Development 108, 605-612 (1990)
Printed in Great Britain ©The Company of Biologists Limited L990
605
Pathways of trunk neural crest cell migration in the mouse embryo as
revealed by vital dye labelling
GEORGE N. SERBEDZIJA
1
*, SCOTT E. FRASER
2
and MARIANNE BRONNER-FRASER
1
1
Department of Developmental and Cell Biology, Developmental Biology Center,
and
2
Department of Physiology and Biophysics, California
College of Medicine, University of California, Irvine, CA 92717, USA
'Author for reprint requests
Summary
Analysis of neural crest cell migration in the mouse has
been difficult due to the lack of reliable cell markers.
Recently, we found that injection of Dil into the chick
neural tube marks premigratory neural crest cells whose
endfeet are in contact with the lumen of the neural tube
(Serbedzjja
et
al. Development
106, 809-819 (1989)). In
the present study, this technique was applied to study
neural crest cell migratory pathways in the trunk of the
mouse embryo. Embryos were removed from the mother
between the 8th and the 10th days of development and
Dil was injected into the lumen of the neural tube. The
embryos were then cultured for 12 to 24 h, and analyzed
at the level of the forelimb. We observed two predomi-
nant pathways of neural crest cell migration: (1) a
ventral pathway through the rostral portion of the
somite and (2) a dorsolateral pathway between the
dermamyotome and the epidermis. Neural crest cells
were observed along the dorsolateral pathway through-
out the period of migration. The distribution of labelled
cells along the ventral pathway suggested that there were
two overlapping phases of migration. An early ventro-
lateral phase began before E9 and ended by E9.5; this
pathway consisted of a stream of cells within the rostral
sclerotome, adjacent to the dermamyotome, that ex-
tended ventrally to the region of the sympathetic ganglia
and the dorsal aorta. A later ventromedial phase was
apparent after E9.5 and continued through E10.5; this
pathway consisted of a thin strand of Dil-labelled cells
along the lateral surface of the neural tube which later
protruded into the rostral sclerotome to form the dorsal
root ganglia and Schwann cells. Those embryos injected
at later stages contained labelled cells only in dorsal
derivatives, suggesting that neural crest derivatives are
populated in a ventral-to-dorsal order. While the overall
pattern and order of neural crest migration was similar
to that previously observed in avians and rats, some
details of the timing and trajectories apparently dif-
fered, most notably the timing of neural crest migration
along the dorsolateral pathway.
Key words: neural crest, migration, mouse, Dil.
Introduction
In vertebrates, the neural crest is a population of cells
that migrates extensively throughout the embryo and
gives rise to a variety of neuronal and non-neuronal cell
types (Horstadius, 1950; Weston, 1970; Le Douarin,
1982).
In avians, experiments using chick-quail chim-
eras or the HNK-1 antibody to identify neural crest cells
indicate that trunk neural crest cells migrate along two
primary pathways (Weston, 1963; Le Douarin, 1973):
(1) a dorsolateral pathway between the dermamyotome
and the epidermis, whose cells give rise to pigment cells
(Rawles, 1947; Serbedzija
et
al.
1989), and (2) a ventral
pathway through the rostral portion of each somitic
sclerotome (Rickmann
et al.
1985; Bronner-Fraser,
1986),
whose cells give rise to the dorsal root and
sympathetic ganglia, the adrenal medulla, and Schwann
cells (Le Douarin, 1982).
Ultrastructural studies of mouse embryos have per-
mitted an analysis of the emigration of neural crest cells
from the neural tube and their initial stages of mi-
gration. These studies have shown that trunk neural
crest cells arise along the dorsal midline of the neural
tube just after fusion of the neural folds, where they
emerge into a cell-free space between the epidermis,
somites and neural tube (Erickson and Weston, 1983;
Sternberg and Kimber, 1986a,
b).
