Single-cell profiling coupled with lineage analysis reveals distinct sacral neural crest contributions to the developing enteric nervous

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

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

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

avian embryo

we compare

the contribut

ions

between

embryonic

vagal

and

sacral neural crest cells

the

ENS

The

results

show that

the two

neural crest

populations

form

partially overlapping but

also

complementary

subset

of neurons and glia

distinct

ganglionic

units

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

highlight the

specific properties of

the

sacral

neural crest

population

in the

hindgut

and have

potential implications for

understanding development

the

complex nervo

us system in the hindgut

environment

that may influence

congenital neuropathies.

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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

interacti

ons

with the

immune

system

and gut microbiota

. 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

highly orchestrated network

physically

and

chemically connected

cells embedded

between

the muscle and mucosa

layers

of the gastrointestinal system

Due to

the ENS’s

vast number

neuron

s and glia

and

its

capacity for

autonomic

regulation

often

referred to as “

second brain”

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

to populate the entire length of the gut.

The vagal neural crest arises

in the CNS adjacent to

somite

at Hamburger Hamilton (HH) stage 10 and fully colonize

the entire length of the gut

embryonic day (E) 8 in the chick

embryo

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

quail

chick chimeric grafts, sacral neural crest cells enter the

hindgut and migrate rostrally to contribute neurons and glia to the post

umbilical gu

The sacral neural

crest arise

posterior

to somite 28 at HH stage 17

fully colonize

the post

umbilical gut

and

expand

in number by E10

5,6

(Fig1

Dysregulation of

ENS

development

is responsible for enteric neuropathies including

Hirschsprung

’s

disease

which

affect

1 in 5000 live births

and is

characterized by hindgut

aganglionosi

that results in

the

lack of gut motility

While the

etiology of Hirschsprung

’s

disease is not completely

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understood

, it

is thought

that insufficient migration

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

destination

Grafting

sacral neural crest cells

in place of ablated vagal neural crest result

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

.

olecular

cues

such as

GDNF

and ET3

are

essential

for

migration

and differentiation

of vagal neural crest during

early

development

. Consistent with this, m

utations in

dnf

dnrb

and

genes

are common in

patients with

Hirschsprung’s disease

, although

phenotypes of these mutations

often

exhibit

a complex

inheritance

pattern and

low

penetrance

ecent studies

hav

e proposed

that

lack of

rostrocaudal

migration of

the

vagal neural crest

may

not

the only cause of

enteric neuropath

ies.

distinction between

the ENS of the

foregut/midgut

versus hindgut

is that

the former arises sole

y from vagal neural

crest

derived cells whereas the latter

populated by both vagal and

sacral neural crest

cells

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?

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

evelopmental programs

may contribute to congenital birth defects.

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To tackle these questions

combine

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

developing

chick e

mbryos

As amniotes, avian

embryos

closely

resembl

human

embryos

at similar developmental stage

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

without the need for transpla

ntation or

mediated

recombination

that

can

result

ectopic expression

his

allows for

precise region

specific lineage

analysis to follow cell fate and

isolate pure populations of cells

derived from vagal

sacral population,

thereby enab

ling

ranscriptional profil

ing

at single cell resolution.

Thus, t

e combined

features

the

chick

embryo and the RIA

retrovirus

provide

a particularly advantageous

system

comparatively

study

the

relative contribution

of the

vagal and sacral neural crest

cells

to the

entire

developing

ENS

for the

first time.

By selective

labeling

each

neural crest

population

, we

demonstrate

that sacral neural crest

cells

form functionally distinct units

rom

the

vagal neural crest,

suggesting

that

these populations

are likely

to have distinct

functions

Single

cell transcriptome analysis show

that

the

sacral neural crest gives

rise

the majority

Schwann cells

and

adrenerg

dopaminergic

/serotonergic neurons

of the post

umbilical gut

whereas

the

pre

umbilical

agal neural crest

the

primary source of

secretomotor

neurons.

