SERdev91.pdf

Development 111, 857-866 (1991)

Printed in Great Britain © The Company of Biologists Limited 1991

857

Vital dye labelling demonstrates a sacral neural crest contribution to the

enteric nervous system of chick and mouse embryos

GEORGE N. SERBEDZIJA

1

*, SCOTT BURGAN

2

, SCOTT E. FRASER

2

and MARIANNE BRONNER-

FRASER

1

Developmental Biology Center, Department of Developmental and Cell Biology, ^Department of Physiology and Biophysics, University of

California, Irvine, CA 92717, USA

*

Author for correspondence

Summary

We have used the vital dye, Dil, to analyze the

contribution of sacral neural crest cells to the enteric

nervous system in chick and mouse embryos. In order to

label premigratory sacral neural crest cells selectively,

Dil was injected into the lumen of the neural tube at the

level of the hindlimb. In chick embryos, Dil injections

made prior to stage 19 resulted in labelled cells in the

gut, which had emerged from the neural tube adjacent to

somites 29-37. In mouse embryos, neural crest cells

emigrated from the sacral neural tube between E9 and

E9.5.

In both chick and mouse embryos, Dil-labelled

cells were observed in the rostral half of the somitic

sclerotome, around the dorsal aorta, in the mesentery

surrounding the gut, as well as within the epithelium of

the gut. Mouse embryos, however, contained consist-

ently fewer labelled cells than chick embryos. Dil-

labelled cells first were observed in the rostral and dorsal

portion of the gut. Paralleling the maturation of the

embryo, there was a rostral-to-caudal sequence in which

neural crest cells populated the gut at the sacral level. In

addition, neural crest cells appeared within the gut in a

dorsal-to-ventral sequence, suggesting that the cells

entered the gut dorsally and moved progressively

ventrally. The present results resolve a long-standing

discrepancy in the literature by demonstrating that

sacral neural crest cells in both the chick and mouse

contribute to the enteric nervous system in the postumbi-

lical gut.

Key words: cell migration, gut, Dil, chick, mouse, enteric

nervous system, neural crest.

Introduction

The enteric nervous system (ENS) comprises the

parasympathetic ganglia of the gut, whose cells are

derived from the neural crest (Yntema and Hammond,

1954;

1955; Andrew, 1971; Le Douarin, 1982; Rothman

and Gershon, 1982; Baetge and Gershon, 1989;

Epperlein

et al.

1990). A major event in the develop-

ment of the ENS is the colonization of the gut by neural

crest cells. This process may shed light on the

mechanisms controlling neural crest cell migration.

Furthermore, it is medically important; improper

migration of neural crest cells leads to birth defects such

as colonic agangliogenesis (Hirschprung's disease), in

which a portion of the bowel lacks enteric ganglia

(Payette

et al.

1988). In avian embryos, quail-chick

chimerae have been used to establish that cells from the

vagal region of the neural crest (arising from the neural

tube adjacent to somites 1 to 7) colonize the bowel to

form enteric ganglia (Le Douarin and Teillet, 1973,

1974).

Quail-chick chimerae also suggest that sacral

neural crest cells (arising from the neural tube adjacent

to and caudal to somite 28) contribute to enteric ganglia

in the postumbilical gut. However, this finding remains

controversial. By examining the differentiation of

morphologically identifiable neurons in explant cultures

of chick embryo gut, Allan and Newgreen (1980) found

no evidence for a contribution of the sacral neural crest

to the gut. The explants were removed from different

rostrocaudal levels of the gut, in the hope that all

regions containing ENS precursors at the time of

explantation would form neurons. Their results sugges-

ted that neural crest cells contribute to the ENS in a

proximal-to-distal sequence, consistent with the colon-

ization of the gut by vagal neural crest cells. Thus, they

concluded that sacral neural crest cells are unlikely to

contribute to the neurons of the gut.

These seemingly contradictory results have been

difficult to resolve due to the different nature and

inherent limitations of the approaches used to study this

question. The quail-chick chimeric technique entails

grafting tissues between different species. Because

some quail cells may be more invasive than chick cells

(Bellairs

et al.

