of 39
Single
-
cell profiling
coupled with lineage analysis
reveals
distinct
sacral neural crest contributions
to the developing
enteric nervous system
Weiyi Tang
,
Jessica Jacobs
-
Li,
Can Li
and Marianne E. Bronner
*
Division of Biology and Biological
Engineering, California Institute of Technology, Pasadena, CA
91125 USA
*Corresponding author
mbronner@caltech.edu
.
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Abstract
During development, t
he enteric nervous system (ENS
)
arises
from neural crest cells
that emerge
from the neural tube
,
migrate to and along the gut,
and
coloniz
e
the entire intestinal tract. While much of
the ENS arises from vagal neural crest
cells
that originate from the caudal hindbrain, there is a second
contri
bution from
the
sacral neural crest
that migrate
s
from the caudal end of the spinal cord to populate
the post
-
umbilical gut.
By coupling
single cell transcriptomics with
axial
-
level specific lineage tracing
in
avian embryo
s
,
we compare
d
the contribut
ions
between
embryonic
vagal
and
sacral neural crest cells
to
the
ENS
.
The
results
show that
the two
neural crest
populations
form
partially overlapping but
also
complementary
subset
s
of neurons and glia
in
distinct
ganglionic
units
.
In
particular, the
sacral neural
crest cells
appear to be the major source of
adrenergic
/dopaminergic
and serotonergic
neurons,
melanocytes and Schwann cells in the post
-
umbilical gut.
In addition to neurons and glia, t
he results also
reveal
sacral neural cre
st contributions to
connective tissue and
mesenchymal cells
of the gut
.
These
finding
s
highlight the
specific properties of
the
sacral
neural crest
population
in the
hindgut
and have
potential implications for
understanding development
of
the
complex nervo
us system in the hindgut
environment
that may influence
congenital neuropathies.
.
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;
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The enteric nervous system (ENS)
, the largest component of the peripheral nervous system,
plays a critical role in regulating gut motility, hormone homeostasis,
as well
as
interacti
ons
with the
immune
system
and gut microbiota
1
. In amniotes,
the ENS consists of
millions of neurons with motor,
sensory, secret
ory
and signal transduction functions
as well as
a larger
number of enteric glia
.
Together
they
form
a
highly orchestrated network
of
physically
and
chemically connected
cells embedded
between
the muscle and mucosa
l
layers
of the gastrointestinal system
2
.
Due to
the ENS’s
vast number
of
neuron
s and glia
and
its
capacity for
autonomic
regulation
,
it
is
often
referred to as “
a
second brain”
3
.
The neurons and gli
a of the ENS originate from the neural crest, a migratory stem cell population
that forms most of the peripheral nervous system. During development,
these
cells migrate from the
central nervous system
(CNS)
into the periphery to colonize
the
intestines. M
uch of the ENS is derived
from “vagal” neural crest cells that arise in the caudal hindbrain, enter the foregut and migrate from
caudal
ly
to populate the entire length of the gut.
The vagal neural crest arises
in the CNS adjacent to
somite
s
1
-
7
at Hamburger Hamilton (HH) stage 10 and fully colonize
s
the entire length of the gut
by
embryonic day (E) 8 in the chick
embryo
4
,
after
undergoing one of the longest migrations of any
embryonic cell type.
However, there is an additional neural crest contribution to the ENS from a
neural
crest
population that arises at the caudal end of the CNS
,
referred to as the “sacral” neural crest. First
observed
by Le Douarin and Teillet
in
quail
-
chick chimeric grafts, sacral neural crest cells enter the
hindgut and migrate rostrally to contribute neurons and glia to the post
-
umbilical gu
t
4
.
The sacral neural
crest arise
s
posterior
ly
to somite 28 at HH stage 17
-
18
,
fully colonize
s
the post
-
umbilical gut
by
E8
and
expand
s
in number by E10
5,6
(Fig1
A)
.
Dysregulation of
ENS
development
is responsible for enteric neuropathies including
Hirschsprung
’s
disease
which
affect
s
1 in 5000 live births
and is
characterized by hindgut
aganglionosi
s
that results in
the
lack of gut motility
7
.
While the
etiology of Hirschsprung
’s
disease is not completely
.
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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
the
ir
destination
.
Grafting
sacral neural crest cells
in place of ablated vagal neural crest result
ed
in
isolated ganglia in myenteric and submucosal plexuses
. However,
the grafted sacral neural crest
’s
contribution to the ENS was insufficient in number
to com
pensate for the lack of vagal
-
derived cells,
suggesting that there are intrinsic differences between these two populations
8
.
M
olecular
cues
such as
GDNF
9
and ET3
10
are
essential
for
migration
and differentiation
of vagal neural crest during
early
development
. Consistent with this, m
utations in
R
et
,
G
dnf
,
E
dnrb
and
E
dn
3
genes
are common in
patients with
Hirschsprung’s disease
11
, although
phenotypes of these mutations
often
exhibit
a complex
inheritance
pattern and
low
penetrance
7
.
R
ecent studies
hav
e proposed
that
lack of
rostrocaudal
migration of
the
vagal neural crest
may
not
be
the only cause of
enteric neuropath
ies.
A
distinction between
the ENS of the
foregut/midgut
versus hindgut
is that
the former arises sole
l
y from vagal neural
crest
-
derived cells whereas the latter
is
populated by both vagal and
sacral neural crest
cells
5
.
