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437
The migration of neural crest cells and the growth of
motor axons through the rostral half of the chick
somite
M. RICKMANN, J. W. FAWCETT
The Salk Institute and The Clayton Foundation for Research, California division,
P.O. Box 85800, San Diego, CA 92138, U.S.A.
AND R. J. KEYNES
Department of Anatomy, University of Cambridge, Downing St, Cambridge,
CB2 3DY, U.K.
SUMMARY
We have studied the pathway of migration of neural crest cells through the somites of the
developing chick embryo, using the monoclonal antibodies NC-1 and HNK-1 to stain them.
Crest cells, as they migrate ventrally from the dorsal aspect of the neural tube, pass through the
lateral part of the sclerotome, but only through that part of the sclerotome which lies in the
rostral half of each somite. This migration pathway is almost identical to the path which presumptive motor axons take when they grow out from the neural tube shortly after the onset of
neural crest migration. In order to see whether the ventral root axons are guided along this
pathway by neural crest cells, we surgically excised the neural crest from a series of embryos,
and examined the pattern of axon outgrowth approximately 24 h later. In somites which
contained no neural crest cells, ventral root axons were still found only in the rostral half of the
somite, although axonal growth was slightly delayed. These axons were surrounded by sheath
cells, which had presumably migrated out of the neural tube, to a point about 50 jan proximal to
the growth cones. With appropriate antibodies we found that the extracellular matrix components fibronectin and laminin are evenly distributed between the rostral and caudal halves of
the somite. Neither of these molecules therefore plays a critical role in determining the specific
pathway of neural crest cells or motor axons through the rostral half of the somite.
INTRODUCTION
The cells of the neural crest are remarkable for the length and complexity of
their migration pathway, and the number of cell types into which they differentiate
(reviewed in Le Douarin, 1982). Perhaps the only comparable structures in terms
of complexity of migratory behaviour are the growth cones of nerve fibres. It is
hardly surprising, therefore, that the mechanisms controlling neural crest migration and axon outgrowth have been two of the most intensively studied
processes in embryogenesis.
Over the years it has been possible to establish which tissues are derived from
neural crest (Horstadius, 1950; Weston, 1963; Johnston, 1966; Noden, 1975), more
Key words: neural crest, somite, axonal guidance,fibronectin,laminin.
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M. RICKMANN, J. W. FAWCETT AND R. J. KEYNES
recently with the technique of chimaeric grafting, in which quail cells are grafted
into chick embryos (Le Douarin, 1973; Le Douarin & Teillet, 1974). The approximate pathway of neural crest migration has also been elucidated, using this and a
variety of other anatomical techniques. Recently, it has been possible to analyse
the migration of crest cells with greater precision using immunohistochemical
methods and monoclonal antibodies which bind selectively to the migrating neural
crest cell surface (Vincent, Duband & Thiery, 1983; Vincent & Thiery, 1984), and
it is a series of experiments using this technique which we report on here.
Some progress has also been made in defining mechanisms which might be
responsible for causing neural crest cells to migrate along their chosen pathways.
In this respect much attention has focused over the last few years on the extracellular matrix component fibronectin, which is present in high concentration in
the pathways followed by neural crest cells (Newgreen & Thiery, 1980; Mayer,
Hay & Hynes, 1981; Duband & Thiery, 19826), is a good substrate for crest cell
migration in culture (Rovasio et al. 1983), and, when coated onto microbeads,
affects their transportation down the neural crest migration pathway (BronnerFraser, 1982).
Shortly after truncal crest cells start to migrate ventrally past the neural tube,
the presumptive motor axons begin to grow out from the neural tube into the
somites. It has recently been reported that these motor nerve fibres grow only
through the rostral half of each somite, and do so initially from only those portions
of the neural tube immediately adjacent to the rostral half of each somite (Keynes
& Stern, 1984). The specificity for this selective outgrowth lies in the cells of the
somite itself, since in embryos with rostrocaudal rotations of the segmental plate
the outgrowth of the motor nerve fibres is still restricted to the half of the somite
which would, in an unoperated animal, have lain rostrally (Keynes & Stern, 1984).
Moreover, ablating the somitic mesoderm with X irradiation results in motor
axons growing out evenly along the length of the neural tube (Lewis, Chevallier,
Kieny & Wolpert, 1981).