1981), it remains possible that donor

858

G. N. Serbedzija and others

quail cells behave differently after transplantation than

host chick cells. However, the explant cultures of Allan

and Newgreen were analyzed for the contribution of the

neural crest to the gut by scoring morphologically

identifiable 'neuroblasts'. The culture conditions may

not have been permissive for the differentiation of all

neuronal types; furthermore, such an assay would have

missed any contribution to the non-neuronal com-

ponents of the ENS by the sacral neural crest.

The use of the monoclonal antibody NC-1 (Vincent

and Thiery, 1984) offers an independent means to

evaluate the contribution to the enteric nervous system.

In avian embryos, Pomeranz and Gershon (1990)

identified a stream of NC-1-positive cells, which

extended from the sacral neural tube to the level of the

gut (Pomeranz and Gershon, 1990). Some of these NC-

1-positive

cells within the gut assumed neuronal

characteristics, suggesting that neural crest cells derived

from the sacral level of the neural tube colonize the gut

and contribute neurons to the developing ENS. Such

immunohistochemical studies, however, only can pro-

vide an indirect assay of cell origin and movement.

Because antibody labelling is performed on fixed

sections of different embryos, it cannot be assured that

the same population of cells are immunoreactive at

different times or in different individuals. This is

particularly problematic in the case of the NC-1

antibody, which recognizes a carbohydrate epitope that

is found on several cell surface glycoproteins (Kruse

et

al.

1984) as well as glycolipids (Newgreen

et al.

1990).

This epitope is found on many, but not all neural crest

cells (Teillet

et

al.

1987) and also is present on numerous

other cell types. An additional complication is that the

NC-1 antibody cannot distinguish between neural crest

cells derived from sacral, vagal or other levels of the

neuraxis. Thus, studies using the NC-1 antibody cannot

resolve this controversy by establishing definitively a

sacral neural crest cell contribution to the gut.

Recently, our laboratory has developed a technique

for labelling premigratory neural crest cells by injecting

a solution of the fluorescent carbocyanine dye, Dil, into

the lumen of the neural tube (Serbedzija

et al.

1989,

1990).

Dil is lipid soluble and becomes incorporated

into the plasma membrane of all cells that it contacts

(Sims

et al.

1974). It is passed to the progeny of the

labelled cells but not to other cells (Honig and Hume,

1986).

The dye does not appear to have any adverse

affects on neuronal or neural crest cell development

(Honig and Hume, 1986; Serbedzija

et al.

1989, 1990),

nor does the dye injection into living embryos itself

appear to alter development (Serbedzija

et al.

1989,

1990).

Injection of Dil into the lumen of the neural tube

offers a method to label premigratory neural crest cells

directly. By controlling the time, site and size of

injection into the sacral region, as well as the period of

development postinjection, this technique provides a

simple and non-deleterious way to study the temporal

and spatial migratory patterns of neural crest cells in a

variety of species without surgical disruption or possible

culture artifacts. Here we use this approach to

demonstrate that cells from the sacral neural crest

populate the developing gut in chick and mouse

embryos.

Materials and methods

Preparation of eggs

White Leghorn chicken embryos were incubated at 37 °C until

they reached stages 14 to 19 (Hamburger and Hamilton,

1951).

The eggs were washed in 70% ethanol, a window was

cut in the shell over the embryo and India ink (Pelikan,

Hanover, FUG) was injected under the blastodisc to aid in

visualization of the embryo. The vitelline membrane was

deflected with a fine tungsten needle to allow access to the

embryo. After dye injection, the egg was sealed with adhesive

tape and returned to the incubator.

Preparation of mouse embryos

Embryos were obtained from CD-I females that had been

mated with BDF-1 males (Charles River) overnight. The

presence of a vaginal plug the following morning indicated

pregnancy, and the date that the plug was observed was

designated as embryonic day zero (E0). Embryos were

removed surgically from anesthetized mothers between late

E9 and early E10 (18 to 33 somites), and placed in dissecting

media consisting of 20 % fetal bovine serum (Hyclone), 79 %

Dulbecco's Modified Eagle's medium (DMEM, Whittaker

Bioproducts) and 1 % penicUlin-streptomycin L-glutamate

mixture (GPS, Whittaker Bioproducts) at 37°C. The embryos

then were loosened and freed from the extraembryonic

membranes, taking care not to damage the blood vessels

within the membranes. Both the embryos and the membranes

were left attached to the placenta for the entire culture

period.