The neural crest cells
are thought to migrate under the epidermis as well as
between the neural tube and somite (Rawles, 1947;
Erickson and Weston, 1983; Erickson, 1986). Unfortu-
nately, once neural crest cells reach the dorsal edge of
the somite, they are no longer distinguishable from the
other cell types in their environment by light or electron
microscopy; hence, little is known about the exact
pathways or timing of neural crest cell migration in
mouse.
606
G. N. Serbedzija, S. E. Fraser and M. Bronner-Fraser
In order to visualize migrating neural crest cells
in
vivo,
several cell marking techniques have been applied
successfully in other species. These approaches include
the transplantation of marked neural tubes into
unmarked hosts (Harrison, 1935; Weston, 1963;
Chibon, 1967; Le Douarin, 1973; Sadaghiani and
Thiebaud, 1987; Krotoski
etal.
1988), microinjection of
labelled neural crest cells into unlabelled hosts (Tan and
Morriss-Kay, 1986) and immunological staining tech-
niques (Vincent andThiery, 1984; Rickmann
etal.
1985;
Bronner-Fraser, 1986; .Erickson
et al.
1989). Neural
tube transplantations are generally not feasible in
mammals because of their
in
utero
development and the
difficulties associated with extensive manipulation at
stages before and during neural crest cell migration.
The HNK-1 antibody, which has been used to identify
neural crest cells and their migratory pathway in avians
(Vincent and Thiery, 1984; Rickmann
et al.
1985;
Bronner-Fraser, 1986), does not specifically recognize
mouse neural crest cells (Holley and Yu, 1987). While
FINK-1 appears to recognize neural crest cells in rat
embryos (Erickson
et al.
1989), interpretation of any
experiments performed with the antibody is hampered
by its incomplete specificity for neural crest cells (Vin-
cent and Thiery, 1984; Bronner-Fraser, 1986) and its
inability to label all neural crest cells (Teillet
etal.
1987;
Serbedzija
et
al.
1989). It has been possible to label and
follow the migration of cephalic neural crest cells in
both the mouse and the rat by orthotopic microinjection
of wheat germ agglutinin labelled neural crest cells (Tan
and Morris-Kay, 1986; Chan and Tarn, 1988). However,
this technique involves extensive manipulation of both
the donor neural crest cells and the host embryo, which
may alter the migratory behavior in an unpredictable
manner. A more direct assay is offered by injection of
gold-conjugated wheat germ agglutinin into the amnio-
nic cavity to label premigratory neural crest cells in
combination with
in vitro
culturing methods (Chan and
Tarn, 1988). The wheat germ agglutinin adheres to cell
surface glycoconjugates (Gesink
et al.
1983), thereby
labelling neural crest cell precursors while they are
contiguous with the ectoderm, prior to neural tube
closure (Smits-van Prooije
et al.
1986). Given that
present culturing techniques allow about
24 h
of normal
development, use of this technique is restricted to the
first day of cephalic neural crest cell migration.
Recently we have 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). Because Dil
is lipid-soluble and hydrophobic, it is incorporated
nearly irreversibly into the plasma membranes of all the
cells it contacts (Sims
et al.
1974). The dye does not
spread from labelled to unlabelled cells, nor does it
appear to have any adverse effects on neuronal or
neural crest cells (Honig and Hume, 1986; Serbedzija
et
al.
1989). Therefore, injection of Dil into the neural
tube lumen offers a simple means to label specifically
only those cells that are in contact with the lumen of the
neural tube. By examining the movement of labelled
cells away from the neural tube, this technique has
allowed a direct analysis of the spatial and temporal
patterns of neural crest cell migration in avians (Serbed-
zija
et
al.
1989).
Here, we utilize Dil-labelling to analyze neural crest
cell migration in the trunk of the mouse. Because
intraluminal injection of Dil can be used to label neural
crest cells at a variety of developmental stages, this
technique circumvents the
24 h
limit imposed by whole
embryo culture methods (New, 1973, 1977; Sadler,
1979;
Sadler and New, 1981). Combining data from
different animals permits an analysis of the overall
pattern of trunk neural crest cell migration in the
mouse, including the pathways of neural crest cell
migration, the duration of neural crest cell emigration
from the neural tube, and the temporal order of
contributions of neural crest cells to their derivatives.
