Download Control of pathfinding by the avian trunk neural crest

63
Development 103 Supplement, 63-80 (1988)
Printed in Grcal Britain © The Company of Biologists Limited 1988
Control of pathfinding by the avian trunk neural crest
CAROL A. ERICKSON
Department of Zoology, University of California, Davis, CA 95616, USA
Summary
We have determined the pathways taken by the trunk
neural crest of quail and examined the parameters
that control these patterns of dispersion. Using antibodies that recognize migratory neural crest cells
(HNK-1), we have found that the crest cells take three
primary pathways: (1) between the ectoderm and
somites, (2) within the intersomitic space and (3)
through the anterior somite along the basal surface of
the myotome.
The parameters controlling dispersion patterns of
neural crest cells are several. The pathways are filled
with at least two adhesive molecules, laminin and
fibronectin, to which neural crest cells adhere tenaciously in culture. The pattern of migration through
the somite may be accounted for in part by the
precocious development of the basal lamina of the
dermamyotome in the anterior half of the somite; this
basal lamina contains both fibronectin and laminin
and the neural crest cells prefer to migrate on it. In
contrast, the regions into which the crest cells do not
invade are filled with relatively nonadhesive molecules
such as chondroitin sulphate. Some of the pathways
are filled with hyaluronic acid, which stimulates the
migration of neural crest cells when they are cultured
in three-dimensional gels, presumably by opening
spaces. Neural crest cells are also constrained to stay
within the pathways by basal laminae, which act as
barriers and through which crest cells do not go.
Therefore, crest pathways are probably defined by
several redundant factors.
The directionality of crest cell migration is probably
due to contact inhibition, which can be demonstrated
in tissue culture. Various grafting experiments have
suggested that chemotaxis and haptotaxis do not play a
role in controlling the dispersion of the crest cells away
from the neural tube.
We have documented the extraordinary ability of
neural crest cells to disperse in the embryo, even when
they are grafted into sites in which they would
normally not migrate. We have evidence that the cells'
production of plasminogen activator, a proteolytic
enzyme, and also the minimal tractional force that
crest cells exert on the substratum as they migrate,
contribute to this migratory ability.
Pathways of migration
the pathways of migration is contingent upon being
able to mark the crest cells in some unambiguous
way.
The earliest studies of trunk crest migration in the
chick that attempted to trace the paths of migration
employed [3H]thymidine as a marker for neural crest
cells (Weston, 1963). Chick embryos were incubated
for 24 h in [3H]thymidine and the labelled neural
tubes excised and grafted into an unlabelled host in
place of its neural tube. The labelled neural crest cells
then migrated, presumably along their normal pathways and could be distinguished from the unlabelled
host tissue. For the most part, Weston's study
mapped the final distribution of crest cells 2-3 days
after they had initiated their migration. Crest cells
If we are to understand what controls the pattern of
crest cell migration, we first must know in detail what
pathways they take so as to examine what physical
and biochemical parameters are associated with these
pathways.
Elucidating the crest cell migratory patterns has not
been an easy matter. Initially the crest cells can be
distinguished from other embryonic cells because
they arise from a localized region, the dorsal side of
the neural tube, and migrate into an extracellular
space. However, as they disperse they mix with the
paraxial mesoderm and then can no longer be distinguished from other embryonic cells. Thus, tracing
Key words: neural crest, pathfinding, extracellular matrix,
chemotaxis, haptotaxis, galvanotaxis.
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C. A. Erickson
were observed (i) in the ectoderm, (ii) aggregated
into what were presumed to be the sensory and
sympathetic ganglia and (iii) dispersed in the somites.
Weston also examined some early stages of crest
cell migration (20h after grafting), but he made no
attempt to trace their precise pathways. The major
conclusion from examining these younger embryos
was that crest migration was enhanced through the
somites. Although not noted at the time, some of his
figures clearly show that the predominant migration
was through the anterior somite (note Fig. 3B in
Weston, 1963). Hindsight has undoubtedly enhanced
our ability to detect such subtleties.
Weston's study and others that were to follow
(Noden, 1975; Johnston, 1966), although important,
had a number of shortcomings. (1) In most early
studies employing grafting of a labelled neural tube,
researchers usually waited at least 20 h for the graft to
heal before fixing and looking for the distribution of
labelled cells. Thus, the very earliest stages of migration were not examined. In the case of the trunk
crest, this turns out to be a critical omission. (2) We
cannot be certain that all the crest cells are initially
labelled with a metabolic marker such as [3H]thymidine. Furthermore, [3H]thymidine becomes diluted
as the cells divide, so its usefulness as a marker can be
short-lived. In the case of the neural crest cells, we do
not know how much they divide as they disperse. (3)
In studies that employ grafting of labelled neural
tubes, the operation itself can disrupt normal tissue
associations at the very time that the crest cells are
invading the paraxial mesoderm. The possibility that
this could alter the normal patterns of migration is
great.
The problem of dilution of the label was circumvented by Le Douarin (1973) who pioneered a permanent marking technique for the crest cells. She
observed that the DNA of the closely related chick
and quail stain differentially with Feulgen. Thus she
could replace a chick neural tube with that of a quail,
allow the crest cells to migrate from the tube, and
then section and stain the chimaeric embryo. The
nuclei of the quail crest cells can be distinguished
from those of the chick host because of their darker,
punctate staining. Using this technique, she confirmed what was known about migratory routes from
Weston's study (Le Douarin & Teillet, 1974).
