DEVELOPMENTAL
BIOLOGY
143,213-217
(1991)
Effects of Mesodermal
Tissues
on Avian Neural Crest Cell Migration
We have used microsurgical
techniques
to investigate
the effects of embryonic
mesodermal
tissues on the pattern
of
chick neural crest cell migration
in the trunk.
Segmental
plate or lateral
plate mesenchyme
was transplanted
into
regions encountered
by neural crest cells. We found that neural crest cells are able to migrate
through
lateral
plate
mesenchyme
but not through
segmental
plate tissue until this tissue diff’erentiates
into a sclerotome.
After this stage,
segmental
migration
is controlled
by the subdivision
of the sclerotome
into a rostra1 and a caudal half: when the
rostrocaudal
orientation
of the sclerotomes
is reversed
by rotating
the segmental
plate 180” about its rostrocaudal
axis,
CL1991 Acukmie Prrss. Inc.
neural crest cells migrate
through
the portion
of the sclerotome
that was originally
rostral.
INTRODlJCTION
The neural crest is a migratory
cell population, which
forms at the dorsal aspect of the neural tube. After closure of the neural tube, neural crest cells emigrate from
its dorsal margin
and proceed along characteristic
pathways
to reach their final destinations.
In the trunk
of avian embryos, neural crest cells migrate along two
primary pathways:
a ventral route through the somite,
including cells that will give rise to dorsal root ganglia,
sympathetic
ganglia, and adrenal chromaffin cells, and
a path under the ectoderm, along which presumptive
melanocytes
migrate (Weston, 1963; LeDouarin,
1982).
Neural crest cell migration along the ventral pathway is
segmental. Like outgrowing
motor nerves (Keynes and
Stern, 1984), migrating
neural crest cells (Rickmann
et
al., 1985; Bronner-Fraser,
1986; Teillet et ul, 1987; Loring
and Erickson,
1987; Newgreen,
1990) are restricted
to
the rostra1 half of each sclerotome. In the case of motor
nerves, this is due to a property of the two halves of the
sclerotome
(Keynes and Stern, 1984; Tosney, 1988a,b)
rather than to intrinsic segmentation
of the neural tube,
though the latter also exists (Keynes and Stern, 1985;
Lumsden and Keynes, 1989). The conclusion that the somite contains segmental information
is based on the
result of a simple experiment:
if the neural tube is rotated 180” about its rostrocaudal
axis, the motor axon
outgrowth
pattern
through
the sclerotome
is unaffected, but if the segmental plate (which gives rise to the
somites) is rotated in the same way the motor axons now
traverse the caudal (original rostral)
halves of the rotated sclerotomes
(Keynes and Stern, 1984). This metamerit pattern may result from the presence of inhibitory cues in the caudal sclerotome.
A peanut lectinbinding glycoprotein
fraction
which inhibits
growth
cones in uifro has been isolated from sclerotomes
(Davies ef cd., 1990).
By analogy with the motor nerves, it is reasonable to
expect that the migration
of neural crest cells will be
subject to the same control mechanisms,
since the dorsal root ganglia must develop in register with the ventral root to generate the adult arrangement.
One of the
aims of this study is to test experimentally
whether
neural crest cells and motor axons follow similar cues. A
second aim is to examine why neural crest cells never
enter other regions of mesenchyme where intercellular
space may be available, such as the segmental plate mesoderm and the lateral plate. We have tested the possibility that these tissues actively inhibit their invasion
by neural crest cells by transplanting
them into the
pathways
of migrating
neural crest cells.
MATERIALS
AND
METHODS
Chick eggs were incubated at 38°C until the embryos
reached stages 11-13 (Hamburger
and Hamilton,
1951).
A window was cut in the egg shell with a scalpel blade
and the yolk was floated with calcium- and magnesiumfree Tyrode’s saline (CMF). Indian ink (Pelikan Fount
India), diluted 1:lO in CMF, was then injected under the
blastoderm
to aid visualization
of the embryo. A standing drop of CMF containing 0.1% trypsin was added to
facilitate
dissection. The vitelline membrane over the
graft site was penetrated with a tungsten needle; the
operations
then were performed,
using a microsurgical
knife (Week, 15” angle) and tungsten needles sharpened
in molten sodium nitrite. A carmine mark was placed at
the rostra1 end of the graft to mark its position. Oper-
214
DEVELOPMENTALBIOLOGY VOLUME143,1991
Fixation
80'
and Embedding
Embryos were fixed in Zenker’s fixative for 1.5-2 hr,
washed in running tap water, and transferred to 70%
ethanol. Embryos were processed by dehydration
in a
graded series of ethanols, clearing in histosol, and finally three changes of paraplast followed by embedding
in fresh paraplast. Sections were cut on a Leitz microtome at a thickness of 10 pm and mounted on albuminized slides.