Materials and methods
Animal
preparation
Embryos were obtained by mating CD-I females with BDF-1
males (Charles River) overnight. The presence of a vaginal
plug the following morning was taken to indicate pregnancy,
and the date that the plug was observed was designated
embryonic day 0 (EO). Embryos were removed surgically
from anesthetized mothers between E8 and E10.5 (8 to 40
somites, respectively), and placed in dissecting media consist-
ing of 20% fetal bovine serum (Hyclone), 79% Dulbecco's
modified Eagle's medium (DMEM, Whittaker Bioproducts),
and 1% penicillin-streptomycin L-glutamine mixture (GPS,
Whittaker Bioproducts) at 37°C. The embryos were then
dissected free of the extraembryonic membranes, taking care
not to damage the blood vessels within the membranes. Both
the embryo and the extraembryonic membranes were left
attached to the placenta for the entire culture period.
Microinjection
of dye
All injections were made with either a 0.25% solution
(weight/volume) in 50% ethanol of l,l-dioctadecyl-3,3,3',3'-
tetramethylindocarbocyanine perchlorate (Dil; Molecular
Probes) or a 0.025
%
solution (weight to volume) in
5
%
ethanol in
0.3
M
sucrose solution. Prior to use, the dye
solution was heated to 37°C and centrifuged to remove any
crystals that might clog the pipet tip. Both the dye and the
micropipets were maintained at 37°C during the injections, to
prevent cold shock to the embryos. The micropipets were
backfilled with the Dil solution and attached to a picospritzer
(General Valve). The injection pipet was inserted into the
lumen of the neural tube (using a micro-manipulator; Marz-
hauser) at either the level of the otic vesicle, or at the level of
the most recently formed somite. In most cases, enough dye
solution was expelled to fill the entire length of the neural
tube lumen. In embryos whose posterior and/or anterior
neuropore had not yet closed, dye was expelled until it could
be seen passing out of the open neuropores.
The potential adverse effects of the injection procedure
itself were examined by comparing embryos injected at E9
with Dil in 50% ethanol with uninjected or saline injection
embryos. In transverse sections of embryos incubated for
24
h,
no morphological differences were obvious among the
three sets of embryos.
Embryo culture
Embryos, with their extraembryonic membranes and placen-
Mouse neural
crest
cell
migration
607
tas attached, were placed
in
15
ml culture tubes containing
2 ml
of
culture media consisting
of
50%
rat
serum,
49%
DMEM,
and 1%
GPS. The culture tubes were gassed with
95%
oxygen
and
5% carbon dioxide
and
rotated
at
30
revs
min~'
at
37°C. Embryos were cultured
for
either 12
or
24 h
before fixation
and
sectioning. Embryos cultured
for
24 h
were gassed
a
second time after
12 h
of
incubation.
Cultured embryos were compared with embryos allowed
to
develop
to
similar stages
in utero
to
ascertain
if
the culture
period itself affected embryonic maturation. Based on the size
of the limb buds and the number of
somites,
embryos cultured
up
to
24 h
appeared similar
to
embryos that developed
in
utero.
In
transverse sections, both sets
of
embryos
had
comparably sized neural tubes and dorsal aortae. Our results
agree with previous studies that have shown that cultured
mouse embryos develop at normal rates for
24 h
after removal
from
the
mother (New, 1973, 1977; Sadler, 1979; Sadler and
New, 1981).
Rat serum
collection
and
preparation
Adult rats were anesthetized
by
inhalation
of
halothane
(Fluothane, Ayerst Laboratories Inc.) and decapitated using
a guillotine. Blood was collected
in
serum separation tubes
(Vacutainer brand SST tubes, Becton Dickinson), and spun
for 30min
at
3400 revs
min"
1
.
The
immediate centrifugation
in SST tubes pelleted the blood cells, allowing
a
clear fibrin
clot to form in the plasma layer. The serum was then decanted
away from the clot and stored
at
—70°C.