In this paper we describe in detail the path taken by the neural crest cells
through the somites, as revealed by staining them with the two monoclonal
antibodies NC-1 (Vincent etal 1983) and HNK-1 (Tucker etal 1984). We report
that neural crest cells demonstrate the same specificity as motor axons, and, like
them, migrate through the rostral half of each somite. We also report the results of
experiments in which we have ablated the neural crest, and examined the subsequent pathway of the motor nerve fibres through crest-free somites. We show
that the motor fibres are not guided through the rostral half of the somite due to
the presence of crest cells within it, since they still grow specifically through the
rostral half when it is completely free of neural crest cells. We also describe the
distribution of the extracellular matrix components fibronectin and laminin, and
speculate on the possible roles of these molecules in guiding neural crest cells and
nerve fibres.
Neural crest migration and axon outgrowth
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MATERIALS AND METHODS
Immunocytochemistry
The embryos were transferred from the egg into a dish filled with PBS (phosphate-buffered
saline without Ca2+ and Mg2"1"), where they were carefully freed from the surrounding
membranes, stretched out and pinned down. Thereafter, the embryos were fixed for 1 h at room
temperature, in a fixative consisting of 1-25 % glutaraldehyde, 1 % paraformaldehyde and 3-5 %
sucrose in O-lM-phosphate buffer at pH7-3. After rinsing in PBS the embryos were encapsulated by placing them into a solution of 20 % BSA (bovine serum albumin) in 0-1 M-sodium
phosphate at pH7-2 which was polymerized with 25 % glutaraldehyde (1 % in final volume).
The resulting blocks were then hardened for 1 h in the original fixative. Since the blocks were
transparent they could be easily trimmed for transverse, horizontal or parasagittal sectioning of
the embryos. Sections were cut either on a vibratome at a thickness between 100 and 150 jum or,
after soaking in 20% phosphate-buffered glycerol, on a freezing microtome at 50 to 100 fjm.
They were collected in wells containing Tris-buffered saline (TBS: 50mmol Tris, 150mmol
NaCl, pH7-6). The immunocytochemical method utilized the avidin-biotin-peroxidase procedure. Free-floating sections were passed through the following incubation steps at room
temperature, while being gently agitated. (1) Non-specific antibody binding was blocked by
incubation for 2-3 h in 0-02 % sodium azide, 0-1 M-L-lysine., 1 % BSA and 1:10 serum (from the
same species as the secondary antibody) in TBS. The same solution was used to dilute both the
primary and secondary antibodies. (2) Without rinsing, the sections were transferred to the
primary antibody for 12-24 h. The following dilutions were used for the various antibodies
employed in this study. Goat anti-chicken fibronectin (Calbiochem, Lot 001428) was diluted
1:2000, rabbit anti-laminin serum (EY labs no. AT 2404, lot no. 020106) 1:100, prediluted
hybridoma ascites containing anti-HNKl antibodies (Becton Dickinson anti Leu-7, no. 7390, lot
no. 60914) 1:50 and monoclonal anti-NCI ascites 1:500. Normal serum and control ascites were
used at the same concentrations as controls. (3) Sections were rinsed in TBS, three changes of
45min each, and then floated onto biotinylated secondary antibody (Vector laboratories),
diluted 1:250, for 6-8h. (4) Rinsed three times in TBS and left in TBS overnight. (5)
Endogenous peroxidase activity was blocked with 0-3% H2O2 in methanol for 30min. (6)
Rinsed three times in TBS, 5min each change. The last two steps were omitted in some
preparations in order to improve the structural preservation of the rather fragile sections. (7)
Incubated with Avidin-coupled horseradish peroxidase complex (Vector laboratories) for 3 h at
1:100 dilution in 1 % BSA in TBS. (8) Rinsed four times, 1 h each change, in TBS. (9) 0-01 %
3,3'-diaminobenzidine tetrahydrochloride (DAB) and 0-03 % H2O2 in TBS for 10-15 min. (10)
Rinsed four times, 10min each change, in TBS. (11) Transferred to PBS. At this stage of the
procedure, some of the sections were mounted on gelatinized slides, air dried, dehydrated in
ethanol and xylene and coverslipped in DPX. In order to obtain higher resolution and better
tissue preservation, in some cases the reaction product was intensified and the sections were
embedded in epoxy resin using the following procedure. (12) 0-01 % OsO4 in PBS for 15 min.