Thus, deciphering
the ontogeny of
hindgut
neuropathy
requires a thorough u
nderstanding of all
neural crest
-
derived
contributions to
the hindgut
and exploring possible
cell fate diversity
between
vagal
and sacral neural crest
populations
.
Open
questions include:
what is the complete complement of cell types 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 neuronal cell types absent in
the pre
-
umbilical region?
A more
complete view of the transcriptional landscape of the sacral compared with
the
vagal neural crest
will
provide a
deeper understanding of the
se different cell populations
and
clarify how dysregulation
of their
d
evelopmental programs
may contribute to congenital birth defects.
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To tackle these questions
,
we
combine
d
single
-
cell transcriptomics with
a recently developed
lineage tracing method utilizing replication
-
incompetent avian (RIA) retroviruses
to specifi
cally label
distinct
neural crest cells
populations i
n
developing
chick e
mbryos
.
As amniotes, avian
embryos
closely
resembl
e
human
embryos
at similar developmental stage
s
,
while being
much more
accessible to
experimental manipulation
than
mammalian embryos
.
RIA retrovir
al infection
permanently expresses a
fluorescence signal in the infected cell lineage
12
without the need for transpla
ntation or
C
re
-
mediated
recombination
that
can
result
in
ectopic expression
.
T
his
allows for
precise region
-
specific lineage
analysis to follow cell fate and
isolate pure populations of cells
derived from vagal
or
sacral population,
thereby enab
ling
t
ranscriptional profil
ing
at single cell resolution.
Thus, t
h
e combined
features
of
the
chick
embryo and the RIA
retrovirus
provide
a particularly advantageous
system
to
comparatively
study
the
relative contribution
s
of the
vagal and sacral neural crest
cells
to the
entire
developing
ENS
for the
first time.
By selective
ly
labeling
each
neural crest
population
, we
demonstrate
that sacral neural crest
cells
form functionally distinct units
f
rom
the
vagal neural crest,
suggesting
that
these populations
are likely
to have distinct
functions
.
Single
-
cell transcriptome analysis show
s
that
the
sacral neural crest gives
rise
to
the majority
of
Schwann cells
and
adrenerg
i
c
/
dopaminergic
/serotonergic neurons
of the post
-
umbilical gut
,
whereas
the
pre
-
umbilical
v
agal neural crest
is
the
primary source of
secretomotor
neurons.
Interestingly
, vagal neural crest cells
in
the post
-
umbilical gut
are
intermediate
in character
between the pre
-
umbilical
vagal
and sacral
population
s
,
suggesting
a strong influence of
environmental
cues
in cell
fate
decision
s
.
In addition, our
results
unc
over
sacral
neural crest
contributions to
connective
tissue
and melanocytes
in the post
-
umbilical
gut
region
.
This
study
expand
s
our
understanding of
the
sacral neural crest, a largely understudied stem cell population
that acts
in close coordination wit
h the
vagal neural crest to form
essential
neuronal and glia
l
cells
of
the hindgut ENS. Our result
s
further
.
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suggest that specific properties of
vagal and
sacral neural crest could be
essential
in
understand
ing
the
nature of
congenital hindgut
neuropathies
.
.
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was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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Results
Population level
RNA
-
seq analysis shows that
the
sacral
and vagal
neural crest population
s
exhibit
distinct
transcriptional profile
s
As a first step in assessing
contributions of
vagal and s
acral neural crest
in the post
-
umbilical
region
,
we
obtained pure populations of each using a novel
replication
-
incompetent avian
(RIA)
retrovirus lineage tracing technique
12,13
for bulk RNA
-
seq analysis
. To th
is
end,
the
neural tube at the
level of the
caudal hindbrain was injected with RIA
retrovirus carrying a YF
P expression cassette
at
Hamburger Hamilto
n
stage
10
(HH10)
to label
vagal neural crest cells,
or
below the
level of somite 2
8
at HH17 to label the
sacral neural crest
. T
he
post
-
umbilical
guts
were
then
harvested
at
embryonic da
y
10
(
E10,
Fig1
A
)
by which t
ime the gut was fully populated by neural crest cells that were undergoing
terminal differentiation
;
after dissociation,
YFP+ cells
were
sorted
using FACS
(Fig1B)
.
Similar regions
from three guts were p
oo
led
as a replicate,
with
each library contai
ning
2000 cells.
Differential gene
expression
analysis
reveals intriguing distinctions between
vagal and sacral
neural crest cells
in the post
-
umbilical gut
at the population level
, suggesting that they
serve distinct
functions.
G
enes
enriched
in
the
sacral
p
opulation
include
Sst1/Sstr
indicating interneuron cell fate,
and
Dbh, Th, Ddc, Pnmt,
and
Slc18
a2
which are
present in
catecholaminergic neurons
and serotonergic
neurons.
Grm3
expression indicates that
g
lutamatergic
character
is also
more
abundant in the sacral
population. In addition, we observed
up
-
regulation of
Gfra3
which is involved
in the GDNF signaling
pathway and
Cxcl12
which is
related to signaling during cell migration (Fig1
C, D
).
C
onversely,
the
vagal
post
-
umbilical
population expresses
the
adrenergic receptor
Adra1b
,
enzyme
Gad1
,
Calb2
and
Nts
consistent with
excitatory motor neuron
fate
.
Additionally, the population expresses
genes related to
.