Immunohistochemistry
FIG. 1. Schematic diagrams summarizing
ments
caudal
tiated”
somite
the microsurgical experiperformed. (A) Segmental plate rotation 180” about its rostroaxis. (B) Graft of segmental plate into the region of “differensomites. (C) Graft of a piece of lateral plate mesoderm into the
region.
ated embryos were incubated in a humidified
atmosphere at 38°C until the time of fixation.
Segmental plate rotations in the rostrocaudal plane. A
length of segmental plate, 200-600 pm long (equivalent
to two to six somites in length), was excised on one side
of the embryo (Fig. 1A). It then was rotated 180” about
its rostrocaudal axis and reimplanted
into the same embryo. The embryos were incubated for l-2 days, by
which time the graft had formed somites and these had
differentiated
into sclerotomes and dermomyotomes,
but in reverse orientation
(Keynes and Stern 1984).
Segmental plate grafts into sclerotome regions. Three
to six somites (with the caudal-most four to six segments rostra1 to the most recently formed somite) were
excised. An equivalent length of segmental plate, from
the middle or caudal portion of its rostrocaudal extent,
was excised from the same or from a donor embryo, and
implanted in their place, either in the same orientation
or after reversal about its rostrocaudal axis (Fig. 1B).
The embryos were incubated for about 9 hr prior to fixation. In a control series of embryos designed to test if
sufficient incubation time had elapsed for neural crest
cells to have entered the graft, three to six sclerotomes
were replaced with a similar number of epithelial
somites and fixed after 6-9 hr.
Lateral plate grafts into sclerotome regions. Four to six
caudal somites were excised. An equivalent length of
lateral plate mesoderm was excised from the caudal
third of the area pellucida of the same or of a donor
embryo (Fig. 1C). The lateral plate explant then was
grafted in place of the excised somites, either without
rotation or after reversal about its rostrocaudal axis.
The embryos were incubated for l-2 days prior to fixation.
The monoclonal
antibody
HNK-1 was used as a
marker for neural crest cells (Tucker et al., 1984). The
sections were immersed in histosol to remove the paraplast, rehydrated through a graded series of ethanols,
and washed in phosphate buffer (PB) or phosphatebuffered saline (PBS). They then were incubated with
culture medium supernatant
(undiluted) from HNK-1
hybridoma cells for 3 hr at room temperature.
After
being washed in PB or PBS, slides were incubated with
rabbit anti-mouse IgM antiserum (Zymed; diluted 1:30
in 0.1% BSA in PBS) for 1 hr, followed by a 1-hr incubation with fluorescein-conjugated
goat anti-rabbit
IgG
serum (Zymed; diluted 1:30). The slides were washed in
PB or PBS, mounted under a coverslip with glycerol
(90% glycerol/lO%
0.5 M sodium carbonate, pH 7.8),
and observed using an Olympus Vanox microscope
equipped with epifluorescence optics. In a series of control slides, the primary antibody was omitted and no
immunoreactivity
was observed in these cases.
RESULTS
Eflects of Rostrocaudal Reversal of the Segmental
on the Pattern of Neural Crest Migration
Plate
After reversal along the rostrocaudal axis (Fig. lA),
the segmental plate heals rapidly and forms epithelial
somites that appear to have a normal morphology
within a few hours after the operation. These somites
soon differentiate
into distinct dermomyotomes
and
sclerotomes. Most of the grafts gave rise to several somites of normal size plus a small somite at the junction
between the rotated and the nonrotated tissue (Fig. 2A).
Embryos were fixed l-2 days after the operation, sectioned, and stained with HNK-1 antibody to visualize
neural crest cells. In contrast to the somites derived
from unoperated
regions of the embryo,
where
HNK-1-immunoreactive
cells were observed only in the
rostra1 half of each sclerotome, neural crest cells in the
sclerotomes derived from the rotated segmental plates
(n = 4) were always observed in the caudal (original
rostral) half of each sclerotome (Fig. 2a). Within the
sclerotomes of the rotated piece, neural crest cells ap-
BRONNER-FRASER
215
AND STERN
FIG. 2. Fluorescence
photomicrographs
of longitudinal
sections through
operated
embryos
stained with the HNK-1
antibody.