After dye injection, embryos were maintained, as described

previously, in culture media consisting of 50% rat serum,

49%

DMEM and 1% GPS (Serbedzija

et al.

1990). The

culture tubes were gassed with 95 % oxygen/5 % carbon

dioxide and rotated at SOrevsmin"

1

at 37°C. Embryos were

cultured for either 12 or 24h before fixation; those cultured

for 24h were gassed a second time after 12h incubation.

Microinjection of dye

All injections were made by pressure with an 0.05 % solution

(weight/volume) of l,l-dioctadecyl-3,3,3',3'-tetramethylin-

docarbo-cyanine perchlorate (Dil, Molecular Probes), made

from an 0.5 % stock solution in 100% ethanol diluted 1:10 in

0.3

M

sucrose. Prior to use, the Dil solution was heated to

37 °C in order to prevent cold shock to the embryos and to

help keep the Dil in solution. The injection micropipets were

back-filled with the Dil solution, attached to a picospritzer

(General Valve) and inserted into the lumen of the neural

tube using a micromanipulator (Marzhauser). In most cases,

the injections were made slightly rostral to the sacral region,

with dye being expelled towards the caudal end of the

embryo. Visual inspection of the embryo confirmed that the

dye was confined to the sacral region of the neural tube, with

no dye entering the truncal or vagal regions. Several embryos

at various stages were injected such that the dye was expelled

rostrally into the truncal and vagal regions. In these embryos,

no Dil-labelled cells were observed in the postumbilical gut.

Histology

All embryos were fixed by immersion in 4%

paraformaldehyde/0.25 % glutaraldehyde in

0.1M

phosphate

buffer solution (PB) overnight at 4°C. The embryos then were

Sacral neural crest cell contribution to enteric nervous system

859

rinsed in 0.1

M

PB and soaked in a 15 % sucrose solution for

12h at 4°C. They were embedded in a 15% sucrose/7.5%

gelatin (Oxoid) solution for 2h at

37

°C and then rapidly

frozen in liquid nitrogen. Serial sections were cut at a

thickness of 25/an on a Zeiss Micron or AO Reichardt

Histostat cryostat. Slides were mounted in Gel/Mount

(Biomeda Corp.) and covered with glass coverslips. The

sections were viewed on an epifluorescence microscope

equipped with a light-intensifying camera (RCA SIT) and an

image-processing system (Imaging Technologies Series 151),

using the Vidlm software package (developed by S. E. Fraser,

G. Belford, J. Stollberg).

Results

Because Dil labels only those cells with which it comes

into direct contact, injection of Dil into the neural tube

lumen selectively labels the neural tube cells, including

premigratory neural crest cells whose endfeet connect

to the lumen (Serbedzija

et al.

1989). By injecting small

amounts of dye into the neural tube at the level of the

hindlimb, it was possible to label sacral premigratory

neural crest cells selectively without labelling cells at

vagal or thoracic levels. The dye spread from the

injection site caudally to the posterior neuropore.

Avian embryos were injected with dye at stages ranging

from 14 to 19 (Hamburger and Hamilton, 1951; 21-38

somite pairs) and were incubated

in ovo

for 24 to 48 h.

Mouse embryos were labelled at E9 to E10 (18-33

somite pairs) and incubated

in vitro

for 12 to 24 h.

Table 1 and Table 2 present the injection and fixation

times for chick and mouse embryos, respectively, the

total number of embryos examined at each stage and a

summary of the results.

The majority of the observations were made in the

region of the hindlimb (chick somite levels 26-32,

mouse somite levels 23-28). In addition, we examined

some of the embryos at the vagal and truncal levels to

ensure that no neural crest cells other than those at

sacral levels were labelled by our localized injections. In

all of the chick and mouse embryos examined, Dil-

labelled cells were not observed in the gut or other sites

of neural crest cell localization at vagal or truncal levels

of the neuraxis. When Dil was injected only into the

trunk, no Dil-labelled cells were observed in the

postumbilical gut.