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neural crest and neuron
al
migration such as
Sema3d
and
Tnc
.
Hes5 and Dll1
expression
indicate
functions for
N
otch/
D
elta signaling
in the vagal
-
post
-
umbilical
population (Fig1C, D).
These results
s
uggest
that vagal and sacral neural crest cells
give
rise to
diverse
cell types
likely reflecting different
functions
14,15
.
Single
-
cell transcriptome profiling of the
chick
ENS
To
understand
the
transcript
ional
profile of
vagal and sacral neural crest
-
derived cells
at single cell
resolution
, we performed viral labeling as described
above
(Fig1A)
and collected
three distinct
ne
ural
crest populations at E10
: vagal neural crest from the pre
-
umbilical gut (vagal
-
pre
; 3 guts
pooled per
replicate
), vagal neural crest from the post
-
umbilical gut (vagal
-
post
; 6 guts
pooled per replicate
) and
sacral neural crest from
the
post
-
umbilical
region (sacral
; 6 guts
pooled per replicate
). After FACS
isolation of YFP+ cells, 4.6k
-
5k cells were sequenced for each replicate, generating a single
-
cell profile
with ~29000 cells
that organized into
13 clusters
16
18
.
To
ascribe
cluster
identity,
we first performed gene
e
xpression heatmap
an
alysis
for top 10 gene markers in clusters 0
-
12
(C0
-
C12)
, marking the most up
-
regulated gene
s
as compared to all other clusters (
Fig2
A).
Next, we selected a group of
genes characteristic of
cell fate to define the dominat
e
cell type in
the clusters
and present them as a dotplot (
Fig2
B)
and feature expression (
Fig3
A)
.
W
e identified
C
0 as
Schwann cells
based on
high level
s of
Pmp22
,
Ednrb
,
Sox10
, and
Plp1
expression
(
Fig2
B,
Fig3
A
Sox10
,
Pmp22
,
Endrb
)
. Consistent with heatmap features,
C
1 is likely to
contain
progenitor
cells
due to its
expression of
Myo9D
, which is associated with p75 signaling, and low expression of differentiation
markers (
F
ig2B
).
C
2 exhibits high level
s
of
t
he
neural crest marker
Ednrb
, progenitor/glial marker
Sox10
,
proliferation marker
Mdk
,
as well as high levels of
Sox8
,
Lmo4
and
Hes5
.
Due to this
gene
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expression profile alongside the
lack
of
Schwann cell markers
, C2 was
considered a
glial progenitor
population
(
Fig2
B)
.
C3 and
C
4
have high
expression of
Elavl4
, a marker for differentiated neurons
,
Ret
representing enteric neurons
,
and the neuropeptide
Npy
, suggesting
that
both clusters
have
Npy
+
inhibitory motor neurons
(
Fig2
B,
Fig3
A
Ret
,
Elavl4
,
Npy
)
.
However,
C
3 exhibits more prominent
Vip
,
Nts
,
Tac1
expression than
C
4, indicating excitatory motor neurons;
C
4 has more cholinergic genes
like
Chrna5
,
and
adrenergic
/
serotonergic genes
such as
Dbh
,
Ddc
,
and
Th
(
Fig2
B,
Fig3
A)
.
Therefore,
C
3 is
likely to represent a secretomotor neuron cluster, while
C
4 is
characteristic of adrenergic neurons,
although neurons with
other functions
are also present
.
Undifferentiated
and proli
ferating
cells are most
abundant in C2 and C6, as they exhibit the highest
Mdk
expression
.
Some cells in C2 and C6 express
Ascl1
, indicating
they may be
neuroblast
s
and/or glioblasts
(Fig2B, Fig3A
Mdk
,
Ascl1
)
.
However,
C2
has glial markers like
Sox8
indicating it is a glial progenitor cluster
.
Due
to high expression of cell cycle
associated genes
like
Top2a
and
Smc2
,
progenitor
cells in
C
6 seem to be actively cycling
(Fig2B)
.
In addition to canonical peripheral nervous system derivatives
, our analysis has
revealed a
range
of cell fates previously
unknown to be derived from the neural crest in the gut. For example,
C5
is
classified as
connective tissue cells
based on
Col1a1
.
C7 is epithelial cells
as
suggested by markers
Krt7
and
Epcam
. Based on expression of
Mlana
,
C
8 is
likely to be composed of melanocytes. C
9
has a
similar expression pattern
to
C
5, but
its
Col18a1
expression
likely reflects
a
vascular
muscle fate.
C
10
appears to
be
smooth muscle
cells
based on
strong expression of
Acta2
,
Actg2
,
Tpm1
,
Myh11
,
Des
, and
Tagln
.
C11 express
es
endothelial markers
Apold1
,
Erg
,
Vwf
, and
Pecam
.
C12 is likely to be
hematopoietic
-
related cells based on
Gsta
3
,
Ly86
, and
Ptpn6
expression
(
Fig2
B
)
.
P
utative cluster identities are presented in
Uniform
M
anifold
A
pproximation and
P
rojection
(UMA
P
,
Fig2
C).
To exclude the possibility that these cell types are a result of
contamination during cell
sorting, we
examined l
ineage tracer (H2B
-
YFP) and viral genome sequences (RIA)
expression
across
.
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the clusters
(
Fig2
B
,
Fig3
A
H2B
-
YFP
,
RIA,
Fig3
B
RIA
)
.
