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Jacobs- Li, Tang
et al
. eLife 2023;12:e79156. DOI: https://doi.org/10.7554/eLife.79156
1 of 26
Single-
cell profiling coupled with lineage
analysis reveals vagal and sacral neural
crest contributions to the developing
enteric nervous system
Jessica Jacobs- Li
, Weiyi Tang
, Can Li, Marianne E Bronner*
Division of Biology and Biological Engineering, California Institute of Technology,
Pasadena, United States
Abstract
During development, much of the enteric nervous system (ENS) arises from the vagal
neural crest that emerges from the caudal hindbrain and colonizes the entire gastrointestinal tract.
However, a second ENS contribution comes from the sacral neural crest that arises in the caudal
neural tube and populates the post-
umbilical gut. By coupling single-
cell transcriptomics with axial-
level-
specific lineage tracing in avian embryos, we compared the contributions of embryonic vagal
and sacral neural crest cells to the chick ENS and the associated peripheral ganglia (Nerve of Remak
and pelvic plexuses). At embryonic day (E) 10, the two neural crest populations form overlapping
subsets of neuronal and glia cell types. Surprisingly, the post-
umbilical vagal neural crest much more
closely resembles the sacral neural crest than the pre-
umbilical vagal neural crest. However, some
differences in cluster types were noted between vagal and sacral derived cells. Notably, RNA trajec-
tory analysis suggests that the vagal neural crest maintains a neuronal/glial progenitor pool, whereas
this cluster is depleted in the E10 sacral neural crest which instead has numerous enteric glia. The
present findings reveal sacral neural crest contributions to the hindgut and associated peripheral
ganglia and highlight the potential influence of the local environment and/or developmental timing
in differentiation of neural crest-
derived cells in the developing ENS.
Editor's evaluation
This paper is useful for researchers in the field of enteric neuroscience and peripheral nervous
system development. The single cell RNA-
sequencing based analysis of the developing chicken
ENS, demonstrates differential cell identity contribution from the sacral and vagal neural crest and
influence of the local distal embryonic environment for final differentiation. A basic classification
scheme of neuronal cell types in the chicken combined with analysis of a more mature embryonic
stages and functional data will however be needed in the future to determine the role of differential
stem cell origin for final neuronal composition in the distal gut of chicken.
Introduction
The enteric nervous system (ENS) is the largest component of the peripheral nervous system and plays
a critical role in regulating gut motility, homeostasis, and interactions with the immune system and
gut microbiota (
Nagy and Goldstein, 2017
). In amniotes, the ENS consists of millions of neurons with
motor, sensory, secretory, and signal transduction functions, as well as a larger number of supportive
enteric glia. Interestingly, enteric glia recently have been shown to retain neurogenic potential via
reentrance into a progenitor-
like state (
Laddach et al., 2023
). Together these diverse neurons and glia
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*For correspondence:
mbronner@caltech.edu
These authors contributed
equally to this work
Competing interest:
See page
21
Funding:
See page 21
Received:
01 April 2022
Preprinted:
09 May 2022
Accepted:
23 October 2023
Published:
25 October 2023
Reviewing Editor:
Kathryn Song
Eng Cheah, University of Hong
Kong, Hong Kong
Copyright Jacobs-
Li, Tang
et al
. This article is distributed
under the terms of the Creative
Commons Attribution License,
which permits unrestricted use
and redistribution provided that
the original author and source
are credited.
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Jacobs- Li, Tang
et al
. eLife 2023;12:e79156. DOI: https://doi.org/10.7554/eLife.79156
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form a highly orchestrated network of physically and chemically connected cells embedded between
the muscle and mucosal layers of the gastrointestinal system (
Fleming et al., 2020
). Due to the vast
number of cells and its capacity for autonomic regulation, the ENS is often referred to as ‘a second
brain’ (
Gershon, 1999
).
The neurons and glia of the ENS arise from the neural crest, a migratory stem cell population,
emigrating from the closing neural tube. This transient population consists of four subpopulations
designated from rostral to caudal along the body axis as cranial, vagal, trunk, and sacral. Best studied
in mouse and chick embryos, much of the ENS is derived from ‘vagal’ neural crest cells that arise in
the caudal hindbrain (adjacent to somites 1–7) at chick Hamburger Hamilton (HH) stage 10, approx-
imately embryonic (E) day 1.5. These cells enter the foregut and migrate caudally to populate the
entire length of the gut by E8 in the chick embryo (
Le Douarin and Teillet, 1973
), as well as giving
rise to nerve-
associated Schwann cell precursors (SCPs) that later invade the gut (
Uesaka et al., 2015
;
Espinosa-
Medina et al., 2017
). However, there is an additional neural crest contribution to the ENS
from the sacral neural crest population (
Le Douarin and Teillet, 1973
). First observed by Le Douarin
and Teillet in quail-
chick chimeric grafts, the sacral neural crest arises caudal to somite 28 at HH 17–18
(E2.5), migrates to the dorsal side of the developing gut, and forms the paired pelvic plexuses and
Nerve of Remak at E3.5 (
Anderson et al., 2006
;
Burns and Douarin, 1998
;
Yntema and Hammond,
1955
;
Figure 1A
). The Nerve of Remak, which is specific to bird, is closely associated with the hindgut
and has been described as a staging ground for many neural crest-
derived cells which migrate to the
gut along extrinsic axons to colonize the post-
umbilical gut by E8 and rapidly expand in number by
E10 (
Burns and Douarin, 1998
;
Pomeranz et al., 1991
).
