Download Regulative interactions in zebrafish neural crest

Development 122, 501-507 (1996)
Printed in Great Britain © The Company of Biologists Limited 1996
DEV4634
501
Regulative interactions in zebrafish neural crest
David W. Raible* and Judith S. Eisen
Institute of Neuroscience, University of Oregon, Eugene, Oregon 97403-1254, USA
*Author for correspondence at present address: University of Washington, Box 357420, Department of Biological Structure, Seattle, WA 98195-7420, USA
(e-mail: [email protected])
SUMMARY
Zebrafish trunk neural crest cells that migrate at different
times have different fates: early-migrating crest cells
produce dorsal root ganglion neurons as well as glia and
pigment cells, while late-migrating crest cells produce only
non-neuronal derivatives. When presumptive earlymigrating crest cells were individually transplanted into
hosts such that they migrated late, they retained the ability
to generate neurons. In contrast, late-migrating crest cells
transplanted under the same conditions never generated
neurons. These results suggest that, prior to migration,
neural crest cells have intrinsic biases in the types of derivatives they will produce. Transplantation of presumptive
early-migrating crest cells does not result in production of
dorsal root ganglion neurons under all conditions, suggesting that these cells require appropriate environmental
factors to express these intrinsic biases. When earlymigrating crest cells are ablated, late-migrating crest cells
gain the ability to produce neurons, even when they
migrate on their normal schedule. Interactions among
neural crest cells may thus regulate the types of derivatives
neural crest cells produce, by establishing or maintaining
intrinsic differences between individual cells.
INTRODUCTION
cells within a forming tissue. For example, differentiated cells
can influence the types of cells undifferentiated precursors may
produce. This type of feedback interaction has been proposed
to occur in the developing retina (Reh, 1992) and, during
neural crest development, in the formation of peripheral
ganglia (Shah et al., 1994). Alternatively, precursor cells can
influence the fates of other types of precursor cells before they
differentiate. For example, interactions between heterologous
precursors occurs during sea urchin development, in which
primary mesenchyme prevents secondary mesenchyme from
forming skeleton (Ettensohn, 1992). A special form of regulative interactions occurs during lateral specification within
invertebrate equivalence groups (reviewed by Greenwald and
Rubin, 1992). In equivalence groups, cells make hierarchical
fate choices based on signaling among group members.
Although cells within the group are initially equivalent, signals
from cells that assume the primary or default fate cause other
members of the group to assume a secondary or alternate fate.
When cells destined to follow the primary fate are removed,
they are replaced by cells that would otherwise follow the
secondary fate.
In systems that undergo regulative development, a cell’s
developmental potential is not simply defined by its fate. Cell
fate represents what a cell will do in its usual environment as
the normal outcome of development. In contrast, cell potential
encompasses all the possible fates a cell may undertake given
appropriate environmental conditions (Weiss, 1939; Slack,
1991). As development proceeds, cells become fate-restricted
so that their progeny express only a subset of possible fates.
A basic premise of developmental biology is that cell fate
decisions result from interplay between intrinsic factors and
signals from the surrounding environment. The neural crest is
a favorite system for studying cell fate specification since it
begins as a population of cells that later forms diverse derivatives including neurons and glia of the peripheral nervous
system, pigment cells and craniofacial mesenchyme, after it
migrates from the neural tube (Horstadius, 1950; Weston,
1970; Le Douarin, 1982). Current models of neural crest development discuss the relative importance of intrinsic and
extrinsic factors in determining cell fate (Weston, 1991;
Anderson, 1994; Le Douarin et al., 1994). In some developing
systems, interactions among cells within a population contribute to cell fate decisions, such that the cells inhibit their
neighbors from following the same developmental pathway;
such interactions can be considered regulative. In the work
described in this paper, we provide evidence that regulative
interactions among zebrafish neural crest cells play a role in
neural crest cell fate decisions.
Ideas about developmental regulation first arose from experiments by Driesch (1892), in which isolated sea urchin blastomeres each developed into a whole organism. Regulative
phenomena also occur at the tissue level, such as in limb regeneration (Bryant et al., 1992) or, with respect to the neural crest,
regeneration of the dorsal neural tube and associated crest after
unilateral ablation (Scherson et al., 1993). At the cellular level,
regulative interactions are involved in the differentiation of
Key words: Danio rerio, neural crest, cell fate, lateral specification,
dorsal root ganglia
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D. W. Raible and J. S. Eisen
Restrictions in fate are defined experimentally by following
individual cells and identifying divisions after which progeny
give rise to a limited set of derivatives. However, restrictions
in fate do not necessarily imply restrictions in cell potential;
that a cell’s presumptive fate is different from its potential was
first recognized by Driesch (1892). Restrictions in potential can
be identified experimentally by challenging cells with new
environments and determining whether they change their
developmental program.