RIA
genomic transcripts were abundantly
expressed in almost all clusters, suggesting these were indeed virally
-
labeled neural crest derived cells.
Some c
ells in
the
p
rogenitor
cluster
(
C1
)
exhibit low
er
YFP and RIA transcripts
; t
hese cells a
lso showed
lower expression for other markers (
So
x10
,
Ednrb
) except for moderate level of
Mdk
,
possibl
y
due to
low overall transcription level
.
Because C10 and C12 general
ly
lack
ed
YFP and RIA transcripts, the two
clusters are likely to be a result of autofluorescence,
and
thus
were eliminated from
downstream analysis.
V
agal and sacral neural crest
differentially
contribute to ENS cell types
Next,
we
explore
d
the relative contribu
tions
of
the
sacral,
and pre
-
and post
-
umbilical
vagal
cell
populations
to
this broad range of
cell types
present in the E10 gut
. According to the UMAP
separated
by populations,
sacral,
post
-
umbilical vagal
and
pre
-
umbilical vagal
populations form distinct
combination
s
of cell types (
Fig4
A, B). Quantification of cluster contributions
reveals a
common
contribution
to
progenitor
cells
(C1)
,
connective tissue
(C5)
,
actively cycling progenitors
(C6),
and
epithelial
cells
(C7)
.
In contrast
,
the remaining
clusters
have primary
contribution from
one or two
populations
(
Fig4
C)
.
For example,
the
Schwann cell cluster
(C0)
is mainly
comprised of sacral
(orange)
and
vagal
(navy)
populations in the post
-
umbilical gut with minimal contribution from the pre
-
umbilical
vagal neural crest cells
(light blue)
(Fig4C)
. This is likely to be a result of non
-
myelinating Schwann
cells located in the intestinal nerve of Remak, present in the post
-
umbilical gut.
The
glial
progenitor
(C2)
population is primarily derived from
pre
-
umbilical
vagal
population. Interestingl
y,
over 75% of
C3
secretomotor
neurons
are
from the
vagal neural crest derived pre
-
umbilical gut, with a small proportion
(20%) from
the
post
-
umbilical vaga
l
population
.
However,
adrenergic
neurons in C4 were restricted
to
the post
-
umbilical gut. Most post
-
umbilical neurons are contributed
to
by
the
sacral neural crest
.
C8
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melanocytes are
only found
in the post
-
umbilical region, with most cells contributed from
sacral neural
crest. C9 is a
vascular smooth muscle
population
that
almost exclusively
arise
s
from sacral neural crest.
While endothelial cells are present throughout the entire gut length,
wit
h
sacral and
post
-
umbilical
vagal
populations
a
s
the major contributors
(
Fig4
C).
In summary,
the
main
cell types derived from the
sacral neural crest
cells
are
Schwann cells,
including those in intestinal nerve of Remak, cholinergic,
adrenergic/
serot
onergic neurons, and
melanocytes. Vagal neural crest
-
derived cells in
the
pre
-
umbilical region
form
a combination of
neuronal progenitor cells and secretomotor neurons.
The high percent of progenitor cells contributed by
vagal
-
pre population
suggests that
cell differentiation is in progress all along the gut
even though
enteric
migration is complete
.
V
agal neural crest cells
in the
post
-
umbilical gut
contribute to most cell fate
s
found in the ENS and
form
intermediate
levels of cells
between
the
pre
-
umbilical
and sacral
(
Fig4
A, B)
.
These results indicate that for
those
vagal neural crest cells
that migrate long distances
, environmental
cues may have
a profound influence
on
cell fate
choice
.
Validation of
marker expression by
vagal
versus
sacral neural crest
using dual r
etroviral lineage
tracing
We next sought to
validate
the
gene expression differences
identified by single cell RNA
-
seq
between sa
cral and vagal neural crest contributions to the gut in differentially labeled cell populations
.
To this end
, we utilized axial
-
level specific retroviral labeling to sequentially mark vagal or sacral neural
crest cells with different fluorophores in the same embryo. For identifying the vagal neural crest, RIA
retrovirus expressing nuclear H2B
-
RFP was injected i
nto the neural tube adjacent to somite 1
-
7.
Embryos were then allowed to develop until HH17, at which time RIA retrovirus carrying H2B
-
YFP
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was injected into the neural tube posterior to somite 28 to label the sacral neural crest. The entire length
of the g
ut was dissected and removed for immunohistochemistry at E10
and stained
with
antibodies to
gene products identified as differentially expressed in our
sc
RNA
-
seq dataset (Fig1A). To gain insight
into the relative contributions of vagal versus sacral neura
l crest to the ENS along the entire length of the
gut, we imaged transverse sections of six subregions along the anterior
-
posterior axis: esophagus (eso),
stomach (sto), pre
-
umbilical small intestine (pre. int), post
-
umbilical small intestine (post.int), c
ecum (ce)
and colon (col) (Fig1A).
The results show that the pre
-
umbilical gut contained only H2B
-
RFP+, but no H2B
-
YFP+,
labeled cells, suggesting that only vagal neural crest cells contributed to the pre
-
umbilical region
(Fig1A). This is consistent with
previous studies using quail
-
chick chimeric systems
4
.