(a) An embryo
in which the segmental
plate was rotated
180” about its rostrocaudal
axis. By the time of fixation,
the segmental
plate had differentiated
into
mature
somites
with dermomyotomes
and sclerotomes.
Neural
crest cells were always
observed
in the original
rostra1
(R) half of each
sclerotome
and were absent from the original
caudal (C) halves. The arrow
indicates
a small somite which formed
at one of the junctions
between graft and host tissue. Because the section is slightly
glancing,
the upper part is through
a slightly
more dorsal level of the neural tube
than the lower part. (b) An embryo
in which a piece of segmental
plate (SP), rotated
180” about its rostrocaudal
axis, was transplanted
into the
left side of the embryo in place of mature
somites. An arrow marks the anterior
border of the graft. Neural crest cells failed to enter the grafted
segmental
plate, whereas
on the unoperated
side, they were observed
within
the rostra1 half of each sclerotome.
(c) An embryo
in which a piece
of lateral plate mesenchyme
was transplanted
in place of mature
somites. A large blood vessel (BV) formed
and was surrounded
by epithelium
and mesenchyme.
Neural crest cells (arrows)
were observed
in an unsegmented
pattern
within
the mesenchyme.
NT, neural tube. The scale bar
in c corresponds
to 45 Km in a, 100 pm in b, and 45 pm in c.
peared to be aligned in two to three distinct rows of cells
oriented in the mediolateral
plane, as they are in unoperated regions of the embryo.
Sepnental
Plate Grafts into Sclerotome
Regions
Embryos
(n = 6) in which sclerotomes
had been replaced with a graft of segmental plate (Fig. 1B) were
allowed to develop for 9 hr after the operation and then
were fixed, sectioned, and stained with HNK-1 antibody. By the time of fixation, the grafted piece had
usually budded off one epithelial somite from its original rostra1 end. Sections (Fig. 2b) revealed that the region of grafted segmental plate that had not segmented
was devoid of HNK-1-immunoreactive
cells in all cases.
After grafting, only about 12-15 hr elapse before the
graft has become segmented into epithelial somites. In
order to improve the chances of neural crest cells confronting the unsegmented portion, the graft was rotated
180” about its rostrocaudal
axis so that the “oldest”
neural crest was opposite the “youngest”
portion of the
grafted segmental plate; the embryos were incubated
until the original rostra1 end of the plate had formed
two to three somites. Under these conditions as well, no
HNK-l-immunoreactive
cells were found within the unsegmented portion of the grafted plate, although many
surrounded
the graft (Fig. 2b).
For a further test of whether the neural crest was able
to enter into a graft within a few hours of the operation,
a control series of embryos (n = 5) had their sclerotomes
replaced with epithelial somites and fixed 9 hr after the
operation. In these cases it was found that HNK-l-immunoreactive
cells were present in the rostra1 halves of
the sclerotomes
formed from the grafted somites.
Lateral
Plate Grafts into Sclerotome Regions
Embryos (n = 6) in which sclerotomes had been replaced with a graft of lateral plate mesoderm (Fig. 1C)
216
DEVELOPMENTAL
BIOLOGY
were allowed to develop for l-2 days and then were
fixed, sectioned, and stained with HNK-1 antibody. The
grafted lateral plate differentiated
into large blood vessels bordered by endothelium
and mesenchyme.
In no
case were any HNK-1-immunoreactive
cells found
within the vessels or among the endothelial cells. However, neural crest cells did enter the surrounding
mesenthyme, where they appeared to be evenly distributed
rather than metamerically
arranged.
In contrast,
regions rostra1 and caudal to the graft displayed the normal pattern of HNK-1 staining in the rostra1 halves of
the sclerotomes
(Fig. 2~).
DISCUSSION
We have examined the influence of various mesoderma1 tissues on the pattern of neural crest cell migration
in the trunk of the avian embryo. The experiments
in
which we reversed a length of segmental plate mesoderm about its rostrocaudal
axis showed, as was expected, that HNK-1-immunoreactive
neural crest cells
are now present in the caudal (original rostral) halves of
the sclerotomes
derived from the rotated mesoderm.
This result is consistent with previous findings (Keynes
and Stern, 1984) for motor nerves growing through the
sclerotome after stage 17 and confirms the idea (Keynes
and Stern, 1985; Rickmann et al., 1985; Bronner-Fraser,
1986; Teillet et ab, 1987; Loring and Erickson, 1987) that
it is the presence of differences between the cells of the
rostra1 and the caudal halves of the sclerotome that is
responsible for the segmental pattern of both neural
crest migration and motor axon outgrowth.