(13) Rinsed four times, 10 min each, in PBS. Subsequently the sections were dehydrated in a
graded series of ethanols and propylene oxide, and then infiltrated with Spurr's low viscosity
embedding medium. The sections were flattened between glass slides, one of which was coated
with a mould release compound, and polymerized at 70°C for one day. Selected sections were
attached to the flat surface of a plastic block, then heated up and detached from the slide. These
sections were resectioned in an ultramicrotome to give semithin sections, which allowed
optimum identification of labelled cells and nerve fibres.
Crest ablations
These were performed on embryos which had been incubated at 38°C for about 48 h, and
which had between 13 and 19 somites. The egg was windowed, a few drops of Hanks balanced
salt solution were dripped onto it, and then about 0-02 ml of india ink (Pelikan fount india
diluted 1:1 with Hanks) was injected under it to render it visible. The vitelline membrane
overlying the embryo was then cut. The operations were performed with electrolytically
sharpened tungsten needles, one in the shape of a hook, and another in the shape of a flat knife
blade. The hook was run up the inside of the neural tube, reopening it, and then the knife was
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M. R I C K M A N N , J. W. F A W C E T T AND R. J. K E Y N E S
used to cut against it, so as to remove approximately the dorsal half of the neural tube. Tissue
was removed in this way from the neural tube opposite the six to eight most caudal somites, and
from about half this length of neural tube opposite the undivided segmental plate. The eggs were
then resealed with Scotch tape and returned to the incubator until they had matured to
Hamburger & Hamilton stage 18 to 21 (Hamburger & Hamilton, 1951). They were then
processed in the same way as described above for unoperated embryos.
RESULTS
HNK-1 and NC-1 staining
We find that the antibodies directed against these antigens bind to the surface of
neural crest cells, the extracellular area around the notochord and to outgrowing
nerve fibres (Vincent etal. 1983; Vincent & Thiery, 1984). We have concentrated
on embryos at three stages of development, stages 13, 15 and 17, which corresponds to 19, 24-27 and 29-32 somites respectively. In any given embryo, there is
a gradient in segmental development along the longitudinal axis, the youngest
stages being found where the segmental plate is dividing into somites, and the most
advanced next to the otic vesicle. At these stages the distribution of neural crest
cells is the same a given number of somites rostral to the region where somites are
forming from the segmental plate, regardless of the actual age of the embryo.
Therefore, we will give a generalized account of the changes in the pattern of the
crest cells with time, using the rostrocaudal developmental gradient of the embryo
as our time axis, instead of describing the appearance of each age of embryo
individually.
We first saw antibody binding to neural crest cells about three somites rostral to
the most caudal somite. These cells are on either side of the midline, dorsal to the
neural tube, and are evenly spread along the longitudinal axis of the embryo.
About three somites rostral to this, the crest cells show the first signs of migration
around the lateral sides of the neural tube. The very first cells to migrate pass
laterally to, and in between, the somites, but the number of migrating crest cells at
Fig. 1. (A) A section, cut slightly obliquely from the horizontal, from a stage-15 chick
embryo, stained with anti-HNK-1. Rostral is to the right, and lateral is above. The lefthand somite is the seventh rostral to the most recently formed somite. These are
therefore young somites, which are not yet fully dissociated into dermomyotome and
sclerotome, and in which there is no obvious morphological divide between the rostral
and caudal halves. The early pattern of neural crest migration is seen here. There are
some crest cells in the intersomitic space, particularly in the more caudal somites, but
there are also some cells which have entered the sclerotome, and become intermixed
with sclerotomal cells. However, all these cells which have entered the somite are
restricted to its rostral half. Bar equals 200 jum. (B) A semithin section taken from the
middle somite in the illustration above. The cells with dark reaction product around
them are neural crest cells, and can be seen to have mingled with the cells of the
somite. (C) Transverse semithin section from the rostral half of the somite 13 or 14
rostral to that most recently formed in a stage-13 embryo, stained with anti-HNK-1.
Dorsal is upwards. Only half of the cross-section of the embryo is included in this
frame; half of the neural tube can be seen on the upper left of the picture. Neural crest
cells can be seen migrating around the neural tube, and through the somite. Some crest
cells are intermixed with the cells of the sclerotome. Bar equals 50/mi.