Immunohistochemistry revealed vagal RIA retrovirus
-
labeled cells that
co
-
expressed acetylcholine
receptor (Fig
5
A
-
C) and HuC/D (E
LAV
) in the pre
-
umbilical (Fig
5
D
-
F), consistent with differentiated
neurons. H2B
-
RFP and P0 double
-
positive Schwann cells were present along the pre
-
umbilical region
(Fig
5
G
-
I), as well as enteric pr
ogenitors or glial cells as determined by Sox10 expression (Fig
5
J
-
L).
Consistent with the scRNA
-
seq, there were only a few sparsely distributed neurons marked by TH
(Fig
5
M
-
O) and DBH (Fig
5
P
-
R), TH+ cells were not observed in the pre
-
umbilical small intesti
ne
(Fig
5
O) and DBH+ cells were absent from the esophagus (Fig2P).
In contrast to the pre
-
umbilical gut, the post
-
umbilical ENS contained both H2B
-
RFP+ cells and
H2B
-
YFP+ cells, indicating a collective contribution from sacral neural crest and some post
-
umb
ilical
vagal neural crest (Fig1A).
Consistent with our scRNA
-
seq data
, neural crest cells from different axial
origins appeared to contribute to distinct neuronal subtypes. Acetylcholine receptor (AchR)
-
positive
cells were more abundant in the vagal
-
deriv
ed population throughout the post
-
umbilical region (Fig
6
A
-
C, inset a1
-
c1) whereas AchR+ cells from sacral neural crest cells were only observed in the colon
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(Fig
6
A
-
C, inset a2
-
c2). Both vagal (Fig
6
D
-
F, inset d1
-
f1) and sacral (Fi
6
D
-
F, inset d2
-
f2) neural c
rest
populations differentiated into HuC/D+ neurons, except in the cecum where HuC/D labeling was more
restricted to vagally
-
derived cells (Fig
6
F, inset f2). Both vagal and sacral neural crest contributed to
Schwann cells and ENS progenitors/glia expressin
g Sox10 and P0 in all subregions of the gut (Fig
6
G
-
I,
P0; J
-
L, Sox10). However, TH+ cells were most frequently derived from sacral neural crest cells in the
colon region (Fig
6
M
-
O, inset n2). These results confirm that a very large proportion of the ENS in
the
colon is derived from the sacral neural crest. DBH+ cells were also more abundant in sacral neural crest
-
derived population (Fig
6
P
-
R), especially in regions highly enriched in sacral neural crest cells, such as
the colon (Fig
6
Q, inset q2) and cecum (Fi
g
6
R, inset r2). These results suggest that vagal and sacral
neural crest cells in the post
-
umbilical gut are distinct rather than functionally redundant, particularly in
the hindgut.
Sub
-
classification of vagal and sacral neural crest
-
derived
neuronal cell types
To
further
probe
for
specific
neurotransmitter
character
i
stic within the
chick ENS
, we
extracted
all cells from secretomotor and neuron
al
clusters (Fig7A’) and clustered the cells into 9
subsets
(Fig7A,
inset of Fig7A’). We examined a variety of
receptors
,
neur
o
transmitters
and neuropeptides
that mark
specific neuronal cell fates
19
and plotted the expression
in
th
e
subsets
(Fig7B).
C1
of this subsetting
is defined as a glial cluster due to its
high and broad expression of
Sox10
,
Pmp22
and
Ednrb
(Fig7B, Fig7C
Sox10
)
. C3 share
s
some similarities with C1,
most likely reflecting
neuronal precursors
as this cluster has
lower
Pmp22
and higher
Mdk
expression
(Fig7B)
.
C0
appears to
be
a precursor for excitatory motor neurons, due to its relatively high expression of
Tac1
,
Penk
,
Gal
and
Vip
, with some cholinergic
transcripts
and
moderately high level of
Mdk
(Fig7B, Fig7C
Tac1
,
Penk
)
. In
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contrast, C7 is identified as excitatory motor neuron
due to abundant expression
of
Calb2
,
Penk
and
Vip
with
possibly
a small number of
intrins
ic primary afferent neurons (
IPANs
)
, identified by
Nog
and
Ntg1
expression
(Fig7B, Fig7C CALB2, PENK)
. C8, a
Tac1
,
Gal
,
Nts
positive cluster
,
is also identified as
excitatory motor neurons
(Fig7B, Fig7C
Tac1
,
Nts
)
.
C
2 is likely to be a combination of excitatory motor
neurons and CALB1
+
IPANs
, with
cholinergic/catecholaminergic properties
(Fig7B, Fig7C
Calb1
)
.
C4 lacks
a
definitive cell marker, but a small percentage of cells
show
high
expression of th
e
inhibitory motor neuron marker
Fut9
(Fig7B)
.
O
ther
inhibitory motor
neuron markers remain low
,
suggesting
these cells
may
not be fully differentiated due to low
Elavl4
expression
(Fig7B)
.
Accordingly,
we ascribed
it
as
inhibitory motor neuron precursor. C5 is another cluster
of
differentiating neuron
s
based on
Ascl1
expression. C6, in contrast, is the cluster with most
Sst
expression, indicating an
interneuron cell fate
(Fig7B, Fig7C
Sst
)
. C6 is also likely to be the major sub
-
cluster with
catecholaminergic properties found
specifically
in
post
-
umbilical
gut
(Fig7C
Pnmt
,
Dbh
)
.