Histology
Embryos were fixed by immersion in 4% paraformaldehyde/
0.25%
glutaraldehyde
in
0.1M
phosphate buffer (PB) over-
night
at
4°C. Embryos were prepared
for
cryostat sectioning
by washing in 0.1
M
PB
for
1
h, and followed by soaking them
in
a 15%
sucrose solution
for
8-12
h
at
4°C. They were
embedded
in
15 %
sucrose and 7.5
%
gelatin (Oxoid), rapidly
frozen
in
liquid nitrogen and serially sectioned
on a
cryostat
(AO Reichert Histostat)
at 30
microns. Sections were
mounted
in
Gel/mount (Biomeda Corp.)
and
covered with
glass coverslips. The sections were viewed immediately on an
epifluorescence microscope through
a
rhodamine filter
set to
visualize
the Dil. The
overall pattern
of the
Dil-labelling
remained relatively stable
for
1
to 2 days; however, fine detail
was lost within 2-3 h.
Results
Premigratory mouse neural crest cells were labelled by
injection
of
Dil into
the
lumen
of
the neural tube.
At
the time
of
our injections,
the
dorsal neural tube
is a
pseudostratified epithelium (Erickson
and
Weston,
1983;
Sternberg
and
Kimber,
1986) in
which
pre-
migratory neural crest cells maintain
an
attachment
to
the lumen of the neural tube. Because of
this,
intralumi-
nal injections of Dil label the premigratory neural crest
cells along with other neural tube cells. Because culture
techniques allow only
24 h
of
normal development,
it
was
not
possible
to
label neural crest cells early
and
follow them over extended periods of development.
To
circumvent this difficulty,
we
have labelled different
embryos at half day intervals and incubated them for 12
or 24h,
so
that all developmental stages between E8.5
to
Ell
were examined. Table 1 presents
the
injection
and fixation times
for all
embryos used
in
this study,
lists
the
total number
of
embryos examined
at
each
stage,
and
summarizes
the
results.
All
observations
were made
at the
level
of
the forelimb (somites 8-12)
and are organized below by stage
of
injection.
Injection at E8
(Table
1:
exp. 1 and 2)
Embryos injected
at
E8 (8-12 somites) and incubated
for
12 h
contained Dil-labelled cells
in the
neural tube
exclusively. This confirmed the ability
of
the technique
to label only cells
in
contact with
the
lumen
of the
neural tube. Transverse sections
of
E8 embryos incu-
bated for
24 h
(approximately 20 somites), showed Dil-
labelled cells between
the
dorsal neural tube
and the
epidermis (Fig. 1A),
as
well
as in the
cell-free space
bordered by the epidermis, the somites and the neural
tube (termed
the
'dorsal wedge').
In
longitudinal sec-
tions,
the Dil-labelled cells did not appear to have any
segmental organization, but instead were present at the
levels
of
both
the
rostral
and
caudal halves
of the
somites.
EXP.
1
2
3
4
5
e
7
e
a
10
11
12
E 3
PERIOD
E
OF INC
9
Ta
J BATON
E1
blel
0
.
Sum
E
mary
1
(ID)'
••••
(13) ^^^^^^H
(33) ••••
(2t) ^^^^^^
(W)
^^™
(171 ^^^^^^™
(21) ^^M
HO
^^^MMi
(11)
^HHH
(12) ^^^^^^M
[10)