A significant advance in our determination of crest
cell migratory pathways was the development of a
monoclonal antibody that recognizes migratory
neural crest cells (Vincent, Duband & Thiery, 1983;
Vincent & Thiery, 1984; Tucker et al. 1984). This
antibody has enhanced our ability to identify neural
crest cells, since fluorescently labelled antibodies are
easier to visualize than quail nuclei against a background of chick cells. Indeed, the distribution of crest
cells becomes unmistakable. Furthermore, this technique requires no grafting, so that it is a noninvasive
method that does not perturb normal development in
any way.
Several laboratories have used the NC-1 antibody
to trace the paths of crest migration in the trunk of the
chick or quail embryo (Rickmann, Fawcett &
Keynes, 1985; Bronner-Fraser, 1985; Loring& Erickson, 1987; Teillet, Kalcheim & Le Douarin, 1987). All
of these studies are in agreement about several
points. (1) The neural crest cells migrate in an
anterior-to-posterior wave beginning in the head. (2)
As the crest cells migrate off the neural tube and
approach a neighbouring somite, they invade the
somite, but only in its anterior half (Fig. I). (3) Some
crest cells collect in the dorsomedial sector of the
sclerotome to form the sensory ganglia, whereas
others migrate through the somite as far as the dorsal
aorta, where they aggregate to form the paravertebral
sympathetic ganglia.
There is still some disagreement about the precise
pathway taken by the crest through the somite. Some
reports suggest that the crest invades throughout
much of the sclerotome in the anterior somite (Rickmann et al. 1985; Bronner-Fraser, 1985). We found,
however, that the crest cells first enter the somite at
the interface between the dermamyotome and sclerotome and migrate along this interface until they reach
the dorsal aorta (Fig. IB; Loring & Erickson, 1987).
Only later are crest cells found in the sclerotome.
These later-appearing crest cells may detach from the
dermamyotome and entering the sclerotome, or may
be intersomitic crest cells (see below) around whom
the sclerotome has dispersed. Finally, other neural
crest cells may enter the sclerotome by migrating
along the ventral root motor fibres that also course
through the anterior somite (see below).
Some of the crest cells migrate between the neural
tube and somite opposite the posterior half of a
somite. What happens to these cells? In a recent
series of elegant grafting experiments, Teillet & coworkers (1987) have shown that these crest cells
migrate either anteriorly or posteriorly along the
neural tube until they arrive at the anterior side of
their own, or the adjacent caudal somite, and then
enter the somite at its anterior aspect.
An additional pathway taken by other crest cells is
an incursion into the intersomitic spaces (Loring &
Erickson, 1987; Teillet et al. 1987; Thiery, Duband &
Delouve'e, 1982). These crest cells are suspected to
reach the dorsal aorta to become part of the paravertebral sympathetic chain. Alternatively some of the
cells in the intersomitic space may mix with crest cells
in the anterior somite as the sclerotome disperses and
obliterates the intersomitic space.
Finally, some crest cells invade the space between
Control of pathfinding by the avian trunk neural crest
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Fig. 1. (A, B). A series of paraffin sections through somite - 9 (i.e. 9 somites anterior to the last-formed somite) of a
30-somite embryo. The sections were incubated with the antibody NHK-1 to identify the neural crest cells. In a section
taken from the posterior portion of this somite (A), the crest cells are not found in the somite. Sections taken through
the anterior portion of the same somite show the neural crest cells spread along the interface between the
dermamyotome (DM) and the sclerotome (Sc). NT, neural tube, NC, neural crest, DA, dorsal aorta, PCV, posterior
cardinal vein, PD, pronephric duct. Bar, 100 ^m. (C). Whole mount preparation of a 28-somite chick embryo labelled
with the HNK-1 antibody photographed at an axial level where neural crest cell migration into the somite is well
underway. The anterior border of each somite is marked by an arrowhead and the anterior-posterior axis is noted. The
crest cells are found only in the anterior half of each somite and are generally aligned in parallel. Bar, 100 jim. (From
Loring & Erickson, 1987).
the somites and ectoderm and ultimately invade the
ectoderm between day 5 and 6 to become the pigment
cells of the skin (Teillet & Le Douarin, 1970; Teillet,
1971). It has never been determined how far laterally
the pigment cell precursors move before they actually
invade the ectoderm. Weston (1963) has suggested
that the crest cells enter the ectoderm just above the
neural tube soon after they begin to migrate. His
results may be due to an artifact of the grafting
technique, since the crest cells may prematurely enter
a breach in the ectoderm made during surgery.
In summary, trunk crest cells migrate (1) laterally
in an extracellular space to form the pigment cells of
the skin, (2) ventrally through an extracellular space
above and alongside the neural tube until the crest
cells enter the anterior portion of the somite and (3)
in the intersomitic space between adjacent somites.
This initial pattern of migration, which limits crest
invasion to the anterior half of each somite, appears
to be responsible for the metameric arrangement of
the sensory and sympathetic ganglia.
I have not considered in this section the pathways
of head crest migration in the chick, since less is
known about the precise pathways taken by these
cells. The studies of Noden (1975,1978) and Johnston
(1966) employing [3H]thymidine or chick-quail chimaeras as markers for the crest may be inaccurate for
the reasons outlined above and should be reexamined
using the more recent antibody markers.
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C. A. Erickson
What, then, are the various factors that determine
these pathways?
Control of pathways
One could conceive of two very different methods of
controlling the migratory pathways taken by neural
crest cells. Either (1) each crest cell is predetermined
to develop into a particular phenotype and it follows
particular cues to get to the correct site or (2) the crest
cells are pluripotential and all crest cells follow the
same cues; those crest cells that migrate first and get
the farthest become the most distal crest derivatives.