Neural crest migration and axon outgrowth
441
this stage is small; about a dozen cells per somite in any 150 jUm section. Very soon
the crest cells start to penetrate into the somite. Despite the fact that the somite
shows no morphological sign of division into rostral and caudal halves at this stage,
the migrating crest cells are all restricted to the rostral half of each somite
(see Fig. 1).
As the somite ages, the number of crest cells seen within it increases rapidly.
These cells are always restricted to the rostral half of the somite, where they
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M. RICKMANN, J. W. FAWCETT AND R. J. KEYNES
occupy the space beneath the dermomyotome and they progressively penetrate
deeper and deeper into the sclerotome, while apparently avoiding the notochord.
This pattern probably reflects the migratory pathway which neural crest cells take
through the sclerotome. Neural crest cells are evenly spread along the dorsal
aspect of the neural tube, but more ventrally they become progressively localized
to the rostral half of the somite, until, around the area where the ventral root
emerges, all the crest cells are restricted to the rostral halves of the somites
(see Figs 2, 3).
About 18 somites anterior to the most recently formed somite, the somite
becomes split morphologically into two halves, separated by a small cleft (von
Ebner's fissure). Slightly before this, about 12 to 15 somites rostral to the most
recently formed somite, the first nerve fibres grow out from the ventral aspect of
the neural tube. As they extend through the sclerotome ventrolaterally towards
the myotome they grow through an environment occupied by both sclerotomal
and neural crest cells. From this stage onwards, until the latest stages we have
studied, the pattern of neural crest staining alters little. The only significant change
is that many antibody-binding cells associate with the outgrowing nerve fibres.
Having migrated through the somite, the neural crest cells accumulate in the
various structures ventral to it, for instance around either side of the aorta. Here,
however, the crest cells are evenly spread along the axis of the embryo.
The pattern of crest migration is rather different in the somites immediately
caudal to the otic vesicle. Here, as described previously (Le Douarin & Teillet,
1974), a large proportion of the neural crest cells migrate between the ectoderm
and the dermomyotome, although some cells do find their way down the lateral
sides of the neural tube, and into the sclerotome. In addition, at cranial levels the
pattern of crest cells becomes complicated by the craniocaudal migration of vagal
neural crest cells.
Control sections, treated with either control ascites, control whole serum, or
with monoclonal antibodies derived from the same parent myeloma line but with a
different specificity, showed no immunoreactivity comparable to that seen with
HNK-1.
Fig. 2. These four sections are taken from a single somite (about no. 18) of a stage-15
embryo, stained with anti-HNK-1; dorsal is above. The sections are roughly equally
spaced through the somite, which is about 180 jtan in length. (A) is from the caudal part
of the somite. Neural crest cells are seen around the dorsal aspect of the neural tube,
and ventral to the sclerotome, but none are seen in between. In (B), the picture is
similar, but there are a few neural crest cells around the region where the ventral root
nerve fibres leave the neural tube, and also, on the right side, a few cells around the
edge of the sclerotome. (C) is taken slightly rostral to the middle of the somite. Neural
crest cells here are present in the lateral part of the sclerotome in larger numbers, and
in (D), which is taken from the rostral part of the somite, there are many crest cells
intermixed with the sclerotomal cells. Note that the restriction of crest cells to the
rostral half of the somite applies only to the region of the sclerotome; crest cells are
evenly spread all along the axis of the embryo both at the dorsum of the neural tube,
and ventral to the somite. Some crest cells can be seen migrating around the aorta. Bar
equals 100 jum.
Neural crest migration and axon outgrowth
443
Embryos with neural crest ablations, stained with NC-1 and HNK-1
These animals were operated on between Hamburger & Hamilton stages 11 to
13 (which corresponds to between 13 and 19 somites), and were killed between
stages 18 and 21, approximately 24 h later. By this time motor nerve fibres have
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M. RICKMANN, J. W. FAWCETT AND R. J. KEYNES
grown out of the neural tube along most of its length. At the time when they were
killed, the embryos looked almost normal, except that many of them had a degree
of kinking of the neural tube around the region of the operation, and usually a
length of neural tube that had not fully closed dorsally.