of experiments
and
results
LOCATION OF
DII-LABELLED CELLS
dNT
X
X
X
(•) ^^^^^"^
8NT
X
X
X
DM
X
SG
X
X
X
DA
X
X
X
DRQ
X
X
X
X
X
X
LB
X
X
DLP
X
X
x
X
X
X
X
dNT = Dorsal surface
of
the neural tube
sNT
= Space between the somite and
the
neural tube
DM = Medial surface
of
the dermamyotome
SG = Sympathetic ganglia
DA = Region around the dorsal aorta
LB = Limb bud
DLP = Dorso-lateral pathway
*
Number
of
embryos in each experiment
608
G. N. Serbedzija, S. E.
Fraser
and M. Bronner-Fraser
Injection at E8.5
(Table
1:
exp. 3 and 4)
In transverse sections of embryos injected at E8.5
(13-16 somites) and incubated for
12
h, many Dil-
labelled cells were located between the dorsal neural
tube and the epidermis, as well as in the cell-free space
of the dorsal wedge (Fig. IB). In longitudinal sections,
Dil-labelled cells had no obvious segmentation to their
rostrocaudal distribution at this stage.
Transverse sections through embryos incubated for
24 h
after injection contained Dil-labelled cells extend-
ing from dorsal of the neural tube to ventral near the
dorsal aorta. Similar to embryos incubated for 12h,
Dil-labelled cells were observed in the dorsal wedge.
Other Dil-labelled cells were observed in the dorsal
portion of the somitic sclerotome between the neural
tube and the medial edge of the dermamyotome, as well
as between the dermamyotome and the epidermis
(Fig. ID). In addition to these dorsally positioned cells,
10-20 Dil-labelled cells per section were observed
further ventrally within the rostral sclerotome, forming
a crescent adjacent to the dermamyotome (Fig. 1C and
D).
Another one to five Dil-labelled cells per section
were observed further ventrad in the region of the
sympathetic ganglia and dorsal aorta. In longitudinal
section, the Dil-labelled cells within the somitic mesen-
chyme were observed exclusively in the rostral half of
each segment (Fig. IE). In contrast, labelled cells
between the dermamyotome and epidermis did not
appear to have a segmental distribution.
Injection at E9
(Table
1:
exp. 5 and 6)
In sections through embryos incubated for
12 h
after
injection, Dil-labelled cells were observed on the lat-
eral surface of the dermamyotome, immediately under
the epidermis (Fig. IF). Dil-labelled cells also were
observed in a thin stream along the lateral surface of the
neural tube, extending from the dorsal portion of the
neural tube to the level of the ventral motor root
(Fig. 2A and B). It was not clear whether these cells
were located within or adjacent to the rostral sclero-
tome, but they were not observed at the level of the
caudal portion of the somites. In addition, individual or
small clusters of Dil-labelled cells were observed in
ventral portion of the sclerotome (Fig. 2A), the region
of the sympathetic ganglia and at the level of the dorsal
aorta.
24h after injection, embryos contained numerous
Dil-labelled cells that appeared to be intermixed with
the somitic sclerotome cells in the region of the forming
dorsal root ganglia (Fig. 2C and D). Dil-labelled cells
were also present in the sympathetic ganglia (Fig. 2C).
Similar to embryos incubated for 12h, only a small
number of Dil-labelled cells were observed around the
dorsal aorta. In addition, 5 to 10 well-separated Dil-
labelled cells were found in each forelimb (Fig. 2B). By
studying serial sections, it was clear that these cells were
spread throughout the forelimb, preceding the motor
axons.
In contrast to embryos injected at E8.5, none of the
embryos injected at E9 contained labelled cells in the
Fig. 1. Sections through embryos labelled with Dil.
(A) Transverse section of an embryo injected at E8 and
incubated for
24
h;
Dil-labelled neural crest cells (arrows)
began to appear on the dorsal surface of the neural tube
(NT).
In the more ventral regions of the embryo, Dil-
labelling was seen exclusively in the neural tube.