Dysregulation of ENS development is responsible for enteric neuropathies such as Hirschsprung’s
disease which affects 1 in 5000 live births (
Amiel et al., 2008
), and is characterized by a paucity or
absence of neurons in the distal colon resulting in potentially lethal obstruction and increased risk of
infection (
Lake and Heuckeroth, 2013
;
Ji et al., 2021
;
Lourenção et al., 2016
). While the etiology
of Hirschsprung’s disease is not completely understood, it is thought that insufficient migration or
proliferation of vagal neural crest precursors results in neuronal deficits, particularly in the hindgut
due to the long distance needed for precursor cells to reach their destination. Grafting sacral neural
crest cells in place of ablated vagal neural crest results in isolated ganglia in myenteric and submu-
cosal plexuses. However, the grafted sacral neural crest’s contribution to the ENS was insufficient
to compensate for the lack of vagal-
derived cells, suggesting that there may be intrinsic differences
between these two populations (
Burns et al., 2000
). Molecular cues such as GDNF (
Young et al.,
2001
) and ET3 (
Nagy and Goldstein, 2006
) are essential for migration and differentiation of vagal
neural crest during early development and mutations in these genes are common in patients with
Hirschsprung’s disease (
Kenny et al., 2010
). However, it is unknown if these genes are involved in the
development of the sacral neural crest.
Recent studies have proposed that lack of rostrocaudal migration of the vagal neural crest may
not be the only cause of enteric neuropathies. A distinction between the ENS of the foregut/midgut
versus hindgut is that the former arises solely from vagal neural crest-
derived cells, whereas the latter
is populated by both vagal and sacral neural crest-
derived cells (
Burns and Douarin, 1998
). Thus, a
complete understanding of ENS ontogeny requires more thorough characterization of possible differ
-
ences between vagal neural crest contributions to the pre-
umbilical versus post-
umbilical gut and
characterization of sacral neural crest-
derived contributions to the hindgut and associated peripheral
ganglia. Open questions include: What cell types are derived from the sacral neural crest? Is the sacral
neural crest population distinct from the vagal, or do they have shared derivatives? Does the post-
umbilical gut possess special cell types absent in the pre-
umbilical region? Defining the transcriptional
landscape of sacral and vagal neural crest-
derived cells along the entire length of the gut holds the
promise of revealing similarities and differences between these populations.
To tackle these questions, we combined single-
cell transcriptomics with a recently developed
lineage tracing method in which replication-
incompetent avian (RIA) retroviruses can be used to infect
specific axial levels of the neural tube of the developing chick embryos to permanently express an
inherited fluorophore (
Tang et al., 2019
). By infecting either vagal or sacral neural crest populations,
RIA retroviral infection permits region-
specific lineage tracing without the need for transplantation
or Cre-
mediated recombination that can result in ectopic expression. This enables transcriptional
profiling of vagal- or sacral-
derived RIA-
labeled ENS cells in the pre- and post-
umbilical gut at
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Figure 1.
Bulk RNA-
seq of vagal and sacral neural crest derived cells in the post-
umbilical enteric nervous system (ENS). (
A
) Schematic diagram
describing experimental procedure for viral labeling. Vagal and sacral neural crest cells were labeled by H2B-
YFP (green) in separate embryos. The post-
umbilical gastrointestinal tracts, including accompanying ganglia, were dissected at E10 for dissociation. (
B
) YFP+ cells from the post-
umbilical region
derived from vagal or sacral neural crest (NC) were sorted via FACS. (
C
) Volcano plot describing differentially expressed genes of sacral (sacral-
post,
blue) and vagal neural crest cells in the post-
umbilical gut (vagal-
post, red). Genes with fold change greater than 2 and p-
value<0.05 are colored. (
D
)
Heatmap highlighting selected genes related to neuronal functions from differential gene expression analysis in sacral and vagal-
post ENS populations
(with two replicates per condition). Genes are ordered based on significance level and fold change.
The online version of this article includes the following figure supplement(s) for figure 1:
Figure supplement 1.
DiI-
labeling of the sacral neural crest.
Figure supplement 2.
Diagram of dissected tissue for single-
cell RNA-
sequencing.