Chicken embryos

Injection at stage 14 (21-23 somites)

Transverse sections through embryos labelled at the 21-

somite stage and incubated for 24 h contained streams

of Dil-labelled cells which extended from the dorsal

neural tube ventrally to the level of the dorsal aorta.

Labelled cells were observed throughout the rostral half

of each somitic sclerotome, except for a region around

the notochord. This migratory pattern was similar to

that observed at trunk levels of the neuraxis (somites

8-28; Serbedzija

et al.

1989). Embryos injected at the

23-somite stage and incubated for 24 h, until they

reached stage 20, contained streams of labelled cells as

described above; in addition, a few labelled cells were

observed extending into the dorsalmost portion of the

dorsal mesentery of the gut (Fig. 1A,B). These labelled

cells were observed in only the most rostral segment of

the sacral gut (at the level of the rostral aspect of the

hindlimb; 26-27 somite level).

Transverse sections through embryos injected at

stage 14 and incubated for 36 h after injection, until they

reached stage 21, contained Dil-labelled cells through-

out the sacral level of the gut. In the most rostral level

of the sacral region, Dil-labelled cells were observed in

both the dorsal and ventral portions of the gut

mesenchyme, as well as within the dorsal portion of the

gut epithelial layer. At the more caudal levels of the

sacral region, Dil-labelled cells were observed only in

the dorsal portion of the gut mesenchyme (Fig. 1C, D).

Table 1.

Summary of experiments and results

EXP.

10

12

13

INJECTION

FIXATION

St14

13

16

18

20

21

22

23 24

(12)

(15)

(14)

(10)

(12) I

(8)

(6)

(5)

Labaltod

oadalngut

* Number oi embryos •xamlrad

860

G. N. Serbedzija and others

Table 2.

Summary of mouse

experiments

and results

EXP.

INJECTION

FIXATION

ES

E10

E11

(10)

(5)

LaMtod

calsingut

* Numtxr of wnbryo* uamlnad

Dil-labelled cells also were observed in the regions of

the condensing dorsal root and sympathetic ganglia.

Transverse sections through embryos incubated for

48 h (stage 23) contained Dil-labelled cells in similar

locations to those described above. By this stage, Dil-

labelled cells were found in the ventral portion of the

gut mesenchyme throughout the sacral region

(Fig. 2A,B).

Injection at stages 15—16 (24-28 somites)

Transverse sections through the sacral region of

embryos injected at stages 15 and 16 and incubated for

24 h, until they reached stage 21, contained many Dil-

labelled cells (approximately 50-100 cells/section) in

the dorsal portion of the gut. In the embryos injected at

stage 15 (24-26 somite pairs), streams of labelled cells

extended from the neural tube to the dorsal portion of

the gut, where several individual Dil-labelled cells were

Fig. 1. Transverse sections through chick embryos labelled with Dil. (A) Section through the most rostral level of the

sacral region (somite 26) of an embryo injected at stage 14 and incubated for 24h, until reaching stage 20. Dil-labelled

cells extend from the dorsal portion of the neural tube (n; long arrows) to the level of the dorsal aorta (a; short arrows).

(B) A higher magnification picture of the section shown in A. Dil-labelled cells are present between the dorsal aorta and

the gut (g), with a few cells present within the most dorsal portion of the gut mesenchyme (short arrows). (C) Section

through the most caudal level of the sacral region (somite 32) of an embryo injected at stage 14 and incubated for 36 h,

until reaching stage 21. Dil-labelled cells are present in the condensing dorsal root ganglia (long arrows) and sympathetic

ganglia, as well as between the dorsal aorta and the gut. (D) A higher magnification picture of the section shown in C; Dil

labelled cells are observed in the dorsal portion of the mesenchyme surrounding the rectum (r; short arrows). (A,C: scale

bar=320/im; B,D: scale bar=100/an)

Sacral neural crest cell contribution to enteric nervous system

861

Fig. 2. Transverse sections

through chick embryos labelled

with Dil. (A) Section through

an embryo injected at stage 14

and incubated for 48 h, until

reaching stage 23. Dil-labelled

cells are present in the gut

mesenchyme (short arrow), in

the dorsal root and

sympathetic ganglia (long

arrows), and in a stream of

cells located ventral to dorsal

aorta (arrowhead). (B) A

higher magnification picture of

the section shown in A.