Interestingly

, vagal neural crest cells

the post

umbilical gut

are

intermediate

in character

between the pre

umbilical

vagal

and sacral

population

suggesting

a strong influence of

environmental

cues

in cell

fate

decision

In addition, our

results

unc

over

sacral

neural crest

contributions to

connective

tissue

and melanocytes

in the post

umbilical

gut

region

This

study

expand

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

cells

the hindgut ENS. Our result

further

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suggest that specific properties of

vagal and

sacral neural crest could be

essential

understand

ing

the

nature of

congenital hindgut

neuropathies

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Results

Population level

RNA

seq analysis shows that

the

sacral

and vagal

neural crest population

exhibit

distinct

transcriptional profile

As a first step in assessing

contributions of

vagal and s

acral neural crest

in the post

umbilical

region

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

end,

the

neural tube at the

level of the

caudal hindbrain was injected with RIA

retrovirus carrying a YF

P expression cassette

Hamburger Hamilto

stage

(HH10)

to label

vagal neural crest cells,

below the

level of somite 2

at HH17 to label the

sacral neural crest

. T

post

umbilical

guts

were

then

harvested

embryonic da

(

E10,

Fig1

)

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

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.

enes

enriched

the

sacral

opulation

include

Sst1/Sstr

indicating interneuron cell fate,

and

Dbh, Th, Ddc, Pnmt,

and

Slc18

which are

present in

catecholaminergic neurons

and serotonergic

neurons.

Grm3

expression indicates that

lutamatergic

character

is also

abundant in the sacral

population. In addition, we observed

regulation of

Gfra3

which is involved

in the GDNF signaling

pathway and

Cxcl12

which is

related to signaling during cell migration (Fig1

C, D

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

migration such as

Sema3d

and

Tnc

Hes5 and Dll1

expression

indicate

functions for

otch/

elta signaling

in the vagal

post

umbilical

population (Fig1C, D).

These results

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

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

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

–

ascribe

cluster

identity,

we first performed gene

xpression heatmap

alysis

for top 10 gene markers in clusters 0

(C0

C12)

, marking the most up

regulated gene

as compared to all other clusters (

Fig2

A).

Next, we selected a group of

genes characteristic of

cell fate to define the dominat

cell type in

the clusters

and present them as a dotplot (

Fig2

and feature expression (

Fig3

e identified

0 as

Schwann cells

based on

high level

s of

Pmp22

Ednrb

Sox10

, and

Plp1

expression

(

Fig2

Fig3

Sox10

Pmp22

Endrb

)

. Consistent with heatmap features,

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 (

ig2B

2 exhibits high level

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

Schwann cell markers

, C2 was

considered a

glial progenitor

population

(

Fig2

C3 and

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

Fig3

Ret

Elavl4

Npy

)

However,

3 exhibits more prominent

Vip

Nts

Tac1

expression than

4, indicating excitatory motor neurons;

4 has more cholinergic genes

Chrna5

and

adrenergic

serotonergic genes

such as

Dbh

Ddc

and

(

Fig2

Fig3

Therefore,

3 is

likely to represent a secretomotor neuron cluster, while

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

and/or glioblasts

(Fig2B, Fig3A

Mdk

Ascl1

)

However,

has glial markers like

Sox8

indicating it is a glial progenitor cluster

Due

to high expression of cell cycle

associated genes

Top2a

and

Smc2

progenitor

cells in

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,

classified as

connective tissue cells

based on

Col1a1

C7 is epithelial cells

suggested by markers

Krt7

and

Epcam

. Based on expression of

Mlana

8 is

likely to be composed of melanocytes. C

has a

similar expression pattern

5, but

its

Col18a1

expression

likely reflects

vascular

muscle fate.

appears to

smooth muscle

cells

based on

strong expression of

Acta2

Actg2

Tpm1

Myh11

Des

, and

Tagln

C11 express

endothelial markers

Apold1

Erg

Vwf

, and

Pecam

C12 is likely to be

hematopoietic

related cells based on

Gsta

Ly86

, and

Ptpn6

expression

(

Fig2

)

utative cluster identities are presented in

Uniform

anifold

pproximation and

rojection

(UMA

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

Fig3

H2B

YFP

RIA,

Fig3

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

rogenitor

cluster

(

)

exhibit low

YFP and RIA transcripts

; t

hese cells a

lso showed

lower expression for other markers (

x10

Ednrb

) except for moderate level of

Mdk

possibl

due to

low overall transcription level

Because C10 and C12 general

lack

YFP and RIA transcripts, the two

clusters are likely to be a result of autofluorescence,

and

thus

were eliminated from

downstream analysis.

agal and sacral neural crest

differentially

contribute to ENS cell types

Next,

explore

the relative contribu

tions

the

sacral,

and pre

and post

umbilical

vagal

cell

populations

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

of cell types (

Fig4

A, B). Quantification of cluster contributions

reveals a

common

contribution

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

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

over 75% of

secretomotor

neurons

are

from the

vagal neural crest derived pre

umbilical gut, with a small proportion

(20%) from

the

post

umbilical vaga

population

However,

adrenergic

neurons in C4 were restricted

the post

umbilical gut. Most post

umbilical neurons are contributed

the

sacral neural crest

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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

from sacral neural crest.