In their classic experiments, Le Douarin & Teillet
(1974) showed that trunk crest cells grafted from one
axial level to another follow the correct pathways for
that axial level. Such experiments suggest that there
are predetermined pathways that the population as a
whole follows. Similarly, Noden (1975) exchanged
crest cells between various levels of the brain and
found that the pattern of crest cell migration
depended upon the environment through which the
grafted cells dispersed.
At present, no evidence supports the notion that
predetermined crest cells follow their own specific
migratory cues. This is not to say that some crest cells
are not predetermined prior to, or soon after, they
initiate migration, since clearly some are (see Barald,
1982; Ciment & Weston, 1982; Girdlestone & Weston, 1985; Barbu, Ziller, Rong & Le Douarin, 1986).
But even in the head crest, where crest cells that will
give rise to skeletal elements are clearly patterned
prior to migration (Noden, 1984), heterotopically
grafted cephalic crest cells will still follow the stereotyped pathways for the axial level in which they have
been grafted, even though ectopic or misplaced
skeletal elements form.
The preponderance of evidence indicates that the
environment is primarily responsible for determining
the patterns of migration. Environmental factors
believed to dictate the migratory pathways are (1)
extracellular spaces that provide a passage way of
least resistance, (2) an adhesive substratum on which
the cells prefer to migrate, bordered by less adhesive
areas into which the crest cells will not go and (3)
basal laminae that may act as barriers and constrain
crest cell migration within certain boundaries. Several or all of these are likely to operate at any one
time to provide redundant cues that assure that these
very important cells will arrive at their appropriate
destination.
Extracellular spaces
Initially crest cells do not invade other embryonic
tissues, but rather they migrate within extracellular
spaces. They migrate in the space between the
ectoderm and somites to become pigment cells of the
skin or they migrate ventrally in the intersomitic
space between somites. The role of spaces is particularly dramatic in the head, where streams of crest
cellsflowaround barriers created by other embryonic
structures. For example, the metencephalic crest cells
migrate laterally and then split into preotic and
postotic streams when they are obstructed by the
invaginating otic placode (Anderson & Meier, 1981).
The anterior mesencephalic crest and diencephalic
crest migrate around the developing optic stalk but
not over the top of it (Noden, 1975), presumably
because of the tight apposition of the overlying
ectoderm.
Most of the spaces into which the crest cells migrate
are filled with the glycosaminoglycan hyaluronic acid
(Toole & Trelstad, 1971; Pratt, Larsen & Johnson,
1975; Derby, 1978). This is a hydrophilic molecule
that expands in water owing to the high density of
negative charges that prevent its compaction. Hyaluronic acid (HA) is responsible, at least in part, for the
development of many spaces in the embryo since it
appears concomitant with spaces and the removal of
HA with hyaluronidase collapses the spaces (Anderson & Meier, 1982; Fisher & Solursh, 1977).
The role of HA in facilitating crest migration is
supported by the observation that when HA is added
to collagen gels, it increases the spaces between
collagen fibrils and increases the speed of movement
of neural crest cells embedded in the gels (Tucker &
Erickson, 1984).
Although spaces are undoubtedly important factors in guiding neural crest cells, the following points
should be kept in mind. (I) Spaces may appear
concomitant with extracellular matrix molecules that
are necessary for sustaining neural crest cell migration. Thus, the stimulus to migration may not be
the space, but rather the appearance of an adhesive
molecule on which to move. (2) Crest cells possibly
do not move into some areas because inhibitory
molecules there prevent crest migration and not
because the space is too small. (3) Finally, crest cells
can get into very small spaces, if given the appropriate substratum on which to migrate. For example, the
space between the dermamyotome and sclerotome is
not large, but crest cells still use this as the primary
path of migration. Thus, it is not clear how large a
migratory 'space' needs to be for a crest cell.
Extracellular matrix substratum
Spaces may very well facilitate migration, but they
are not sufficient, since the cells must be able to
adhere to a substratum in order to generate the
tractional force to move.
Several types of extracellular matrix molecules that
Control of pathfinding by the avion trunk neural crest
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Fig. 2. Frozen sections through somite —9 of a 25-somite embryo stained with an antibody to laminin, which identifies
the distribution of basement membranes in the developing somite. In the posterior portion of the somite a still intact
basement membrane borders the medial edge of the somite, whereas in the anterior portion this medial basement
membrane has begun to break down and a second basement membrane is being deposited (arrowheads) beneath the
developing dermamyotome (DM). NT, neural tube; Sc, sclerotome; N, notochord.
might facilitate crest cell adhesion have already been
localized in the crest migratory pathways. These
include fibronectin (Newgreen & Thiery, 1980;
Thiery et al. 1982; Mayer, Hay & Hynes, 1981),
laminin (Rickmann et al. 1985; Rogers, Edson,
Letourneau & McLoon, 1986), collagen I and III (von
der Mark et al. 1976; our unpublished results),
cytotactin (Grumet, Hoffman, Crossin & Edelman,
1985) and the glycosaminoglycans hyaluronic acid
and chondroitin sulphate (Derby, 1978; Pintar, 1978;
Kvist & Finnegan, 1970). Furthermore, the distribution of fibronectin and laminin is temporally and
spatially correlated with the migration of the crest
cells through the anterior somite. These molecules
appear in the basal lamina of the dermamyotome on
which the crest cells appear to move. Basal lamina
develops first, however, in the anterior somite
(Fig. 2; Loring & Erickson, 1987), the site of initial
crest migration.
Although the presence of these various molecules
is spatially and temporally correlated with crest
migratory pathways, such correlative evidence does
not predict the behaviour of the cells on particular
matrix molecules.