Ousters of Dil-labelled cells

are present in both the dorsal

and middle portions of the gut

mesenchyme (short arrows).

(C) Section through an embryo

labelled at stage 16 and

incubated for 36 h, until

reaching stage 22. Dil-labelled

cells are located throughout

the dorsal and ventral portions

of the gut mesenchyme

(arrows), as well as in a stream

extending from the

sympathetic ganglia to the gut

(arrowheads). (D) A higher

magnification of the section

shown in C; Dil-labelled cells

appear throughout the gut

mesenchyme (short arrows)

and within the gut epithelium

(long arrow). (A: scale

bar=320^m; C: scale

bar=200^m; B,D: scale

bar=100/im)

recognizable (schematized in Fig. 4A). In sections

through embryos injected at stage 16 (26-27 somite

pairs),

the streams of Dil-labelled cells extended

further ventrally into the gut than in embryos injected at

stage 15, perhaps reflecting the fact that the host was

slightly older at the time of fixation. A few individual

Dil-labelled cells were identified in the middle and

ventral portions of the gut.

In transverse sections through embryos injected at

either stage 15 or 16 and incubated for 36 h after

injection, until they reached stage 22, Dil-labelled cells

were found in the dorsal root ganglia and sympathetic

ganglia, as well as throughout the mesenchyme of the

gut. A few Dil-labelled cells (1-10 cells/embryo) were

also observed in the gut epithelium (Fig. 2C,D;

schematized in Fig. 4B). Embryos injected at stage 16

contained fewer labelled cells in the more rostral

regions of the sacral gut than embryos injected at stage

15.

This suggests a rostrocaudal order to the contri-

bution of sacral neural crest cells to the gut, consistent

with the rostrocaudal maturation of the embryo.

Injection at stage 17 (29-32 somites)

Transverse sections through the sacral region of

embryos injected at stage 17 and incubated for 24 or

36 h, until reaching stages 23 and 24, respectively,

contained Dil-labelled cells (approximately 25-50 cells/

section) only in the more caudal levels of the gut.

Labelled cells were located within the gut mesenchyme,

as well as intercalated into the gut epithelium (similar to

Fig. 2C,D; schematized in Fig. 4B). Dil-labelled cells

also were present in dorsal root and sympathetic

ganglia, in groups of cells around the dorsal aorta and

underneath the ectoderm on the dorsolateral pathway.

No Dil-labelled cells were found in the most rostral

segments of the sacral gut, although labelled cells were

present in other sites of neural crest cell localization as

described above.

862

G. N. Serbedzija and others

Injection at stage 18 (33-36 somites)

Embryos injected at this stage and incubated for 24 or

36 h contained only limited numbers of labelled cells in

the gut (less than 12), with the majority observed in the

ventral portion of the caudal regions of the gut

(schematized in Fig. 4C). These cells were more dimly

labelled than those found in embryos injected at stage

17.

Because this appears to be the last stage at which

injection of dye into the neural tube yields labelled cells

in the gut, it is likely that these dimly labelled cells

emigrated from the neural tube shortly after injection of

Dil,

thereby limiting the time for dye incorporation.

However, the distribution of Dil-labelled cells outside

the gut was similar to that described above.

Injection at stage 19 (37-40 somites)

Embryos labelled at this stage contained no Dil-

labelled cells in the gut. This suggests that all

premigratory neural crest cells destined to contribute to

the gut had already left the neural tube by stage 19. Dil-

labelled cells were present in the dorsal root and

sympathetic ganglia and underneath the ectoderm

along the dorsolateral pathway.