While endothelial cells are present throughout the entire gut length,

wit

sacral and

post

umbilical

vagal

populations

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

agal neural crest cells

in the

post

umbilical gut

contribute to most cell fate

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

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

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

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

Immunohistochemistry revealed vagal RIA retrovirus

labeled cells that

expressed acetylcholine

receptor (Fig

C) and HuC/D (E

LAV

) in the pre

umbilical (Fig

F), consistent with differentiated

neurons. H2B

RFP and P0 double

positive Schwann cells were present along the pre

umbilical region

(Fig

I), as well as enteric pr

ogenitors or glial cells as determined by Sox10 expression (Fig

L).

Consistent with the scRNA

seq, there were only a few sparsely distributed neurons marked by TH

(Fig

O) and DBH (Fig

R), TH+ cells were not observed in the pre

umbilical small intesti

(Fig

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

C, inset a1

c1) whereas AchR+ cells from sacral neural crest cells were only observed in the colon

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(Fig

C, inset a2

c2). Both vagal (Fig

F, inset d1

f1) and sacral (Fi

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

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

P0; J

L, Sox10). However, TH+ cells were most frequently derived from sacral neural crest cells in the

colon region (Fig

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

R), especially in regions highly enriched in sacral neural crest cells, such as

the colon (Fig

Q, inset q2) and cecum (Fi

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

further

probe

for

specific

neurotransmitter

character

stic within the

chick ENS

, we

extracted

all cells from secretomotor and neuron

clusters (Fig7A’) and clustered the cells into 9

subsets

(Fig7A,

inset of Fig7A’). We examined a variety of

receptors

neur

transmitters

and neuropeptides

that mark

specific neuronal cell fates

and plotted the expression

subsets

(Fig7B).

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

some similarities with C1,

most likely reflecting

neuronal precursors

as this cluster has

lower

Pmp22

and higher

Mdk

expression

(Fig7B)

appears to

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

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

)

2 is likely to be a combination of excitatory motor

neurons and CALB1

IPANs

, with

cholinergic/catecholaminergic properties

(Fig7B, Fig7C

Calb1

)

C4 lacks

definitive cell marker, but a small percentage of cells

show

high

expression of th

inhibitory motor neuron marker

Fut9

(Fig7B)

ther

inhibitory motor

neuron markers remain low

suggesting

these cells

may

not be fully differentiated due to low

Elavl4

expression

(Fig7B)

Accordingly,

we ascribed

inhibitory motor neuron precursor. C5 is another cluster

differentiating neuron

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

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

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

Pnmt

and

Ddc

(Fig1C, D).

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scussion

The enteric nervous system regulates

critical

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

Hirschsprung’s

disease

7,11

. However, the

role

the

sacral neural crest

, which

coloniz

the hindgut in close

coordination with the vagal neural crest,

has been

largely und

erstudied

in enteric neuropathies

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

cell types arising from

vagal

versus

sacral

axial

levels at

single cell resolution.

blation and heterotopic graf

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

aganglionic hindgut model cre

ated by surgically removing the caudal part of vagal neural crest and

replacing

with quail sacral cells

found that

quail sacral cells migrated into the hindgut with a small

increase in number of neurons

. This

suggest

that sacral neural crest cells do not require the vagal

population to migrate

Reciprocally, w

hen

vagal neural crest cells are grafted to the sacral region, they

migrat

earlier and produc

a larger neuronal population t

han

the endogenous

sacral neural crest cells

However, other studies suggested a more prominent environmental effect,

such

that interchanged vagal

and sacral neural crest cells migrated according to the local

environment

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

results

using axial

level specific

labeling provide

a complementary

approach

address

question

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

. In addition,

the

sacral

neural crest

expresses high levels of

Sox10

mediated

Cdh19

, and well as

Pax

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

. 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

and migration pattern

expose the cells

to differenti

environments

, leading to

diverg

ent

cell fates and functions.