An approach used by our laboratory and others to
study crest cell behaviour on extracellular matrix
(ECM) molecules is to culture neural crest cells on
tissue culture plastic to which individual or combinations of known ECM components have been
adsorbed or conjugated (Fig. 3). Such studies have
shown that crest cells flatten and spread more on
fibronectin and laminin than on any other ECM
component yet tested (Newgreen et al. 1982; Rovasio
et al. 1983; Erickson & Turley, 1983; Newgreen,
1984). In fact, when 'lanes' of either fibronectin
(Rovasio et al. 1983) or laminin (Newgreen, 1984) are
painted on a tissue culture plastic dish, the crest cells
will stay on the fibronectin or laminin lanes and not
venture onto the less-adhesive plastic. Furthermore,
the crest cells migrate faster and are more directionally persistent on fibronectin than any other ECM
molecule tested (Erickson & Turley, 1983).
Crest cells also adhere to, and migrate on, denatured collagen in vitro (Newgreen, 1982; Erickson
& Turley, 1983) as well as through native collagen
gels (Davis, 1980; Tucker & Erickson, 1984).
The glycosaminoglycans, on the other hand, are
either nonadhesive or lowly adhesive for trunk neural
crests. Crest cells are unable to adhere to hyaluronic
acid (Fisher & Solursh, 1979; Erickson & Turley,
1983), but, as mentioned above, hyaluronic acid can
enhance neural crest cell migration when it is added
in low concentration to collagen gels (Tucker &
Erickson, 1984). Thus hyaluronic acid may function
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C. A. Erickson
Fig. 3. Neural crest cells grown for 18 h on individual matrix components that have been adsorbed or derivatized to the
plastic substratum with no serum or embryo extract in the medium. The neural tube, from which the neural crest cells
have immigrated, is located at the margin of each micrograph. (A) Neural crest cells on tissue culture plastic. This is a
relatively less adhesive substratum than others such as fibronectin and the cells are somewhat rounded. (B) Neural crest
cells grown in medium containing 25 ^g fibronectin (FN). The cells are considerably flatter and have dispersed further
away from the neural tube than when grown on plastic alone. (C) Neural crest cells grown on chondroitin sulphate (CS)
that has been derivatized to a tissue-culture plastic substratum. Cell morphology and behaviour is similar to that on
plastic alone. (D) Only a few neural crest cells have migrated from the neural tube when hyaluronic acid (HA) has been
derivatized to a plastic substratum. x990. (From Erickson & Turley, 1983).
not as a substratum for migration, but as a facilitator
of migration by opening spaces.
Chondroitin sulphate and chondroitin sulphate
proteoglycan (CSPG) when tested alone or in combination with other ECM molecules, reduce spreading
and adhesiveness of neural crest cells to their substratum (Newgreen et al. 1982; Newgreen, 1982;
Erickson & Turley, 1983; Tucker & Erickson, 1984).
Interestingly, regions that are avoided by the crest in
the trunk (Newgreen et al. 1982) and the head
(Brauer, Bolender & Markwald, 1985) are high in
CSPG. Newgreen, Scheel & Kastner (1986) have
direct evidence from tissue culture studies that the
CSPG present in the perinotochordal space is probably responsible for inhibition of crest invasion into
that region. These data suggest that ECM molecules
may function not only to stimulate neural crest
migration, but also to inhibit it. Thus, an effective
means of controlling the pathways of migration may
be to prevent the cells from invading into regions
adjacent to the pathway (see Morris, 1979).
A recent approach to identifying the role of various
ECM components is to perturb their function by the
injection of specific antibodies or peptides into the
embryo that compete with the binding of cells to a
particular ECM molecule. Such studies have at least
corroborated the conclusions derived from tissue
culture studies (see Tucker, this volume). Boucaut et
al. (1984) injected a decapeptide that competitively
inhibits fibronectin function into the mesencephalon
at the time that neural crest cells in that region initiate
migration and found that morphogenetic movement
ceases. Similarly, Bronner-Fraser (1985) injected the
CSAT antibody, which recognizes a receptor for
fibronectin and laminin, into the mesencephalon and
showed a marked reduction in cranial crest migration.
Control of pathfinding by the avian trunk neural crest
Curiously, none of these injections appears to
affect trunk crest migration as dramatically as in the
head, if at all. This could be because the controls of
crest migration in the trunk are different to those in
the head. Alternatively, because the embryonic architecture is so different in the trunk, the trunk crest may
simply not encounter enough antibody to affect their
migration.
Neurones
Crest cells may also associate with other cell types
that act as migratory pathways. During the early
stages of crest migration in the trunk, a large population of crest cells begin to accumulate in the
dorsomedial corner of the sclerotome. Since dorsal
root motor neurones are also known to grow into the
anterior portion of the sclerotome (Keynes & Stern,
1984; Rogers et al. 1986), they were a logical choice
for directing crest migration into the sclerotome and
perhaps even trapping the presumptive dorsal root
ganglion cells there.
Double-labelling experiments identifying both
neural crest cells and ventral root motor fibres show
that the two are co-distributed (Rickmann et al. 1985;
Loring & Erickson, 1987). Indeed the crest cells
appear enmeshed in the tangle of ventral motor
neurones (Fig. 4). Furthermore, the ventral root
fibres grow into the sclerotome just hours prior to the
time that crest cells accumulate in the sclerotome.
We believe that crest cells adhere to and perhaps
are even arrested in their migration by the neurones.
Of course, neural crest cells and ventral root
neurones could be independently following the same
environmental cue and may not be guided by each
other. In support of a causal relationship, we have
preliminary evidence that removal of the ventral root
neurones prior to their emergence from the neural
tube results in a reduction in size of the crest-derived
sensory ganglia and a loss of neural crest cells along
the ventral root fibres at that axial level (S. A. Scott,
J. F. Loring and C. A. Erickson, unpublished data;
see also Landmesser & Honig, 1986). Conversely,
ablation of neural crest cells prior to their exit from
the neural tube does not affect motor neurone outgrowth (Rickmann et al. 1985). We do not know why
the crest cells might have a stronger affinity to the
neurones than the surrounding environment.