(B) Transverse section of an embryo injected at E8.5 and
incubated for
12
h; Dil-labelled neural crest cells were
present in the cell-free space (arrows) bordered by the
neural tube (NT), the adjacent somite (S) and the ectoderm
(the dorsal wedge). (C) Transverse section of an embryo
injected at E8.5 and incubated for 24h; A stream of
individual Dil-labelled cells (arrows) was observed in the
rostral sclerotome immediately adjacent to the medial
surface of the dermamyotome (DM). Labelled cells were
also present on the dorsal surface of the neural tube (NT),
in the cell-free space (arrowhead) and along the dorsal
lateral pathway (long arrow). (D) Transverse section of an
embryo injected at E8.5 and incubated for 24h; Dil-labelled
cells were present in the dorsal wedge (arrow heads) and
along the dorsal lateral pathway (long arrow). In addition,
labelled cells were observed adjacent to the dermamyotome
(DM),
within the rostral sclerotome and at the level of the
sympathetic ganglia (SYM; arrows). (E) Longitudinal
section at the level of the forelimb through an embryo
injected at E8.5 and incubated for
24
h;
In the region just
ventral to the cell-free space, Dil-labelled cells (arrow)
were seen entering the rostral (R), but not the caudal (C)
portion of the somite. (F) Longitudinal section through the
tenth somite of an embryo injected at E9 and incubated for
12
h;
Dil-labelled cells following the dorsolateral pathway
(arrow) were found on the lateral surface of the
dermamyotome (DM), just under the ectoderm.
(Rostrocaudal level of section: A=somite 10, B=somite 8,
C=somite 11, D=somite 9. A,B,C,D: scale bar=125^m;
E: scale bar=100/jm; F: scale bar=75fim).
sclerotome immediately adjacent to the dermamyo-
tome.
Injection at E9.5
(Table
1:
exp. 7 and 8)
Transverse sections through embryos injected at E9.5
and incubated for
12
or
24 h
contained Dil-labelled cells
in the dorsal root ganglia (Fig. 3A) and the forelimbs,
in addition to Dil-labelled motor axons. Dil-labelled
cells also were seen on the lateral surface of the
dermamyotome. In contrast to embryos injected at
earlier stages, embryos injected at E9.5 contained no
Dil-labelled cells in the region of the sympathetic
ganglia or the dorsal aorta.
Injection at E10
(Table
1:
exp. 9 and 10)
Transverse and longitudinal sections through embryos
incubated for 12 or
24 h
after injection contained a few
Dil-labelled cells in the dorsal portion of the dorsal root
ganglia, and on the lateral surface of the dermamyo-
tome, under the epidermis (Fig. 3B).
Injection at E10.5
(Table
1:
exp. 11 and 12)
Sections through embryos injected at E10.5 contained
no Dil-labelled cells external to the neural tube
(Fig. 3C). Dil-labelled motor axons were the only
labelled objects observed external to the neural tube.
Mouse neural crest cell migration
609
Fig.
2.
Transverse
and
longitudinal sections through
embryos injected
at E9 and
incubated
for
12
or
24
h.
(A) A
transverse section
of
an embryo incubated
for
12
h;
A
thin
stream
of
Dil-labelled cells (short arrows) was seen along
the lateral surface
of the
neural tube (NT) down
to the
level
of
the ventral root (long arrow).
In
addition,
a
single
labelled cell was observed
in the
ventral portion
of the
sclerotome (arrow head). (B) The same section described
in
A viewed with both brightfield
and
fluorescence.
(C) A
transverse section
of
an embryo incubated
for
24
h;
Dil-
labelled cells protruded into
the
sclerotome
in the
region
of
the forming dorsal root ganglia (arrow heads). Dil-labelled
cells were also seen
in the
limb
bud (LB) and in the
region
of
the
sympathetic ganglia (short
and
long arrows,
respectively).
(D) In a
glancing longitudinal section
of the
somites
at the
level
of
the forelimb through
an
embryo
incubated
for
24h; Dil-labelled cells were seen
in the
rostral
(R) but not the
caudal
(C)
portion
of the
somite
(arrows). (Rostrocaudal level
of
section:
A and
B=somite
11,
C=somite 10. A-D: scale bar=100/«n).
Fig. 3. Sections through embryos injected
at
E9.5, E10
and
E10.5.
(A)
Longitudinal section
at the
level
of the
forelimb
through
an
embryo injected
at
E9.5
and
incubated
for
24h;
Dil-labelled cells were seen
in the
condensing dorsal root
ganglia (DRG).