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

–

By f

ocusing transcriptome analysis on

the

small intestine in mice at postnatal day 21, one study

identified 12 distinct neuronal classes categorized by

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

, 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

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

(Fig7B)

ompared with these studies done with

postnatal and adult tissue, w

e observed more

clusters with progenitor

/precursor

identity

(

Fig2

Fig7B)

which is

not

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

, which are distinct from anterior vagal derivatives.

Our analysis

reveals that

gene e

xpression pattern

are markedly different according to

axial

level

confirming the results from previous studies

within

proximal

versus

distal colon

. This important

conclusion suggests

that the ENS is not uniformly distributed

throughout

the gut

but varies from

proximal to distal

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

the post

umbilical

ENS is distinct from that of pre

umbilical ENS,

with major

diffe

rences largely

resulting from

the

differential contributions

the

sacral neural crest. In

addition

the

differentiation program of vaga

derived

neural crest

in the post

umbilical gut

different from that of the

pre

umbilical vagal

population,

suggesting th

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

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

76000

for

1.5 hours to get a viral stock

tittered

about 10

pfu/ml, aliquoted and stored at

until use. Viral

solution was

supplemented with 0.3

l of 2% food dye (Spectral Colors, Food Blue 002, C.A.S# 3844

9) as an indicator

, injected to fill

the neural tube between somite 1

7 at

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

ere

supplied with Ringer’s Solution

(

.9% NaCl, 0.042%KCl, 0.016%CaCl

•

O wt/vol,

pH7.0

ealed with surgical tape

, and incubated at

until embryonic day 10.

Gut cell dissociation

and

Fluorescence

Activated Cell Sorting (FACS)

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

PBS and

loose

fit

homogenized in

Accumax

solution

(

EMD Millipore

)

400

l of Accumax

tissue

mixture

was

aliquoted

into 1.7 ml Eppendorf tubes

and

shake

at 37

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

l in 50ml, PH7.5, ThermoFisher)

was added to quench the reaction. The dissociated cells

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were passed throu

m cell strainer (

Corning

) and collected by centrifug

ing

500g

for 11mins at

. The cells

were

resuspended in HBSS

BSA

, supplemented

AAD Viability Staining Solution

(Biolegend # 420404, 500 TESTS)

, and sorted for

YFP+, viable single cells

usi

Sony

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,

biological replicates were processed, with each replicate containing

YFP+

cells from

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

with

50 milli

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

and

mapped to Galgal6 genome using

Bowtie2

DESeq2

analysis was

performed to find differential expressed genes between vagal and sacral neural crest at post

umbilical

regions

generated by

HTseq

count

Differential gene expression was presented using

olcano

lot

(

coloring genes with Fold

Change>

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

post

umbilical

gut,

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

and

kallisto

0.46.2 &

bustools

0.40.0 pipelines

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

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

mins at 4

and

washed with PBS for three times. Pre

and post

umbilical regions were separately incubated in

15%

sucrose

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

°C

until

the gelatin was dissolve

soaked

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

ections were

washed with 1xPBS

for

10 minutes

and

3 times. After the washed, sections were incubated with

secondary antibody for

minutes

at room temperature.

List of p

rimary antibod

ies

used

1:20

chicken anti

chR ratIgG2a,

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mAB270

(

DSHB

Antibody Registry ID: AB_5318

)

;

1:500 Mouse anti

HuC/D IgG2b

(

Invitrogen

Cat#A21271

)

;

1:20

Chicken anti mouse

IgG1, IE8 (

DSHB

Antibody Registry ID: AB_2078498

)

;

1:500

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

)

:1000

goat

anti

mouse IgG

647

(

Invitrogen A21240

1:1000

donkey

anti

rat

647

(

Abcam ab150155

)

, 1:1000

goat

anti

bbit

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

o M.E.B. We

thank

. 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

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

We thank Dr.

Michael L

Piacentino

Dr.

Erica J.

Hutchins

and Prof. A

ngelike

tathopoulos

for the helpful discussion on the manuscript.

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igure legends

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The copyright holder for this preprint (which

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