Antibody staining of horizontal sections of the embryos revealed that crest cells
had migrated longitudinally from the edge of the ablated region so as to fill in
partially the area from which neural crest had been removed. Thus, although the
original operation had removed the dorsal half of the neural tube over a distance
of more than 8 somites, dorsal root ganglia were only completely absent over a
length of 2 to 4 somites, and often only 1 or 2 somites were entirely free of crest
cells. In all we produced nine embryos with successful crest ablations, and these
had between 1 and 4 somites which were both completely clear of crest cells, and
also contained motor axons. These were usually surrounded by somites in which
there were very few crest cells. There were also regions in four of the embryos in
which there were no axons growing from the neural tube, usually where the tube
had been largely cut away. In general it was not difficult to identify axons, crest
cells and presumptive Schwann cells, since the crest cells have a very distinctive,
irregular shape, whereas axons, and the cells surrounding them, have a smooth
outline.
The motor axons were always found in the rostral half of the somite, even in
somites which were entirely free of crest cells (see Fig. 4). They appeared to be
following an entirely normal pathway, which, in the region of the somites, is
virtually identical to the migration pathway of the neural crest cells. The axons
growing through crest-free somites were not entirely naked. From the neural tube
to approximately 50 ^m behind their growing tips they were encased by cells with a
smooth outline, which showed NC-1 and HNK-1 immunoreactivity. These are
almost certainly presumptive Schwann cells, which are stained by both antibodies
in normal animals. We think that these cells probably migrated out from the neural
tube and down the nerve fibres, as described in previous publications (Harrison,
1924; Yntema & Hammond, 1954; and see Horstadius, 1950). Certainly, we could
see no neural crest cells migrating from the dorsal aspect of the neural tube to the
Fig. 3. Horizontal section of a stage-17 embryo, stained with anti-HNK-1. Rostral is to
the left, and the leftmost somite is two somites caudal to the otic vesicle. The section is
taken at the level of the notochord, which can be seen running between the somites
over most of the section. The more rostral somites are clearly divided in two, and the
HNK-1 staining (neural crest cells and nerve fibres) is restricted to the more rostral of
the two halves. More caudally, the plane of section goes through the neural tube, and
here the ventral roots can be clearly seen. Bar equals 200 /an. (B) This is a section from
the same embryo as in (A), but the section is more dorsal, so as to cut through the
neural tube about half way through it. The plane of section is slightly off the horizontal,
so the lower side is slightly more dorsal than the upper. Rostral is to the left. Neural
crest cells are almost evenly distributed along the axis of the animal dorsally, but start
to form into segmental groups more ventrally. Bar equals 200 jum. (C) A horizontal
semithin section from a stage-18 embryo taken just ventral to the level of the notochord. The neural crest cells are clearly restricted to the rostral half of each somite.
Crest cells are intermingled with unstained cells. Bar equals 100 [jm.
Neural crest migration and axon outgrowth
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M. RlCKMANN, J. W. FAWCETT AND R. J. KEYNES
ventral root axons. The growth cones in crest-free somites were clearly naked, and
not in contact with any antibody-staining cells; we saw growth cones in the
sclerotome, and on the myotome. The fibres looked essentially normal, but we
formed the impression that the growth cones were rather more spread out in crestfree somites than in somites which contained crest cells. The axons in operated
somites were generally slightly retarded in their development (see Fig. 5). In
several pairs of somites, there were no crest cells on one side and an incomplete
B
Neural crest migration and axon outgrowth
447
crest ablation on the other; here the nerve fibres appeared to have grown slightly
further from the neural tube in the somite which still contained some crest cells.
The somites adjoining those which were completely free of neural crest cells often
had only a very small number of crest cells in them, and no dorsal root ganglion. In
these somites, the crest cells were generally closely associated with the nerve
fibres.
Fig. 5. This is a transverse section taken from an animal which was operated on to
remove its neural crest. All the neural crest was successfully removed from the right
side, but some remained on the left. On the left, crest cells can be seen migrating along
their normal pathway through the somite. On the right, there are no crest cells, and the
nervefibresgrowing out from the ventral aspect of the cord are seen. Bar equals 50 /«n.
Fig. 4. (A) Horizontal section from an embryo which had a neural crest ablation at the
15-somite stage, was killed at stage 19, and stained with anti-NC-1. Only the middle
somite on either side is entirely free of neural crest cells; the surrounding somites have
small numbers of stained cells within them (arrowed). Some erythrocytes have also
shown up because they contain an endogenous peroxidase. The pattern of outgrowth
of the ventral root nerve fibres is essentially normal in the crest-free somites, being
restricted to the rostral half of the somite. The nervefibresappear thick in this picture,
because they are surrounded by sheath cells proximally, which stain with anti-NC-1.