Zebrafish neural crest cells express tissue-specific markers
and display characteristic cell behaviors, revealing that they
have become specified, before reaching their final locations
(Schilling and Kimmel, 1994; Raible and Eisen, 1994). They
also undergo lineage restrictions to produce precursors that
give rise to a single derivative type. Although fate restrictions
are indicative of cell specification, they say nothing about
restrictions in potential, since specification may be conditional
(Davidson, 1990; Kimmel et al., 1991). In this paper, we
suggest that regulative interactions among neural crest cells
play a role in how they become specified. By transplanting
individual cells from different neural crest subpopulations, we
demonstrate that they have different intrinsic biases in the
types of derivatives they will make. We find that after ablation
of specific subpopulations of neural crest cells, remaining
neural crest cells are able to compensate and generate derivatives they normally never produce. Although two defined populations of neural crest cells have different intrinsic biases in
the types of derivatives they will produce, under appropriate
conditions they can both produce the same derivative types,
suggesting they initially have the same developmental
potential. We propose a model where cell fate decisions are
influenced by interactions among neural crest cells, and draw
parallels to lateral specification in invertebrate equivalence
groups.
MATERIALS AND METHODS
Animals
Embryos were obtained from the zebrafish colony at the University
of Oregon, and were staged by hours post-fertilization at 28.5°C (h;
Kimmel et al., 1995). Chorions were removed with watchmaker
forceps and living embryos were mounted for observation between
coverslips held apart by spacers (Raible et al., 1992). When necessary,
embryos were immobilized in a dilute solution of tricaine methylsulfonate (Sigma).
Cell ablation
Embryos were mounted in 1.2% agar so that neural crest cells could
be visualized under Nomarski (DIC) optics (Raible et al., 1992). Premigratory neural crest cells were removed by aspiration with a pipette
whose tip was manually broken to a diameter of about 20 µm. The
suction pipette was inserted into the embryo through a hole produced
manually with fine glass needles. Alternatively, neural crest cells were
ablated by laser-irradiation as described previously (Eisen et al.,
1989). Irradiated cells were observed for 5-10 minutes to ensure that
they did not recover.
Single-cell transplantation
Transplants of single neural crest cells were performed essentially as
described for individual motoneurons (Eisen, 1991; Eisen and Pike,
1991). Briefly, donor embryos were labeled at the 2-8 cell stage with
lysinated rhodamine dextran (10×103 Mr; Molecular Probes). Labeled
donor and unlabeled host embryos were mounted side by side in agar.
Individual neural crest cells were removed from segments 6-8 of
donor embryos by gentle suction using a micropipette whose tip was
manually broken to a diameter of about 20 µm. The micropipette was
withdrawn from the donor embryo, monitored to ensure the removal
of a single crest cell and inserted into the host embryo. The cell was
then expelled with gentle pressure onto the dorsolateral aspect of the
neural tube at the level of segments 5-8 of host embryos. The time of
migration onset for the transplanted cell was established by monitoring host embryos at half-hour intervals. Progeny of transplanted cells
were identified at 2 and 3 days of development.
Intracellular labeling and antibody staining
Neural crest cells were labeled by intracellular injection with lysinated
rhodamine dextran (10×103 Mr; Molecular Probes) as described
(Raible et al., 1992). Labeled cells were monitored using low light
level, video-enhanced fluorescence microscopy and images were
captured on a Macintosh IIci using the Axovideo program (Axon
Instruments; Myers and Bastiani, 1991).
For whole-mount antibody staining, embryos were fixed overnight
in 4% paraformaldehyde at 4°C and rinsed several times with glassdistilled H2O. Embryos were incubated for 1 hour in blocking buffer
(PBS with 1% BSA, 1% DMSO, 0.1% Triton-X 100), then incubated
overnight in primary antibody at 4°C. Embryos were rinsed in wash
buffer (PBS with 0.1% Triton-X 100), incubated overnight in horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary
antibody (Sternberger) at 4°C, rinsed in wash buffer, and incubated
overnight in mouse PAP (Sternberger) at 4°C. Embryos were then
rinsed in wash buffer followed by 0.1 M phosphate buffer, and then
incubated in 50 µg/ml diaminobenzidine with 0.01% H2O2 in 50 mM
phosphate buffer to develop the HRP reaction product.
For antibody staining of sections, embryos were oriented in blocks
of 1.5% agar in 5% sucrose and then incubated overnight in 30%
sucrose. Cryostat sections were incubated for 30 minutes in blocking
buffer, 2 hours in primary antibody, and 1 hour in fluorescein-conjugated secondary antibody (Cappell). The neuron-specific anti-Hu
monoclonal antibody (Marusich et al., 1994) was obtained from
Michael Marusich at the University of Oregon. The zn-5 monoclonal
antibody (Trevarrow et al., 1990) was obtained from Ruth BreMiller
at the University of Oregon.
