. and Y.O. wrote the manuscript with help from B.S. and
A. J. Macpherson, and contributions from all authors.
Competing interests The authors declare no competing interests.Additional information
Supplementary information is available for this paper at https://doi.org/10.1038/s41586-020-
1975-8.
Correspondence and requests for materials should be addressed to Y.O. or V.P.
Peer review information Nature thanks John Cryan, Michael D. Gershon, Sven Petterson and
Harry Sokol for their contribution to the peer review of this work.
Reprints and permissions information is available at http://www.nature.com/reprints.