Mouse embryos

Injection at late E9 (18-20 somites)

Transverse sections through embryos injected at this

stage and incubated for 12 h contained a few Dil-

labelled cells in the rostral portion of the somitic

sclerotome, suggesting that neural crest cells at this

level were just beginning their migration. No labelled

cells were present in the gut.

After 24 h of incubation, embryos injected at the 20-

somite stage contained a few labelled cells in the dorsal

portion of the gut in the sacral region of the embryo

(Fig. 3A). In addition, labelled cells were seen in the

rostral sclerotome and around the dorsal aorta, similar

to the distribution observed in the trunk (Serbedzija

et

al.

1990).

Injection at early E9.5 (21-23 somites)

In transverse sections through the sacral region of

embryos injected at early E9.5 and incubated for 12 h,

no labelled cells were observed in the gut. Dil-labelled

cells were observed in the rostral sclerotome, at the

level of the dorsal aorta and in the sympathetic ganglia.

Embryos incubated for 24 h contained Dil-labelled

cells in the gut (approximately 25 cells/embryo). The

majority of these cells were located in the dorsal portion

of the gut, although a few labelled cells were observed

more ventrally (Fig. 3B).

Injection at mid E9.5 (24-26 somites)

Embryos injected at 24- to 26-somite stage and

incubated for 24 h contained many Dil-labelled cells

(25-75 cells/embryo) in the sacral gut (Fig. 3C,D).

Most of these labelled cells were located further

ventrally than labelled cells in embryos injected at

earlier stages. Labelled cells also were observed within

the dorsal gut epithelium. There were no Dil-labelled

cells observed in the gut rostral to the hindlimb bud.

Dil-labelled cells were observed in the region of the

dorsal root and sympathetic ganglia, around the dorsal

aorta, and along the dorsolateral pathway. The number

of brightly labelled cells was reduced in comparison to

the number observed in embryos injected at E9; the

number of dimly labelled cells increased. This reduced

dye content is consistent with the possibility that the

Dil-labelled cells were proliferating rapidly; alterna-

tively, these dimly labelled cells may have emigrated

from the neural tube shortly after injection of Dil into

the neural tube, thereby limiting the amount of dye that

they could incorporate. The dimmer Dil-labelled cells

tended to be further rostrad than the brightly labelled

cells.

Injection at late E9.5 (27-29 somites)

Embryos injected at the 27- to 29-somite stage

contained no Dil-labelled cells in the gut. Dil-labelled

cells were observed in other neural crest derivatives, as

described above.

Discussion

We have used a vital dye, Dil, to determine if cells from

the sacral neural crest contribute to the development of

the enteric nervous system (ENS) in chick and mouse

embryos. By injecting Dil into the lumen of the neural

tube at the level of the hindlimb, the premigratory

sacral neural crest population was labelled selectively,

without labelling vagal or truncal neural crest cells. In

chick embryos, Dil-labelled cells were observed in the

rostral half of the somitic sclerotome, around the dorsal

aorta, in the mesentery surrounding the gut, as well as

within the epithelium of the gut. The pattern of Dil-

labelling was similar in the mouse embryo, although

consistently fewer cells were observed. These results

clearly support the conclusion of studies using chick/

quail chimerae (Le Douarin and Teillet, 1974) that the

sacral neural crest contributes to the developing ENS.

By injecting Dil into chick embryos at a variety of

stages, we were able to infer the times during which

neural crest cells contributed to the enteric nervous

system in sacral regions. The sacral neural crest cells

that populate the gut emerge from the neural tube

between stages 17 to 19. This is supported by two

observations: (1) Dil-labelled cells were found in the

gut only for injections prior to stage 19, and (2) sacral

neural crest cells were first observed external to the

neural tube at stage 17. We also were able to ascertain

the pattern in which neural crest cells populate the gut.