(B)
Similar section through
an
embryo
injected
at
E10
and
incubated
for
12
h; Dil-labelled cells
were seen
in the
dorsal root ganglia (DRG; short arrows)
and along
the
dorsolateral pathway (long arrows).
(C) Transverse section
of an
embryo injected
at
E10.5
and
incubated
for
12
h; Dil-labelled motor root fibers (arrow)
and what may
be a
ventral root sheath cell (arrow head)
were
the
only labelled objects observed external
to the
neural tube. (Rostrocaudal level
of
section: C=somite
10.
A: scale bar=125j/m; B: scale bar=40fjm;
C:
scale
bar=100^m).
Pathways
of
neural crest cell migration inferred from
Dil-labelling
Based on the pattern of Dil-labelling described above
at the level of the forelimb, we infer that neural crest
emigration from the neural tube begins at E8.5 and
continues through E10. Labelled cells appear to mi-
grate along the dorsolateral pathway throughout this
period (Fig. 4B-D). Migration along the ventral path-
ways appears to occur in two overlapping phases. The
first phase occurs between E8.5 and E9.5. Labelled
cells leave the neural tube and enter the dorsal wedge, a
cell-free space adjacent to the neural tube, ectoderm
and dorsal somite (Fig. 4A). From this site, individual
labelled cells move through the rostral sclerotome
adjacent to the dermamyotome, to populate the sym-
pathetic ganglia and the adrenergic derivatives around
the dorsal aorta (Fig. 4B and C). Starting at E9 and
proceeding through E10, a second phase of migration
takes place; labelled cells appear as a thin stream along
the lateral surface of the neural tube, opposite the
rostral half of the sclerotome (Fig. 4C and D). Later,
these cells protrude into the sclerotome, where they
condense into dorsal root ganglia. In addition, other
cells appear to migrate along the neural tube, then
move laterally through the rostral sclerotome into the
limb bud. Based on their final location, neural tube
Fig.
4.
Schematic representation
of the
pathways
of
trunk
neural crest cell migration
at the
level
of the
forelimb.
(A)
At
approximately E8.5, neural crest cells emerge from
the dorsal portion
of
the neural tube (NT) into
the
cell-free
space bordered
by the
neural tube,
the
somite
and the
ectoderm.
(B) At
E9, neural crest cells
can be
seen along
two pathways:
(1) a
dorsolateral pathway between
the
dermamyotome
and the
epidermis (DLP),
and (2) a
ventral
pathway between
the
neural tube
and the
dermamyotome.
The ventral pathway consists
of
two overlapping phases
of
migration. Initially, neural crest cells appear only under
the
medial surface
of
the dermamyotome (DM). These early
migrating cells extend along
the
ventrolateral portion
(VL)
of
the
sclerotome
to the
level
of
the dorsal aorta (DA).
(C)
By
E9.5, migrating neural crest cells
are
apparent along
the lateral surface
of the
neural tube (NT) (i.e.
the
ventromedial (VM) portion
of the
sclerotome).
A few
neural crest cells
are
present along
the
ventrolateral portion
(VL)
of
the sclerotome,
as
well
as in the
region
of the
ventral derivatives.
In
addition, neural crest cells migrate
along
the
dorsolateral pathway (DLP).
(D) By
E10, neural
crest cells
no
longer migrate along
the
ventrolateral portion
(VL)
of
the ventral pathway,
but are
present along
the
ventromedial portion (VM)
and the
dorsolateral pathway
(DLP).
origin, and association with the ventral motor axons,
the cells that migrate into the limb bud are likely to be
Schwann cells. Labelled cells are not found in ventral
derivatives in embryos labelled with Dil at E9.5 or
later. Dil-labelled cells are found in progressively more
dorsal derivatives with later injections, suggesting that
neural crest cells contribute to their derivatives in a
ventral-to-dorsal order (Fig. 5). Neural crest cell emi-
gration from the neural tube appears to be complete by
E10.5.