Bar equals l/im. (B) A horizontal section from another crest-ablated embryo. The
neural tube is at the lower edge of the frame. The left-hand somite is entirely crest free,
and the nerve fibres are all in its rostral half. The plane of focus is through the tips of
the axons, which are not covered by sheath cells. The somite on the right has one
neural crest cell in the plane of focus (arrowed). Bar equals 50jum. (C) Transverse
section through the rostral half of a somite from an animal with a neural crest ablation.
Only half of the cross-section is included in this picture. The nerve fibres are clearly
shown growing ventrolaterally through the crest-free somite, taking much the same
pathway as neural crest cells would normally follow. There are no crest cells in this
somite migrating down from the dorsal aspect of the neural tube. Bar equals 50 fjm.
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M. RICKMANN, J. W. FAWCETT AND R. J. KEYNES
Staining for fibronectin
Our results on the distribution of fibronectin are essentially in agreement with
previous studies (Newgreen & Thiery, 1980; Mayer et al 1981; Duband & Thiery,
19826). Fibronectin is present at the earliest developmental stage we studied. The
molecule is rather ubiquitous in its distribution, but initially it is concentrated in
the extracellular spaces between structures; thus there is a high concentration of
fibronectin around the neural tube, and around the somites. Later, there is also
fibronectin staining within the somite, throughout the sclerotome, although its
concentration always remains highest around the neural tube, notochord, and
between the somites (see Fig. 6). At no time is there any difference in staining
between the rostral and caudal halves of the somite.
Staining for laminin
The amount of laminin in the youngest embryos which we examined is probably
rather low, since we were only able to see any staining by using high concentrations of anti-laminin, which resulted in a certain amount of non-specific background antibody binding. Up to the stage when the myotome begins to
differentiate, the distribution of laminin present resembles that of fibronectin. The
molecule is mainly found in the extracellular space around the neural tube, and
between the somites (see Fig. 7A,B). In later stage embryos, from stage 17
onwards, strong laminin staining is seen in three main regions: around the
developing myotome, around motor axons, and around the dorsal root ganglia
(see Fig. 7B,C). The staining associated with axons does not extend up to their
growing tips, and only seems to appear as they mature. We think that this staining
is not associated with the axons themselves, but is on the surfaces of differentiating
Schwann cells. Certainly adult Schwann cells secrete laminin and other basal
lamina components (Cornbrooks etal. 1982; Cornbrooks etal. 1983). The laminin
staining associated with the dorsal root ganglia appears late, and is not really
marked until stages 22-26, the oldest embryonic stages at which we have
performed laminin immunohistochemistry. The laminin is found only in the
capsule of the ganglion, and associated with the sensory nerve fibres. The myotomal cells appear to produce laminin, resulting in clear longitudinally oriented
tubes of reaction product. Outgrowing motor axons come into contact with the
myotome, but laminin seems to appear in large quantities at the myotome cell
surfaces some time after the first growth cones have reached them, The myoblasts
migrating from the myotome into the limb (Chevallier, Kieny & Mauger, 1977) do
not stain for laminin, and neither do any other cells in the path of the first nerve
fibres growing into the limb.
DISCUSSION
In the chick embryo, truncal neural crest cells are first detectable on the dorsal
aspect of the neural tube about three or four somites rostral to the region where
somites begin to differentiate from the segmental plate. Here they pause briefly,
Neural crest migration and axon outgrowth
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Fig. 6. These illustrations show the pattern offibronectinstaining in the chick embryo.
(A) is from a stage-16 embryo stained with anti-fibronectin antibody. The strongest
staining is seen around the neural tube, notochord, and around the dermomyotome,
particularly laterally. There is also less intense fibronectin staining throughout the
sclerotome The neural tube and dermomyotome are unstained. Bar equals 50/an.
(B,C) are photomicrographs of the same section, taken at different planes of focus.
The embryo was fixed at stage 15, and this is about the 10th somite rostral to the most
recently formed somite; the somite is younger than that illustrated in (A), since it has
not yet fully dissociated into sclerotome and dermomyotome. In (B) the plane of focus
goes through the intersomitic cleft, showing the intense reticular staining for fibronectin here. In (C) the plane of focus is on the centre of the somite; there is dark
staining around the somite, but little inside it. Bars equal 100/an.