Dil-labelled cells first were observed in the rostral and

dorsal portion of the gut at stage 21. This coincides with

the time when cells immunoreactive with the NC-1

antibody, which recognizes some neural crest cells, are

evident in the avian gut at the sacral level (Pomeranz

and Gershon, 1990). Paralleling the maturation of the

embryo, there was a rostral-to-caudal sequence in

which neural crest cells populated the gut at the sacral

level. In addition, Dil-labelled cells appeared to

populate the gut in a dorsal-to-ventral sequence,

Sacral neural crest cell contribution to enteric nervous system

863

Fig. 3. Transverse sections through mouse embryos labelled with Dil. (A) Section through an embryo injected at the 20-

somite stage and incubated for 24 h. A Dil- labelled cell is located in the dorsal portion of the gut mesenchyme (dgm;

arrow).

For orientation, the gut epithelium is labelled g. (B) Section through an embryo injected at the 22-somite stage and

incubated for 24h. A Dil-labelled cell can be observed in the ventral portion of the gut mesenchyme (vgm; arrow).

(C) Section through an embryo injected at the 24-somite stage and incubated for 24 h. Three Dil-labelled cells are present

in the dorsal portion of the gut mesenchyme (arrows). (D) A higher magnification of the section shown in C. (A,B,D:

scale bar=50/im; C: scale bar=100/«n)

864

G. N. Serbedzija and others

ROSTRAL CAUDAL

*

\O©/ \COI

B

suggesting that the cells entered and colonized the gut

dorsally, spreading progressively ventrally. Embryos

fixed at early stages had Dil-labelled cells only in the

dorsal portion of the gut, whereas embryos fixed at late

stages contained labelled cells only in the ventral gut

(schematized in Fig. 4). The finding that progressively

later injections result in Dil-labelled cells in more

caudal and ventral locations suggests that the gut is

populated by neural crest cells in a rostrocaudal and

dorsoventral sequence.

In the mouse, there were fewer Dil-labelled neural

crest cells in the sacral gut than in chick embryos,

although the overall distribution pattern was similar. By

injecting embryos at various stages, we were able to

infer that murine neural crest cells first emigrate from

the sacral neural tube 2-4 somites rostral to the most

recently formed somite in the mid-E9 embryo. By late

E9.5 (27-29 somite pairs), those cells destined to

populate the gut have already left the sacral neural

crest. Similar to the chick embryo, there appeared to be

a rostrocaudal and dorsoventral sequence of neural

crest cell population of the mouse gut. There are two

Fig. 4. A schematic representation of the distribution of

labelled cells in both the rostral and caudal portions of the

gut (g; the dorsal aorta is indicated by an a) in chick

embryos labelled with Dil at progressively later stages and

incubated for 24 h. While the observed pattern of cell

distribution was similar for both the chick and the mouse,

the number of Dil-labelled cells observed was far greater

in the chick than in the mouse. (A) Embryos injected at

stage 15 contained Dil-labelled cells only in the more

rostral region of the sacral gut. These labelled cells were

present only in the dorsal portion of the gut mesenchyme.

(B) Embryos injected at stage 17 contained Dil-labelled

cells throughout the rostral and caudal regions of the sacral

gut. In the rostral region, Dil-labelled cells were present in

both the dorsal and ventral portion of the gut

mesenchyme, as well as within the dorsal gut epithelium.

In the caudal region, labelled cells were present only in the

dorsal portion of the gut mesenchyme. (C) Embryos

injected at stage 18 contained Dil-labelled cells only in the

ventral portion of the gut mesenchyme in the rostral region

of the sacral gut, whereas the Dil-labelled cells were

present in both the dorsal and ventral portion of the gut

mesenchyme, as well as within the dorsal gut epithelium.

(D) Embryos injected at stage 19 contained no Dil-labelled

cells in the rostral regions of the sacral gut, and only a few

labelled cells in the caudal regions. These Dil-labelled cells

were present only in the ventral portion of the gut

mesenchyme.

possible explanations for the reduced number of

labelled cells observed in the mouse compared to the

chick: (1) there may be fewer numbers of sacral neural

crest cells in the enteric nervous system of the mouse, or

(2) there may be equivalent numbers of sacral neural

crest cells in both enteric nervous systems, but the

mouse enteric nervous system may arise from fewer

progenitor cells.

