either remain within its immediate vicinity or
cross in a stepwise manner to form the submucosal plexus and eventually the mucosal ENS
(fig. S6). Only NG- and G-type progeny have the
potential to colonize the submucosal plexus and
the mucosa while myenteric and submucosal
neurons are generated sequentially from plexus-specified neurogenic precursors, which are restricted to the respective ganglionic layer (fig. S6).
This hierarchical model integrates early stages
of ENS development, dominated by enteric neu-
ron and glia progenitor allocation in the prospec-
tive myenteric plexus, with later events relating to
the organization of the ENS within the 3D space
of the intestinal wall. Given the different strat-
egies employed for the migration of ENS progen-
itors in the small intestine and the colon (28), it
will be interesting to explore the underlying dif-
ferences in the assembly and organization of
neural networks in the two parts of the gastro-
intestinal tract. Our work also suggests that
ontogenetic clonal units organized within colum-
nar spaces along the gut constitute fundamental
elements in the functional architecture of the
ENS. It is currently unclear whether the in-
creased propensity for synchronization of clo-
nally related myenteric neurons is due to the
preferential formation of chemical or electrical
synapses or common input from lineally un-
related neurons. In addition, it remains unknown
whether sister neurons located in different layers
of the ENS also show increased synchronization.
Despite these limitations, allocation of lineally
related enteric neurons into parallel and overlapping clonal units provides an opportunity to
integrate the physiological activity of neuronal
ensembles along the longitudinal and radial axis
of the gut and contributes to the functional integration of the ENS.
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The transcriptome sequencing data for all single cells have been
deposited in the European Molecular Biology Laboratory–
European Bioinformatics Institute ArrayExpress database under
accession number E-MTAB-5553 ( www.ebi.ac.uk/arrayexpress/
experiments/E-MTAB-5553). We thank members of the Pachnis
laboratory and T. Vogt for discussions and comments on the
manuscript, J. Bornstein for educational discussions about ENS
function, T. Margrie and D. Burdakov for lab space and advice
relating to the Ca2+ imaging experiments, A. Sesay for advice
and support with scRNA-seq, and the staff of the Biological
Research Facility and the Flow Cytometry Science Technology
Platform of the Crick Institute for advice and expert help. P.V. B.
was supported by Fonds voor Wetenschappelijk Onderzoek
(FWO-Flanders; grants G.0510.10 and G.0921.015). W.B. was a
recipient of a postdoctoral fellowship from FWO-Flanders. C.P.
acknowledges the support of the UK Biotechnology and Biological
Sciences Research Council (BBSRC) Gut Health and Food Safety
Programme Grant (BB/J004529/1). Work in V. P.’s laboratory
is supported by the BBSRC (BB/L022974/1), the UK Medical Research
Council (MRC), and the Francis Crick Institute (which receives funding
from the MRC, Cancer Research UK, and the Wellcome Trust). The
supplementary materials contain additional data.
Materials and Methods
Figs. S1 to S7
Tables S1 and S2
Movies S1 to S4
20 January 2017; accepted 10 April 2017
726 19 MAY 2017 • VOL 356 ISSUE 6339 sciencemag.org SCIENCE
Fig. 5. Coordinate activity of sister neurons. (A and B) Fluorescence micrograph of a live preparation of
myenteric plexus (including part of an RFP+ clone) loaded with Fluo-4 (green) at baseline (A) or during a
train of electrical pulses (300 ms, 20 Hz, 2 s) (B). Analyzed neurons are indicated by arrows. Neurons 5, 6, and
10 belong to the RFP clone, and RFP– neurons were used as lineally unrelated controls. Scale bar: 50 mm.
(C) Fluo-4 tracings (bottom) of neurons after single-pulse electrical stimulation conveyed to three
different interganglionic nerve strands (top). F, arbitrary fluorescence; F0, F at time point 0. (D) Baseline
intracellular Ca2+ levels in RFP+ and RFP– neurons (two-tailed Student’s t test, P > 0.05). (E) Maximal
amplitude of Ca2+ transients induced by an electric pulse train in RFP+ and RFP– neurons (two-tailed
Student’s t test, P > 0.05). (F) Proportion of neuronal pairs that display coordinate Ca2+ responses to
single electrical pulses [one-way analysis of variance (ANOVA), P < 0.0001; Dunnett’s multiple comparison
test, ***P < 0.001 versus pairs of RFP-labeled sister neurons]. (G) Proportion of sister pairs present in
the same ganglion compared to sister pairs located in different ganglia that display coordinate Ca2+ responses
to single electrical pulses (two-tailed Student’s t test, P > 0.05). Error bars in (D) to (G) indicate SEM.