449
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M. RlCKMANN, J. W. FAWCETT AND R. J. KEYNES
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Fig. 7. These pictures illustrate the pattern of anti-laminin staining at different stages
of somitic development. (A) is a horizontal section from a stage-13 embryo, around
somite number 8. The staining pattern is very similar to that of fibronectin at this stage,
being concentrated around the somites and neural tube. Bar equals 100/mi. (B) is a
transverse section from a stage-17 embryo. There is intense laminin staining around the
neural tube and the dermomyotome, and faint staining throughout the rest of the
somite. However, there is no staining at all in the neural tube. Bar equals 50/mi. (C)
This is a horizontal section from a stage-26 embryo, taken through the level of the
developing dorsal root ganglia. Two ganglia are surrounded by laminin staining, as are
fascicles of axons going leftward towards the neural tube. To the right of the picture,
the myotomes are darkly stained. Bar equals 100 jian. (D) A section from the same
embryo, through the level of the ventral roots. The ventral root axons, surrounded by
anti-laminin-stained sheath cells, are coming out of the neural tube on the left of the
frame, the outside of which is also stained. The heavily stained myotome is on the
right. Bar equals 50/an.
Neural crest migration and axon outgrowth
451
before beginning to migrate ventrally and ventrolaterally. Their subsequent migration takes them almost throughout the body to parent all the neuronal and glial
cells of the autonomic and sensory ganglia, the adrenal medullary cells, the
Schwann cells, and the melanocytes of the skin (reviewed by Le Douarin, 1982).
All the crest cells which migrate from the dorsal aspect of the neural tube, except
for the progenitors of the melanocytes, have to pass down the lateral aspect of the
neural tube, and then through the region of the somites. Our findings on the
earliest migratory pathway of neural crest cells through the somites are similar to
those reported in previous publications on the subject (Weston, 1963; Le Douarin
& Teillet, 1974; Thiery, Duband & Delouvee, 1982; Vincent & Thiery, 1984). We
find that the very first crest cells to migrate ventrally, which are few in number,
take a pathway down either side of the neural tube, and then go around the
somites, either medially, laterally, or between them. However, shortly after this
first phase of crest migration, the neural crest cells start to migrate between the
dermomyotome and sclerotome, and through the sclerotome itself, in which
regions they are restricted to the rostral half of each somite. They then progressively populate the rostral half of the sclerotome. The specific restriction of the
migration of neural crest cells to the rostral half of the somite has not been noted
by previous authors. There has been some controversy in the past as to the extent
to which migrating crest cells pass through the sclerotome. On this point our
results are in general agreement with those of Weston (1963), who described
neural crest cells migrating through the sclerotome, but are somewhat at variance
with those of Thiery and others, who have suggested that most neural crest cells
migrate around the somite, circumventing the sclerotome. However, crest cells are
only seen in the more lateral regions of the sclerotome; they never come close to
the notochord, and in fact neural crest cells in culture seem to be repelled by
explanted notochord (Newgreen, Scheel & Kastner, 1985).
A few hours after neural crest migration has begun, the first motor nerves begin
to grow out of the neural tube, and through the sclerotome of the somite towards
their targets. The motor nerves do not grow out evenly along the length of the
neural tube, but instead emerge opposite the rostral half of each somite, and it is
specifically through this half that they grow (Keynes & Stern, 1984). At the time
when the first axons grow out from the neural tube, there are many crest cells
around the lateral aspect of the tube, aligned with the rostral half of the somite, in
the sclerotome.
Since the distribution of crest cells so exactly matches the pathway of motor
axon growth, we were obviously interested to find out if there is any causal
relationship between the two; are the axons guided specifically through the rostral
half of the somite by neural crest cells? In order to test this hypothesis, we
operated on a series of chick embryos to remove their neural crest. We allowed
these embryos to survive to a stage when motor axon outgrowth is well under way
in normal animals, and then stained them with HNK-1 or NC-1, in order to
demonstrate both the crest cells and the axons. We saw several somites which were
quite free of crest cells, but through which motor nerve fibres were growing. These
452
M. RICKMANN, J. W. FAWCETT AND R. J. KEYNES
motor axons were always restricted to the rostral half of the somite. The presence
of crest cells is not, therefore, a prerequisite for this specific axon outgrowth.