The segmental plate appears to discourage the invasion of neural crest cells. HNK-1-immunoreactive
cells
were never seen in grafts of segmental plate mesoderm,
although they surrounded the grafted piece. Our control
experiments show that sufficient time had elapsed after
the operation for neural crest cells to enter a permissive
graft, since after 9 hr they invaded the rostra1 halves of
the sclerotomes that developed from grafted epithelial
somites. In contrast to the segmental plate grafts,
neural crest cells were able to enter lateral plate mesoderm grafts. The pattern of neural crest cell distribution in the lateral plate mesenchyme was uniform, suggesting that all portions of the mesenchyme are permissive substrates for migration.
These results suggest that the mesenchyme of the
segmental plate, but not that of the lateral plate mesoderm, inhibits neural crest cell migration. The selective
inhibition by the segmental plate tissue may assure that
neural crest cells do not invade regions of the embryo
until the proper time. In normal embryos, neural crest
cells do not invade the lateral plate region. Why then
were HNK-1 immunoreactive cells found within grafted
VOLUME
143.1991
lateral plate? One possible explanation is that since
neural crest cells never encounter the lateral plate during their normal migration, this tissue does not require
inhibitory
properties for neural crest cells. Alternatively, the transplanted lateral plate may assume some
somitic properties as a result of interactions with adjacent structures after grafting. Fraser (1960) has shown
that transplantation of a neural tube into the lateral
plate mesenchyme can elicit segmentation of this tissue.
Furthermore, in the caudal portion of the embryo, segmental plate and lateral plate mesenchyme cells share a
common lineage (Stern et al, 1988). Thus, the permissive
nature of the grafted lateral plate mesenchyme for migrating neural crest cells may reflect its acquisition of
some somite-like properties.
The present study demonstrates that, in avian embryos, trunk neural crest cells are largely guided by cues
intrinsic to their surrounding tissues. The metameric
pattern of neural crest cell migration is controlled by
rostrocaudal differences in the somitic sclerotome. A
number of molecular differences have been noted between rostra1 and caudal somites. The caudal half of the
sclerotome contains peanut lectin binding glycoproteins
of 48 and 55 kD (Stern et aZ.,1986, Davies et al., 1990) and
T-cadherin (Ranscht and Bronner-Fraser, 1991). In the
rostra1 half of the sclerotome, a i’O-kDa protein (Tanaka
et ab, 1989), butyrylcholinesterase
activity (Layer et al.,
1988), cytotactin/tenascin/Jl
(Tan et ab, 1987; Mackie et
ah, 1988; Stern et al, 1989), and an HNK-1 bearingglycolipid (Newgreen et al., 1990) have been found. In addition, many other differences in the polypeptide composition between rostra1 and caudal somite portions have
been demonstrated by two-dimensional gel electrophoresis (Norris et al., 1989). Although peanut lectin receptors
in the caudal sclerotome cause axon collapse (Davies et
al., 1990), a functional role has yet to be demonstrated
for molecules expressed at the correct time to affect
neural crest cell migration. One possibility is that the
molecules that are inhibitory for neural crest migration
in the caudal sclerotome are similar to those present
within the segmental plate.
This study was funded by USPHS Grant HD 15527 and a grant from
the Muscular
Dystrophy
Association
to M.B.F. and by a travel grant
from the Wellcome
Trust to C.D.S. M.B.F. is a Sloan Research
Fellow.
REFERENCES
BRONNER-FRASER,
M. (1986). Analysis
of the early stages of trunk
neural crest cell migration
in avian embryos
using monoclonal
antibody HNK-1.
Dm
Biol.
115, 44-55.
DAVIES, J. A., COOK, G. M. W., STERN, C. D., and KEYNES, R. J. (1990).
Isolation
from chick somites of a glycoprotein
fraction
that causes
collapse of dorsal root ganglion
growth
cones. Neurcm 4,11-20.
FRASER, R. C. (1960). Somite genesis in the chick. III. The role of
induction.
J. Exp. Zool. 145, 151-167.
217
BRONNER-FRASER AND STERN
HAMBURGER, V., and HAMILTON, H. L. (1951). A series of normal
stages in the development of the chick embryo. J. Mwphol.
88,49-92.
KEYNES, R. J., and STERN, C. D. (1984). Segmentation in the vertebrate
nervous system. Nuturr
(Lmdo~~,l310,786-‘789.