Together, these results confirm the differential gene analysis in
post
-
umbilical vagal
and sacral
bulk transcriptome analysis. In general, vagal neural crest cells are the primary source of motor neurons,
as indicated
by
upregulation of
Calb2
and
Nts
in vagal
-
post, whereas sacral neural crest cells give rise to
adrenergic
and sero
tonergic
neuronal types, as
reflected
by high fold change in
Dbh
,
Th
,
Pnmt
and
Ddc
(Fig1C, D).
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Di
scussion
The enteric nervous system regulates
critical
g
astrointestinal functions including digestion,
hormone secretion and immune
interactions
.
Abnormal ENS development can lead to
enteric
neuropathies including Hirschsprung’s disease,
characterized by
lack of motility and obstruction.
Studies have suggested
a critical role
for
the
vagal neural crest in
the
etiology
of
Hirschsprung’s
disease
7,11
. However, the
role
of
the
sacral neural crest
, which
coloniz
es
the hindgut in close
coordination with the vagal neural crest,
has been
largely und
erstudied
in enteric neuropathies
.
To
better
understand
the derivatives of the
sacral neural crest
and
their
coordination with the vagal neural crest
during
ENS
development
,
here we
examine
the
diversity
of
cell types arising from
vagal
versus
sacral
axial
levels at
single cell resolution.
A
blation and heterotopic graf
t
ing
experiments have previously been used to study t
he interplay
between the
vagal and sacral
population
s but
have
led to contradictory
interpretations
.
While s
ome
studies
concluded
that vagal and sacral neural crest exhibit autonomous migration properties
independent of the environment
, others
suggested a
role for
environmental
influences
.
On the one hand
,
an
aganglionic hindgut model cre
ated by surgically removing the caudal part of vagal neural crest and
replacing
it
with quail sacral cells
found that
quail sacral cells migrated into the hindgut with a small
increase in number of neurons
. This
suggest
ed
that sacral neural crest cells do not require the vagal
population to migrate
8
.
Reciprocally, w
hen
vagal neural crest cells are grafted to the sacral region, they
migrat
e
earlier and produc
e
a larger neuronal population t
han
the endogenous
sacral neural crest cells
19
.
However, other studies suggested a more prominent environmental effect,
such
that interchanged vagal
and sacral neural crest cells migrated according to the local
environment
20
.
Consistent with this,
combining chick gut before neural crest colonization with chick or quail neural crest
revealed
that sacral
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neural crest cells
can
colonize the colorectum independent of the vagal neural
crest, but require the
hindgut environment to differentiate
21
.
O
ur
results
using axial
-
level specific
labeling provide
a complementary
approach
to
address
th
e
s
e
question
s
by
compar
ing
transcriptome between vagal and sacral neural crest for the first time
. We
show that
sacral and vagal
-
derived ENS cell types
are not equ
ivalent
.
For example,
we find
Tnc
expression to be
specific
to the
vagal neural crest
(
Fig1C, D
), consistent with its r
equirement for
migration into the hindgut by changing the extracellular microenvironment
22
. In addition,
the
sacral
neural crest
expresses high levels of
Sox10
-
mediated
Cdh19
23
, and well as
Pax
3
and 5
-
HT3 for
innervation and
neuronal maturation in the pelvic ganglion
24,25
.
Ret
is also known to be upregulated in
vagal neural
crest cells to mediate more invasive
behavior
than in sacral neural crest
26
. In the hindgut,
distinct properties of vagal and sacral neural crest suggest that they are likely to play disparate roles in
enteric neuropathies. Indeed, diverse timeframe
s
and migration pattern
s
expose the cells
to differenti
al
environments
, leading to
diverg
ent
cell fates and functions.
Previous
studies have provided
valuable
information
regarding
gene expression, cell fate
divergence, gene regulatory networks and chromatin landscape
of vagal
-
derived neural crest cells
14,15,27
29
.
By f
ocusing transcriptome analysis on
the
small intestine in mice at postnatal day 21, one study
identified 12 distinct neuronal classes categorized by
a
combination of neurotransmitters
;
the authors
found
Pbx3
to be
a key gene for differentiation. Our results h
ave
identified most neurotransmitte
r genes
found in the murine system with the
excep
tion of
NOS1+ nitrergic neurons, GAD2+ GABAergic
neurons, or SLC17A6+ Glutamatergic neurons
14
, which may deve
lop at a later time point than studied
here
(Fig7B)
.
Another study, utilizing
RAISIN RNA
-
seq, identified 21 neurons and 3 glial clusters in
the
mouse small intestine and colon according to subset
s
of neurotransmitters.
Neuronal and glial
subsets we identi
fied generally
agree with
this
,
showing an overlapping group of sensory neuron
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markers such CCK, VIP, SST, NOG and NMU
29
(Fig7B)
.
C
ompared with these studies done with
postnatal and adult tissue, w
e observed more
clusters with progenitor
/precursor
identity
(
Fig2
C,
Fig7B)
,
which is
not
un
expected
, given that our analysis utilizes the gut from embryonic stages
.
While previous
studies
did not separate vagal from sacral contributions
, the small intestine and colon
are
likely to be
composed of
both
vagal
-
post and sacral population
s
, which are distinct from anterior vagal derivatives.
Our analysis
reveals that
gene e
xpression pattern
s
are markedly different according to
axial
-
level
,
confirming the results from previous studies
within
proximal
versus
distal colon
29
. This important
conclusion suggests
that the ENS is not uniformly distributed
throughout
the gut
but varies from
proximal to distal
.