Boundaries
Crest cells may be restrained to particular highways
by adhesive molecules on which they prefer to adhere
and by borders of nonadhesive or less adhesive
molecules, such as chondroitin sulphate proteoglycan, into which they will not go. In addition, most of
the crest pathways are bordered by epithelia that rest
on basal laminae. Such epithelia include the epider-
69
mis, the neural tube, the undersurface of the dermamyotome, a portion of the medial surface of the
somite and the larger embryonic blood vessels such as
the dorsal aorta. It has been suggested that the basal
lamina delimits early crest migration by acting as an
impenetrable barrier (e.g. see Weston, 1982; Erickson & Weston, 1983).
To test this idea, neural crest cells were cultured on
basal laminae derived from placental amnions (see
also Gehlsen & Hendrix, 1987) or grafted into chick
neural tubes or epidermis, where they were forced to
confront a basal lamina. Under no circumstance did a
neural crest cell penetrate a basal lamina (Fig. 5;
Erickson, 1987). Rather, the crest cells flatten and
spread upon contact with the basal lamina, suggesting
that the basal lamina is indeed a barrier to migration.
Alternatively, the basal lamina may represent a
substratum to which crest cells prefer to adhere and
will not detach once they have made contact, since it
is composed of ECM molecules that are particularly
adhesive for the neural crest, notably fibronectin and
laminin (Newgreen, 1984). In either case, the basal
lamina may act as yet another element, perhaps
redundant, in the environment, that determines
boundaries for crest cell migratory pathways.
Control of directionality
Neural crest cells must receive some directional cues
as they migrate, because the crest pathways, as they
have just been described, define position only.
Specifically, some stimulus must direct the neural
crest cells to disperse from their point of origin;
furthermore, once crest cells enter a pathway they
must know whether to turn right or left.
Many mechanisms may impose directional movement on embryonic cells. These include positive and
negative chemotaxis, haptotaxis, galvanotaxis and
contact inhibition. Each of these has been tested for
its possible role in neural crest morphogenesis. Such
studies in toto suggest that contact inhibition is the
most probable control of directional movement.
Haptotaxis and chemotaxis
At least two mechanisms employ gradients that could
direct crest cells laterally and ventrally: these are
haptotaxis and chemotaxis.
Haptotaxis is the ability of cells to move up an
adhesive gradient (Carter, 1967), owing at least in
part to the more rapid detachment from the less
adhesive end of the substratum (Harris, 1973). A
gradient of adhesiveness extending from the neural
tube, both laterally and ventrally, is an attractive
mechanism for directing the crest to lateral and
ventral positions.
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C. A. Erickson
Alternatively, a diffusible chemotactic molecule,
which has its source in either lateral or ventral
positions, might attract crest cells. Chemotaxis has
been implicated in other developmental phenomena,
including the migration of primordial germ cells
(DuBois, 1968) and growing axons (Lumsden &
Davies, 1983; Gundersen & Barrett, 1979).
The presence of a preestablished gradient, such as
the two suggested above, was tested by a series of
grafting experiments (Erickson, 1985). When neural
crest cells were placed either ventrally (Fig. 6) or
laterally (Fig. 7) in the crest migratory pathways, the
grafted cells dispersed radially and some of them
migrated back toward the neural tube. This migration
toward the neural tube ceases when the grafted crest
cells reach an oncoming stream of host crest cells.
Since crest cells can move in the reverse direction
along established migratory pathways, it is probable
that a preestablished gradient of either an adhesive or
chemotactic nature, does not exist in the chick
embryo.
Negative chemotaxis
Negative chemotaxis has been proposed to direct
dispersion of amphibian neural crest cells away from
the neural tube (Twitty, 1949). Studies by Twitty and
his associate Niu (Twitty & Niu, 1948, 1954) suggested that neural crest cells produce a factor that causes
their mutual repulsion when it accumulates in highenough concentrations. They observed, for example,
that if aggregates of neural crest cells were sucked
into a capillary tube and one end sealed, the crest
cells at the sealed end dispersed, whereas those at the
open end did not move. In another experiment, crest
cells grown beneath a coverslip were more dispersed
Fig. 4. Double label with HNK-1
(A,C) and E/C8 (B,D) to identify
neural crest cells and ventral root
fibres, respectively. (A,B) A section
through the anterior portion of
somite 17 of a 30-somite chick
embryo. Neural crest cells are found
along the undersurface of the
dermamyotome as well as in the
dorsomedial portion of the
sclerotome (arrowheads). (A) The
emerging ventral root fibres are
superimposable on the neural crest
cells found in the sclerotome
(arrowhead). (C,D) A section
through the axial level two somites
anterior to that pictured in A,B.
More HNK-1-stained cells are seen in
the developing sensory ganglion, on
the ventral root and along the dorsal
aorta. The ventral root fibres (B,
arrowheads) have now reached the
dermatome/myotome. Again, crest
cells in the sclerotome are
colocalized with these fibres. Bar,
100nm. (From Loring & Erickson,
1987).
Control of pathfinding by the avian trunk neural crest
71
Fig. 5. (A) Transmission electron micrograph of a thin section through a chick neural tube fixed 24 h after receiving a
graft of pigmented neural crest cells into the lumen of the neural tube. x720. (B) Higher magnification of the area
indicated by arrow b in A, showing that a pigmented neural crest cell has reached the basal surface of the neural tube.