KEYNES, R. J., and STERN, C. D. (1985). Segmentation and neural development in vertebrates. 2’rend.s Neurosci. 8, 220-223.
LAYER, P., ALBER, R., and RATHJEN, F. (1988). Sequential activation of
butyrylcholinesterase in rostra1 half somites and acetylcholinesterase in motoneurones and myotomes preceding growth of motor axons. Developtnent
102, 387-396.
LEDOUARIN, N. M. (1982). “The Neural Crest.” Cambridge IJniv. Press,
Cambridge.
LORING, J., and ERICKSON, C. (1987). Neural crest cell migration pathways in the chick embryo. Den Bid. 121, 230-236.
LUMSDEN, A., and KEYNES, R. (1989). Segmental patterns of neuronal
development in the chick hindbrain. N&tre (Lorrdo?~~ 337,424-429.
MACKIE, E. J., TUCKER, R. P., HALFTER, W., CHIQUET-EHRISMANN, R.,
and EPPERLEIN, H. H. (1988). The distribution of tenascin coincides
with pathwags of neural crest cell migration. Dewlopwnt
102,237250.
NEWGREEN, D., POWELL, M. E., and MOSER, E. (1990). Spatiotemporal
changes in HNK-l/L2 glycoconjugates on avian embryo somite and
neural crest. Dev. Bid. 139, 100-120.
NORRIS, W’. E., STERN, C. D., and KEYNES, R. J. (1989). Molecular differences between the rostra1 and caudal halves of the sclerotome in
the chick embryo. Develo~mmt 105,541-548.
RANSCHT, B., and BRONNER-FRASER, M. T-cadherin expression alternates with migrating neural crest cells in the trunk of the avian
embryo. Dclvkqm~ent,
in press.
RICKMANN, M., FAWCETT, J. W., and KEYNES, R. J. (1985). The migration of neural crest cells and the growth of motor axons through the
rostra1 half of the chick somite. J Entbryol.
Exp Morphol.
90, 437455.
STERN, C. D., FRASER, S. E., KEYNES, R. J., and PRIMMETT, D. R. N.
(1988). A cell lineage analysis of segmentation in the chick embryo.
104, Suppl., 231-244.
STERN, C. D., NORRIS, W. E., BRONNER-FRASER, M., CARLSON, G. J.,
FAISSNER, A., KEYNES, R. J., and SCHACHNER, M. (1989). Jl/tenascin-related molecules are not responsible for the segmented pattern of neural crest cells or motor axons in chick embryo. Dwdoy
Development
nlent
107, 309-320.
STERN, C. D., SISODIYA, S. M., and KEYNES, R. J. (1986). Interactions
between neurites and somitc cells: Inhibition and stimulation of
nerve growth in the chick embrpo. J. Embrrryol. Exp. Morphol.
91,
209-226.
TAN, S.-S., CROSSIN,K. L., HOFFMAN, S., and EDELMAN, G. M. (1987).
Asymmetric expression in somites of cytotactin and its proteoglycan ligand is correlated with neural crest cell distribution. Proc.
Nrrtl. Aad.
Sri. USA 84, 7977-7981.
TANAKA, H., AGATA, A., and OBATA, K. (1989). A new membrane antigen revealed by monoclonal antibodies is associated with axonal
pathways. Dev. Bid. 132, 419-435.
TEILLET, A. M., KALCHEIM, K., and LEDOUARIN, N. M. (1987). Formation of the dorsal root ganglia in the avian embryo: Segmental origin and migratory behavior of neural crest progenitor cells. Drv.
Bid.
120, 329-347.
TOSNEY, K. W. (1988a). Proximal tissues and patterned neurite outgrowth at the lumbosacral level of the chick embryo: Deletion of the
dermamyotome. &?I. Bid. 122, 540-588.
TOSNEY, K. W. (1988b). Proximal tissues and patterned neurite outgrowth at the lumbosacral level of the chick embryo: Partial and
complete deletion of the somite. Dea Biol. 127, 266-286.
TUCKER, G. C., AOYAMA, H., LIPINSKI, M., TURSZ, T., and THIERY, J. P.
(1984). Identical reactivity of monoclonal antibodies HNK-1 and
NC-l: Conservation in vertebrates on cells derived from the neural
primordium and on some leukocytes. Cell Difer: 14,223-230.
WESTON, J. A. (1963). A radioautographic
analysis of the migration
and localization of trunk neural crest cells in the chick. De/,. Bid. 6,
279-310.