T
aken t
ogether, the present results
suggest that there are
different
developmental programs
for
vagal
versus sacral neural crest population
.
Our results
help explain
sacral
neural crest
cannot
completely compensate for the loss of vagal neural crest
.
The c
ell
composition
of
the post
-
umbilical
ENS is distinct from that of pre
-
umbilical ENS,
with major
diffe
rences largely
resulting from
the
differential contributions
of
the
sacral neural crest. In
addition
,
the
differentiation program of vaga
l
-
derived
neural crest
in the post
-
umbilical gut
is
different from that of the
pre
-
umbilical vagal
population,
suggesting th
at
environmental factor
s have a large influence on cell fate
.
Our study highlights the
contributions of the
sacral
neural crest, particularly to the hindgut,
the
region
most affected in
Hirschsprung’s disease
c
haracterized by colonic agangliogenesis
.
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Methods
Retroviral labeling and chick embryology
H2B
-
YFP (#96893)
and
H2B
-
RFP (#92398)
obtained from Addgene were
cloned into the RIA plasmid
between Not1 and Cla1 sites. RIA
-
H2B
-
YFP/RFP was transfected into D
F1 cells (
ATCC, Manassas,
VA; #CRL
-
12203, Lot number 62712171, Certificate of Analysis with negative mycoplasma testing
available at ATCC website
) using PEI standard transfection protocol. DF1 cells were maintained in
Gibco Dulbecco's Modified Eagle Medium
(
DMEM
)
supplied with 10% FBS for 4 days, with 12ml of
supernatant collected per day. The supernatant was concentrated using ultracentrifuge for
at
76000
g
for
1.5 hours to get a viral stock
tittered
about 10
7
pfu/ml, aliquoted and stored at
-
80
°
C
until use. Viral
solution was
supplemented with 0.3
μ
l of 2% food dye (Spectral Colors, Food Blue 002, C.A.S# 3844
-
45
-
9) as an indicator
, injected to fill
the neural tube between somite 1
-
7 at
HH
Stage
10 to label vagal
neural crest and/or posterior to somite 28
at HH Stage 17
to label sacral neural crest
in ovo
. Embryo
s
w
ere
supplied with Ringer’s Solution
(
0
.9% NaCl, 0.042%KCl, 0.016%CaCl
2
2H
2
O wt/vol,
pH7.0
),
s
ealed with surgical tape
, and incubated at
37
°
C
until embryonic day 10.
Gut cell dissociation
and
Fluorescence
-
Activated Cell Sorting (FACS)
A
t embryonic day 10
, the g
astrointestinal tract was dissected from chick embr
yos and washed with
Ringer’s solution
. Pre
-
and post
-
umbilical regions were separated, broke into pieces in chilled
D
PBS and
loose
-
fit
homogenized in
Accumax
solution
(
EMD Millipore
)
.
400
μ
l of Accumax
-
tissue
mixture
was
aliquoted
into 1.7 ml Eppendorf tubes
and
shake
n
at 37
°
C
for 12mins.
After dissociation, c
hilled
Hanks
Buffered Saline Solution (HBSS)
supplemented by BSA (125mg in 50ml,
Sigma; 0.2% w/v
) and 1M
HEPES (500
μ
l in 50ml, PH7.5, ThermoFisher)
was added to quench the reaction. The dissociated cells
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were passed throu
gh
a
70
μ
m cell strainer (
Corning
) and collected by centrifug
ing
at
500g
for 11mins at
4
°
C
. The cells
were
resuspended in HBSS
-
BSA
, supplemented
7
-
AAD Viability Staining Solution
(Biolegend # 420404, 500 TESTS)
, and sorted for
YFP+, viable single cells
usi
ng
Sony
SY
3200 cell
sorter at the Caltech Flow Cytometry Facility.
Bulk
transcriptome analysis
For vagal neural crest in
the
post
-
umbilical regions, sacral neural crest at post
-
umbilical regions,
2
biological replicates were processed, with each replicate containing
YFP+
cells from
3
embryos. 2000
cells per replicate were
lysed to generate cDNA library using
SMART
-
Seq v4 Ultra Low Input RNA Kit
(
Takara Bio
). The library was sequenc
ed
with
50 milli
on
single end reads
with
50 bp length
using
HiSeq
2500
at the
Millard and Muriel Jacobs Genetics and Genomics Laboratory
Caltech
.
Sequencing
reads
were
trimmed using
cutadapt
30
and
mapped to Galgal6 genome using
Bowtie2
31
.
DESeq2
32
analysis was
performed to find differential expressed genes between vagal and sacral neural crest at post
-
umbilical
regions
generated by
HTseq
-
count
33
.
Differential gene expression was presented using
V
olcano
P
lot
(
coloring genes with Fold
Change>
2
and p value<0.05
) and Heatmap2 provided by
the Galaxy platform
.
Because there were more upregulated genes in the sacral than vagal
-
post population
s, we annotated
genes related
to neuronal function
for sacral population, genes related
to neuronal function
as well as
genes with
top fold
-
change and top significance
in vagal
-
post population.
Single
-
cell
transcriptome analysis
and data processing
For
vagal neural crest in pre
-
and post
-
umbilical
gut,
sacral neural crest
in
post
-
umbilical
gut,
2
biological replicates were
obtained
. E
ach
replicate of pre
-
umbilical gut was pulled from 3 embryos; each
replicate containing
post
-
umbilical gut was pulled from
6 embryos
.