However, this is not to say that there is no interaction between nerve fibres and
crest cells. In somites in which the crest removal was incomplete, the few
remaining crest cells tended to be closely associated with the axons, indicating that
axons and crest cells may be mutually adherent. Moreover, the ventral root axons
in crest-ablated segments seemed to be growing more slowly than those in somites
which contained crest cells. One cannot be sure that this was not simply due to
non-specific effects of the operation, but it could be that the outgrowing axons
often do adhere to neural crest cells, and this hastens their outgrowth. However,
whatever the possible interactions of presumptive motor axons and neural crest
cells, it seems very likely that both crest cells and axons are able to respond to the
same cues in the somitic environment; there may be one or more molecules which
are distributed differently in the two halves of the somite and which are attractive
or repulsive to both axons and crest cells.
The antigen recognized by HNK-1 antibodies is found on a family of related
molecules, among which are myelin-associated glycoprotein, LI and N-CAM
(Kruse etal. 1984), and Jl (Kruse etal. 1985). However, in the published studies of
the immunohistochemical localization of N-CAM (Thiery, Duband, Rutishauser
& Edelman, 1982) there is no evidence of any specific staining of neural crest cells.
Moreover, since there was no differential distribution of HNK-1 immunoreactivity
in the sclerotome during the migration of neural crest cells, it appears unlikely that
any molecule on the sclerotomal cells bearing the HNK-1 epitope determines the
path of neural crest cells or motor axons through the rostral half-sclerotome. In
recent years a good deal of evidence has accumulated to show that both neural
crest cells and growing axons can have their pathways and rates of growth
influenced by basal lamina components. The time of onset of neural crest migration closely follows the time of appearance of fibronectin at high concentration
in the migratory pathway (Newgreen & Thiery, 1980; Mayer etal. 1981; Duband &
Thiery, 1982/?; Mitrani & Farberov, 1982; Duband & Thiery, 1982a), and neural
crest cells in tissue culture migrate particularly well on fibronectin, an effect which
is blocked by anti-fibronectin antibodies (Rovasio et al. 1982). Moreover, latex
microspheres, which will normally migrate with crest cells when injected into their
pathway, will not migrate when coated with fibronectin or laminin (neural crest
cells themselves are fibronectin and laminin negative) (Bonner-Fraser, 1982).
Fibronectin, therefore, may well play at least a permissive role in the control of
neural crest migration. Nerve fibres grow very well in vivo on basal lamina
(McMahan, Edgington & Kuffler, 1980; Keynes, Hopkins & Huang, 1984; Scherer
& Easter, 1984; Fawcett & Keynes, 1985), and in vitro both fibronectin and laminin
are good growth substrates for many types of axon (Rogers et al. 1983; Manthorpe
etal. 1983; Edgar, Timpl & Thoenen, 1984; Lander, Fujii & Reichardt, 1985). We
therefore stained a series of chick embryos with anti-fibronectin and anti-laminin
antibodies, to see whether either molecule was restricted to the rostral half of the
somite. However, it appears that both are evenly distributed between rostral and
Neural crest migration and axon outgrowth
453
caudal halves. Fibronectin is present in large amounts around the sclerotome, and
also in smaller amounts within it, at the times when crest cells migrate and motor
nerve fibres grow out; whereas laminin is initially distributed around and between
the somites, and is subsequently associated with the presumptive Schwann cells
and with the developing myotome. Certainly, neither laminin nor fibronectin
shows any preferential localization in the rostral half of the somite. Our feeling,
therefore, is that fibronectin, and possibly laminin, may need to be present in
order for crest cells to migrate normally; but since both fibronectin and laminin are
equally distributed in the rostral and caudal halves of the somite (as is N-CAM,
another possible candidate for guiding axons and crest cells (Thiery et al. 1982)),
there must be one or more other factors present which guide axons and crest cells
specifically through the rostral half-somite.
We are indebted to Dr Jean-Paul Thiery for his gift of anti-NC-1 antibody. We would like to
thank Steve Pfeiffer and Jim Rokos for their skilled technical assistance. We also thank Dr
Marianne Bronner-Fraser for her help and encouragement, and Dr W. M. Cowan for his
support, and his many helpful suggestions on the manuscript. R.J.K. was in receipt of a
Wellcome Trust travel grant when this work was done.
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(Accepted 24 September 1985)