X9600. (C) High magnification of B, demonstrating that the grafted cell has spread on the basal lamina of the neural
tube but has not penetrated it. X24800. (From Erickson, 1987).
than the cells that escaped out the sides. In both
situations, the capillary tube and the coverslip presumably 'trapped' the putative repulsive molecule or
limited its diffusion away.
Attempts to duplicate these experimental results
with either quail (Erickson & Olivier, 1983) or
amphibian crest cells (R. E. Keller, personal communication) have not been successful. We found, for
example, that quail crest cells are less dense under a
coverslip because of accelerated cell death. In addition, there appeared to be no difference in the
extent of dispersion of crest cells dependent upon
their position in a capillary tube. Finally, we drew
aggregates of neural crest cells into capillary tubes of
varying diameters. As the diameter of the tube is
decreased, one would predict that the chemotactic
factor would accumulate faster and the cells would be
more dispersed; in fact, the opposite occurred.
Oalvanotaxb
Recently, there have been numerous reports that
embryonic cells, such as neural crest cells and sclerotome cells, respond to an imposed electrical field by
migrating toward the negative pole (Nuccitelli &
Erickson, 1983; Stump & Robinson, 1983; Cooper &
Keller, 1984). The threshold for this response is
surprisingly low, as little as lOmVmm" 1 for somite
cells (Erickson & Nuccitelli, 1984). It is also known
that embryonic epithelial cells can create electrical
fields (e.g. Jaffee & Stern, 1979; Lindemann &
Voute, 1976) by pumping ions across themselves.
We have found that currents emanate from isolated
quail neural tubes that are in the correct orientation
and of sufficient magnitude to direct crest cell migration. Furthermore, they arise at the appropriate
axial levels and at the correct time (Fig. 8; Erickson
& Nuccitelli, 1986). However, we do not yet know if
these same currents exist in the intact embryo and,
more importantly, if they have any role in neural crest
motility. Even if they do exist, the field strength must
drop off dramatically some distance from the neural
tube, perhaps owing to resistivity in the extracellular
matrix, because our grafting experiments described
C. A. Erickson
above show that crest cells can move counter to their
usual direction of migration until they meet the
oncoming host crest.
Contact inhibition
Still the most attractive explanation for directed
motility of neural crest cells is contact inhibition,
since at the very least observed crest cell behaviour or
experimental manipulations do not disprove it. Contact inhibition of movement is the result of contact
between two locomotory cells, in which the contacting cell ceases lamellipodial activity (socalled contact
paralysis) at the point of contact, and generally
retracts and moves off in another direction (Abercrombie & Heaysman, 1954; Abercrombie, 1970).
In tissue culture, this phenomenon has two effects
on population dynamics: (1) cells will move radially
away from regions of highest concentration to regions
where cells are sparsest and (2) elongate cells will
align in parallel arrays (Erickson, 1978). Crest cells in
the embryo similarly disperse from their point of
highest concentration at the neural tube toward the
periphery where there are no other cells occupying
their pathway. Furthermore, crest cells are often
aligned in parallel during their early stages of migration when we can observe them best (Bancroft &
Bellairs, 1976; Tosney, 1978).
Several investigators (Newgreen et al. 1979; Rovasio et al. 1983) have clearly shown that neural crest
cells in tissue culture migrate directionally away from
the point of highest concentration although neither
study demonstrated contact paralysis owing to the
magnification of their filming conditions. Similarly,
Gooday & Thorogood (1985) provide indirect evidence that crest cells display contact inhibition using
the nuclear overlap assay developed by Abercrombie
& Heaysman (1954). 1 have used time-lapse cinemicrography combined with differential interference
optics to observe contact behaviour of neural crest
cells. These studies have unequivocally shown that
the lamellipodium of a neural crest cell displays
contact paralysis when it contacts the side of another
crest cell (Erickson, 1985).
There is at least one instance in which contact
inhibition cannot explain the directional migration of
neural crest cells. Keller & Speith (1984) filmed
pigment cells as they migrated beneath the skin of the
axolotl embryo. These cells are unerringly persistent
in their directional migration and yet they do not
apparently contact each other. Perhaps galvanotaxis
can explain this directional migration.
With the advent of a variety of fluorescent vital
dyes, it may now be possible to label premigratory
neural crest cells in chick or mammalian embryos and
actually observe neural crest migration in vivo.
Migratory capability
One of the extraordinary features of neural crest cells
is that they are migratory no matter where they are
placed in the early chick embryo, even in ectopic sites
Fig. 6. (A) A section through a chick embryo fixed 24 h after receiving a graft of pigmented crest cells adjacent to the
notochord. The graft was placed originally at somite level 26 of a 26-somite embryo. This micrograph shows that the
grafted cells moved laterally along the endoderm and dorsal aorta (arrows) and dorsally between the neural tube and
somite. NC, neural crest; S, somite. Bar, 100//m. (B) A cross-section of a chick embryo 18 h after receiving a graft in
the ventral pathway at the level of the last somite. The grafted cells (arrows) have moved laterally along the dorsal
aorta and dorsally along the surface of the somite. The extent of dorsal migration of the grafted cells coincides with the
ventral distribution of the host crest cells. Bar, 50 j.im. (From Erickson, 1985).
Control of pathfinding by the avian trunk neural crest
73
" •*•••
.40 S•- "24**.*
^
Fig. 7. Sections through a 27-somite chick embryo that received a graft of pigmented crest cells lateral to the last somite
(X marks the site of graft placement) and was then fixed 7h later. These sections are taken through the middle of the
somite (A), near the anterior margin (B) and through the intersomitic space (C). The grafted cells moved both medially
and laterally along the somite (S) and ectoderm (E) and are spread along the intersomitic blood vessel (BV) in C. NT,
neural tube; M, mesonephric duct; DA, dorsal aorta. Bar, 50fzm. (From Erickson, 1985).