After FACS for viable
YFP+
cells,
.
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4600
-
5000 cells
per replicate
were used for library preparation by the SPEC at Caltech.
The library was
sequenced on
NovaSeq S4 lane with 2x150bp reads
by Fulgent Therapeutics
.
To process fastq raw data,
standa
rd ENSEMBL galgal6 reference database was used. Single
-
cell level gene quantification was then
performed using
Cellranger
v3.1.0
16
and
kallisto
0.46.2 &
bustools
0.40.0 pipelines
17
with default
parameters. Gene count matrices from all the samples were combined and only cells with more than 200
genes detected were kept for th
e downstream analysis. To further remove potential doublet cells,
DoubletFinder
2.0.3 package was used.
Gene counts were normalized and scaled using Seurat v3.2.0
18
.
The first 30 principal components from PCA analysis were used to find neighbors with
Findneighbors
function b
efore cell clustering with
FindClusters
function (resolution = 0.2). UMAP dimensionality
reduction was performed using RunUMAP function with uwot
-
learn selected for the parameter
umap.method.
Immunohistochemistry
and imaging
Gastrointestinal tracts
were
dissected and
fixed in 4% PFA in PBS
(PH7.5)
for
25
mins at 4
°
C
and
washed with PBS for three times. Pre
-
and post
-
umbilical regions were separately incubated in
15%
sucrose
at
4°C
overnight
and in gelatin at 37°C
for 2 hours.
Gut segments were
embedded in
gelatin
solution, flash
-
frozen with liquid nitrogen, and mounted with
Tissue
-
Tek O.C.T compound (Sakura
#4583)
for
sectio
ning
(
Microm
HM550 cryostat).
Gut
sections were incubated in 1xPBS at
42
°C
until
the gelatin was dissolve
d
,
soaked
in
0.3% vol/vol Triton
-
X100 in 1xPBS
for permeabilization. B
locking
buffer
was prepared in
1xPBS with 5% vol/vol normal donkey serum
and
0.3% vol/vol Triton
-
X100.
Sections were
incubated with
primary antibody at 4
°
C overnight
S
ections were
washed with 1xPBS
for
10 minutes
and
3 times. After the washed, sections were incubated with
secondary antibody for
45
minutes
at room temperature.
List of p
rimary antibod
ies
used
:
1:20
chicken anti
A
chR ratIgG2a,
.
CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted May 9, 2022.
;
https://doi.org/10.1101/2022.05.09.491197
doi:
bioRxiv preprint
mAB270
(
DSHB
Antibody Registry ID: AB_5318
09
)
;
1:500 Mouse anti
HuC/D IgG2b
(
Invitrogen
-
Cat#A21271
)
;
1:20
Chicken anti mouse
P0
IgG1, IE8 (
DSHB
Antibody Registry ID: AB_2078498
)
;
1:500
R
abbit
anti
Sox10
(
Millipore
-
Cat# AB5727
)
;
1:500
Rabbit anti
Tyrosine Hydroxylase
(
Millipore
-
Cat# AB152
);
1:500
Rabbit anti
DBH
(
Immuno Star
-
Cat #:22806
)
.
List of
secondary antibodies used:
1:1000 donkey anti
-
mouse
IgG2b
647
(
Invitrogen A31571
)
,
1
:1000
goat
anti
-
mouse IgG
1
647
(
Invitrogen A21240
),
1:1000
donkey
anti
-
rat
Ig
G
647
(
Abcam ab150155
)
, 1:1000
goat
anti
-
ra
bbit
Ig
G
647
(
Invitrogen A21245
)
. Sections were imaged
with
Zeiss AxioImager.M2 with Apotome.2
. Images
were cropped and magnified for representation.
Acknowledgement
This work was supported by
R01DE027568 and
R35NS111564
t
o M.E.B. We
thank
Dr
s
. Igor
Antoshechkin and Vijaya Kumar
and t
he Millard and Muriel Jacobs Genetics and Genomics Laboratory
at California Institute of Technology
for their guidance and support in
bulk
RNA
-
sequencing
.
We thank
Jamie Tijerina and Rochelle D
iamond
from the
Beckman Institute
Flow Cytometry Facility
for their
help
with
the
FACS.
We thank
Dr. Sisi Chen, Jeff Park
, Prof. Matt Thomson
and
SPEC at Caltech for
their
dedicated
support in optimization and guidance in single
-
cell RNA
-
sequencing. We tha
nk
Dr. Fan
Gao
and
Bioinformatics Resource Center in the
Beckman Institute at Caltech
for
guiding us through
single
-
cell transcriptomic analysis. We appreciate the help from
Prof. Carlos Lois for
kindly
sharing
equipment
with us to perform
RIA concentratio
n.
We thank Dr.
Michael L
.
Piacentino
,
Dr.
Erica J.
Hutchins
and Prof. A
ngelike
S
tathopoulos
for the helpful discussion on the manuscript.
.
CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted May 9, 2022.
;
https://doi.org/10.1101/2022.05.09.491197
doi:
bioRxiv preprint
F
igure legends
.
CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted May 9, 2022.
;
https://doi.org/10.1101/2022.05.09.491197
doi:
bioRxiv preprint