We were not able to determine the phenotypes of the

Dil-labelled cells in our sections, because preservation

of Dil requires fixation with paraformaldehyde and

glutaraldehyde, which is incompatible with most anti-

body staining. Previous investigators have noted NC-1-

positive neuronal and non-neuronal cells in the sacral

portion of the gut (Pomeranz and Gershon, 1990);

however, the axial level of origin of these cells could not

be ascertained. In addition to the labelled cells in the

mesenchyme surrounding the gut, we observed labelled

cells in the epithelium of the gut in both mouse and

chick embryos. The fate of these cells is not clear,

although they may represent neural crest cells interca-

lating into the epithelium. Previously, we noted a

similar phenomenon, with neural crest cells invading

the epithelium of the dermomyotome in the trunk of the

chick embryo (Serbedzija

etal.

1989) Unfortunately, we

cannot follow the fate of these Dil-labelled cells for

more than two days because the dye becomes progress-

ively diluted with prolonged development. Further-

more, in the mouse, the short length of the culture

period makes long-term identification of the labelled

cells unfeasible.

In addition to enteric ganglia, sacral neural crest cells

contribute to the dorsal root ganglia, sympathetic

Sacral neural crest cell contribution

to

enteric nervous system

865

ganglia,

and

aortic plexuses

at

the

level

of

the

hindlimb.

Furthermore, later emigrating Dil-labelled cells were

observed underneath

the

epidermis, where pigment

cells will differentiate. These derivatives

and

pathways

of migration

are

similar

to

those

of

truncal neural crest

cells.

Thus,

it

appears that sacral neural crest cells give

rise to

the

same derivatives

as

trunk neural crest cells,

with

an

additional contribution

to the

enteric nervous

system.

Both sacral

and

vagal neural crest cells invade

the

mesentery

of the gut to

contribute

to the

enteric

nervous system whereas truncal neural crest cells

do not

normally enter

the

dorsal mesentery. Grafting exper-

iments suggest that this differential ability

to

enter

the

gut

is an

inherent property

of the

neural crest cells

derived from different axial levels. When

a

neural tube

from

the

vagal region

is

transplanted

in

place

of a

trunk

neural tube,

the

vagal neural crest cells

not

only migrate

appropriately

for

their

new

environment (contributing

to

the

dorsal root ganglia, sympathetic ganglia

and

adrenal medulla

of

the

host),

but

also enter

the gut (Le

Douarin

and

Teillet,

1974;

Smith

et

al.

1977). Because

trunk neural crest cells

do not

normally enter this

region, this suggests

the

presence

of an

inherent

difference

in

neural crest cells from trunk

and

vagal

levels.

It has not yet

been established

if a

similar

situation exists

for

neural crest cells derived from sacral

levels.

However,

its

seems likely that

the gut

mesen-

chyme represents

a

non-permissive substrate

for

trunk

neural crest cells,

but a

permissive

one for

vagal

and

sacral neural crest cells. Numerous extracellular matrix

molecules, such

as

fibronectin, laminin, collagens,

hyaluronate,

etc. are

distributed along neural crest

migratory pathways (Pintar,

1978;

Newgreen

and

Thiery, 1980; Krotoski

et

al.

1988; Perris

et

al.

1991a,b).

In

the

lethal spotted mouse,

a

build-up

of

extracellular

matrix components appears

to

restrict migration

of

neural crest cells into

the

post-umbilical bowel (Payette

et al.

1988), suggesting that

the

excess

or

lack

of

some

critical matrix component

may be

critical

for

this

differential ability.

Our results support

the

idea that sacral neural crest

cells contribute

to the

postumbilical

gut,

as

originally

proposed

on the

basis

of

quail-chick chimeric grafts

(Le

Douarin

and

Teillet, 1974).

The

negative result

of

Allan

and Newgreen (1980)

may

have resulted from

any

of a

number

of

factors.

For

example, sacral neural crest cells

may contribute only non-neuronal cells

to the

gut,

or

they

may

give rise

to

neuronal types that

are not

readily

detectable

by the

toluidine blue staining used

in

their

study.

The

positive results presented here conclusively

demonstrate that sacral neural crest cells contribute

to

the developing

gut in

both chick

and

mouse embryos.

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