Suigc 12 (46 h, 16 somites)
Fig. 8. Current pattern measured
around the tube of a chick embryo
at stage 12 using the vibrating
probe. Somites were carefully
removed with sharpened tungsten
needles to allow access to the
neural tube epithelium. The current
pattern detected along the length of
the neural tube and in cross section
is shown in the lower figure. A,
anterior; P, posterior; D, dorsal; V,
ventral. (From Erickson &
Nuccitelli, 1986).
(Erickson et al. 1980). We found, for example, that
crest cells, even after they have differentiated into
pigment cells, can be grafted back into the dorsal
migratory pathway and will resume migration. Furthermore, crest cells can be grafted into sites where
they would normally not be found, such as in the
chick limb mesenchyme, and will disperse in the limb
and even migrate out of the limb and into the region
adjacent to the developing kidney and the dorsal
aorta (Fig. 9). This latter dispersal cannot be
accounted for by simple growth of the limb, since
many of the grafted cells escape from the limb.
Conversely, other embryonic cells will not migrate
on the crest migratory pathway. Chick heart fibroblasts and limb bud mesenchyme stay as intact
aggregates when grafted into the dorsal crest pathway. Even limb bud mesenchyme grafted into the
limb will not mix with the host mesenchyme.
The only cell type tested that appears to have some
of the dispersal properties of the neural crest is a
mouse tumour cell line, sarcoma 180 (S-180). After
grafting S-180 cells into the crest pathways, they were
found in many of the same areas to which the crest
would normally migrate (Fig. 10; Erickson et al.
1980), although some of this dispersal may be passive
in nature (see Bronner-Fraser, 1982). In fact, these
experiments testing the migratory potential of S-180
cells should be reinvestigated in light of our current
understanding of neural crest cell pathways.
Such experiments demonstrate that crest cells have
migratory capabilities that exceed those of almost all
other embryonic cells. The basis for this exceptional
dispersal capability is not yet known, but I will
present a few possibilities.
Protease activity
Numerous tumorigenic cells lines that are notoriously
invasive produce high levels of proteolytic enzymes,
74
C. A. Erickson
Fig. 9. Low magnification of a chick embryo fixed 48 h after receiving a graft of pigmented neural crest cells into a
stage-21 hindlimb bud. (B) High magnification of boxed area in A. Arrowheads indicate pigmented crest cells that have
migrated from the limb into the ventral crest pathway and are now distributed in the dorsal mesentery and around the
developing kidneys and adrenal cortex. Bars, 25 fim. (From Erickson et at. 1980).
A<a
••*.?•'>•-!<••;
1QA
Fig. 10. (A) Low magnification through a section of a chick embryo fixed 24 h after receiving a graft of sarcoma-180
cells into the crest pathway at the level of somite 26 in a 27-somite host. (B) High magnification of box I in A. The
sarcoma-180 cells (arrowheads) are distributed along the ventral root fibres and in the region of the dorsal root
ganglion. (C) High magnification of boxed area 2 in A. Sarcoma-180 cells (arrowheads) are distributed ventrally around
the dorsal aorta and in the dorsal mesentery. Bars, 25;*m. (From Enckson el al. 1980).
Control of pathfinding by the avian trunk neural crest
75
including collagenases and plasminogen activator
(PA; e.g. Mignatti, Robbins & Rifkin, 1986). PA is a
neutral serine protease that converts plasminogen to
plasmin. Both plasmin and PA itself can degrade
extracellular matrix components (Quigley, 1979;
Fairbairn et al. 1985; Sheela & Barrett, 1982; Laug,
de Clerck & Jones, 1983; Sullivan & Quigley, 1986).
Degradation of the ECM might allow cells to tunnel
through a dense matrix. Furthermore, Chen, Olden,
Bernaud & Chu (1985) have shown that proteases are
locally secreted in adhesion plaques and may be
important for releasing cell adhesions, allowing the
cell to take another step forward. The secretion of
proteases might allow the crest cells to move more
readily. Finally, a number of morphogenetic events
are associated with the localized action of the PA/
plasmin system, including the colonization of the
bursa of Fabricius by haemopoietic precursors
(Valinksy, Reich & Le Douarin, 1981) and the
involution of mammary epithelium (Ossowski, Biegel
& Reich, 1979).
Valinsky & Le Douarin (1985) have identified PA
activity in cephalic neural crest cells. Similarly, we
have found that trunk crest cells in chick do indeed
produce higher levels of PA activity than somite cells
isolated from the same age embryos. Furthermore, as
the crest cells differentiate into pigment cells, they
produce an increasingly higher level of PA activity.
PA, in preliminary experiments, appears to be important in migration, since inhibitors of PA activity
(NPGB, 10~ 5 M; benzamidine, 2 X 1 0 ~ 3 M ; leupeptin,
5 X 1 0 ~ 5 M ) reduce crest migration in tissue culture
(Fig. 11). We are presently using antibodies to
chicken PA to perturb crest migration in the embryo.
Reduced tractional force
Harris & co-workers (1980, 1981) demonstrated that
fibroblasts exert a tractional force on their substratum
as they locomote and that this force is sufficiently
strong to deform a silicone rubber sheet or a collagen
lattice. Indeed this force can be so strong that a
fibroblast will tear its deformable substratum and
consequently be immobilized (Tucker, Edwards &
Erickson, 1985). However, cells that are normally
locomotory in the embryo or in the adult, such as
neurones, lymphocytes and macrophages, do not
exert a strong or even a measurable tractional force.
These investigators suggest that low tractional force
permits migration and that the excessive tractional
forces exerted by fibroblasts may have a role in tissue
modelling and patterning, rather than migration.
We tested the possibility that neural crest cells
might also exert a low tractional force that could
explain their migratory capability (Tucker et al.
1985). When crest cells are grown on a silicone rubber
sheet they do not deform their substratum. Likewise
Fig. 11. Neural tubes were embedded in threedimensional collagen gels into which were incorporated
benzamidine (2X1(T 3 M, B) or leupeptin ( 5 X 1 0 " 5 M , C).
Compared to the control (A), neural crest migration was
greatly reduced when inhibitors of plasminogen activator
were added to the cultures. Bar, 1 mm.
they do not deform a collagen gel unless the concentration of collagen falls below 250/igml"1. On the
other hand, those embryonic cell types that do not
migrate in the embryo, such as somite cells, not only
76
C. A. Erickson
Stage of neural crest migration
Length of pathway*
Crest cells at medial edge
of somite
128 ± 1 9
Crest cells distributed
131 ± 36
along dermatome/myotome
Crest cells at dorsal aorta
143 ± 2 3
deform such substrata, but can even tear a collagen
gel and their dispersal is arrested.
An interesting possibility is that crest cells are able
to use various extracellular matrices as migratory
substrata owing to their minimal fractional force,
whereas other embryonic cells may destroy the substratum because of their excessive fractional force.
While this remains a fascinating possibility, we have
no further data to support this notion, and it remains
a difficult one to test.
Passive movement
Recent experimentation suggests that not all of the
crest dispersion is due to active, lamellipodial driven
migration (Noden, 1984; Bronner-Fraser, 1982). Such
studies have pointed out that the enormous distances
traversed by the crest are partially due to the growth
of the surrounding mesenchyme in which the crest
cells disperse.
In a review, Noden (1984) has summarized three
different studies in which host chick ectoderm, paraxial mesoderm or neural crest cells were replaced
with the equivalent quail tissue. When the grafted
cells were followed in successive stages, the lateral
expansion of the ectoderm, underlying mesoderm
and the crest cells progressed at the same rate. Thus,
in the head, once the crest cells have managed to
Fig. 12. Path length does not change during
migration under dermatome/myotome.
Camera-lucida tracings showing the
distribution of neural crest cells at successive
stages in their dispersal across the width of
the somite and the average length of the
neural crest pathway (from medial edge of
the dermamyotome to the dorsal aorta) at
each stage of migration. There is no
statistical difference in the length of the
pathway as the crest cells move from one
end to the other, suggesting that the crest
cells must move actively in relationship to
the somite, rather than being transported
passively due to the growth of the somite.
(From Loring & Erickson, 1987).
spread over the mesoderm, they may continue their
ventral dispersion by more or less riding along on the
underlying mesoderm as it grows. Similarly, BronnerFraser (1982, 1985) has shown that Latex beads
injected into the cavity of the somites are passively
dispersed and accumulate in the sclerotome lateral to
the dorsal aorta, where the sympathetic ganglia
develop. This dispersion of the beads is probably due
to the growth and expansion of the sclerotome
(Gasser, 1979).
The situation in the trunk is quite different to that
in the head. In the trunk, crest cells migrate into the
somite and have even traversed the somite and
reached the dorsal aorta during a short period when
the somite itself has not grown (Fig. 12; Loring &
Erickson, 1987). Thus, although the growth of embryonic structures undoubtedly accounts for the large
distances that crest derivatives are found from the
dorsal neural tube, the patterns of migration that are
established early are likely due to active movement.
Conclusions
The patterns of crest cell migration in the trunk of the
avian embryo have been delineated recently owing to
the development of a monoclonal antibody that
recognizes migratory neural crest cells. The detailed
Control of pathfinding by the avian trunk neural crest
analysis of these pathways should now allow us to
ascertain the environmental controls for these pathways.
Still unknown are all the environmental macromolecules that may guide neural crest cells. A particularly fruitful avenue of investigation, in this regard,
may be embryonic mutants in which the environment
through which the crest cells move is atypical. For
example, recent investigations of the mutant lethalspotting (Is), in which the bowel is developmental^
abnormal due to the absence of the crest-derived
enteric ganglia, suggest that the environment of the
mutant's terminal gut cannot support invasion of
neural crest cells (Rothman, Nilaver & Gershon,
1982; Rothman & Gershon, 1984). If this is true, the
Is/Is mutant may allow us to identify the extracellular
matrix components that sustain neural crest cell
migration (Gershon & Rothman, 1987).
Likewise the perturbation of the crest pathways
with monoclonal antibodies to specific matrix molecules may prove useful. Results from such studies,
however, need to be interpreted cautiously since a
negative result does not necessarily discount the role
of that molecule in crest morphogenesis.
The pathways of crest migration in the head are not
nearly as defined as those in the trunk but we now
have the tools to study cephalic crest migration as
well. It will be particularly intriguing to see if the
paraxial mesoderm organized as somitomeres has the
same ability to organize crest migration (see Anderson & Meier, 1983) as the somites do in the trunk.
Finally, we know little about what confers the
extraordinary migratory abilities on the neural crest
cells. Indeed we have little information about the
invasive behaviour of many cells, including lymphocytes and metastasizing cancer cells. The rapidly
expanding list of cellular oncogenes that are associated with cellular transformation may eventually
provide the clues for the controls of invasive behaviour of normal cells during development.
I would like to thank Dr D. W. Phillips for a critical
reading of this manuscript. Research reported here is
supported by N1H grant DE05630. The author is also the
recipient of an NIH Research Career Development Award.
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