RESULTS
Intrinsic differences between neural crest cells are
revealed by cell transplantation
Zebrafish trunk neural crest cells at the same axial level that
migrate at different times produce different derivatives (Raible
and Eisen, 1994); this characteristic can be used to define two
different cell types. There are fewer neural crest cells in
zebrafish than in tetrapod embryos, with only 10-12 cells per
trunk segment, but in other respects, zebrafish trunk neural
crest cells are similar in the migration pathways they follow
and the derivatives they make (Raible et al., 1992). Earlymigrating crest (EMC) cells constitute a group of 5-8 cells per
hemisegment positioned on the dorsolateral aspect of the
neural tube. At the level of somite 7 in the embryo, EMC cells
begin to migrate between 16.5 and 18h on a medial path
between the somite and neural tube, and generate all types of
neural crest derivatives, including dorsal root ganglion (DRG)
neurons, glial cells and pigment cells. In contrast, latemigrating crest (LMC) cells are positioned medially on the
dorsal neural tube, begin to migrate on the medial pathway
after 18h, and give rise to glial cells and pigment cells but do
Zebrafish crest cell interactions
Table 1. Cell transplantation reveals intrinsic biases
Cell type
EMC
LMC
EMC
Native EMC†
Host stage
(h)
Migration times
of transplanted cells (h)
Cells producing
DRG neurons (%)
15-16
15-16
18-19
−
18.5-20.5 (19.25)
17.5-20.5 (19.1)
21-23.5 (22.1)
16.5-18
−
4/21 (19)
0/30 (0)*
0/39 (0)*
22/63 (35)
EMC cells and LMC cells were removed from hosts just before beginning
to migrate. EMC cells from 16.5h donor embryos and LMC cells from 18h
donor embryos were transplanted into hosts of ages indicated. Of 191 cells
transplanted, 94 survived to generate neural crest derivatives. Cells recover 34.5 hours after transplantation before they begin to migrate, and this
characteristic was used to examine the fates of EMC cells under two different
environmental conditions. The range and average migration times (parenthesis)
were determined by monitoring 8-10 transplanted cells for each experimental
condition at half-hourly intervals to establish when cells first entered the
medial migration pathway. As described for native non-neuronal clones
(Raible and Eisen, 1994), transplanted non-neuronal clones consisted of
pigment and glial cells. Native EMC cells that produce DRG neurons also
often produce pigment or glial cells; this was true for 3 of the 4 transplanted
EMC cells described in the first line of this table. *, significantly different
from EMC cells transplanted into 15-16h hosts with P<0.01. Although the
proportion of transplanted EMC cells in 15-16h hosts that generated neurons
is not significantly different from native EMC cells (P=0.08), there may be a
trend that transplanted cells are less likely to generate neurons. †Data for
native EMC cells, which have been labeled but not transplanted, are from
Raible and Eisen (1994).
not generate DRG neurons. Still later, neural crest cells migrate
on a lateral path between the somite and overlying ectoderm;
these cells are not considered further in this study. Both EMC
and LMC cells migrate on the same pathway, so any differences in their migratory environments are temporal. We can
thus define two different cell types: EMC cells, some of which
produce DRG neurons, and LMC cells which do not.
To test the roles of intrinsic and environmental factors in
shaping EMC and LMC cell fates, we transplanted individual
cells so that they migrated under the same conditions. If the
difference between EMC cells and LMC cells is due to changes
in environmental signals, EMC cells should adopt LMC fates
if their migration is delayed and should fail to produce DRG
neurons. In contrast, if the difference between the two cell
types is intrinsic, then delaying the migration of EMC cells
should have no effect on their fates. When EMC cells and LMC
cells are challenged with the same environmental conditions,
they behave differently (Table 1). EMC cells transplanted into
15-16h hosts (‘early’ hosts) migrated 1-2.5 hours after host
EMC cells. Under these conditions, transplanted EMC cells
still generated neurons even though native LMC cells
migrating along side them at the same time and on the same
pathway did not. Fig. 1 shows the labeled progeny of a single
transplanted neural crest cell in an embryo that has been fixed,
sectioned and stained with the neuron-specific anti-Hu
antibody. The transplanted cell generated progeny that contributed to a dorsal root ganglion of the host embryo and differentiated as neurons. In contrast, LMC cells never make
neurons when transplanted to early hosts under the same conditions (Table 1). By the criteria set above, these results
suggest that the difference in fates between transplanted EMC
and LMC cells reflect intrinsic differences, since they behaved
differently in the same environment. These intrinsic differences already exist within the premigratory neural crest, since
cells were transplanted before they began to migrate.
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Table 2. Cell ablation reveals a hierarchy of fates
Cells ablated
EMC
EMC
Type of ablation
Cells migrating
before 18h
DRG normal
Aspiration
Laser
9/9
−
15/15
6/6
EMC cells were ablated either by aspiration or with a laser microbeam at
16.5 hours. After aspiration, embryos were monitored at 18h to determine
whether cells had entered the migration pathway. In all embryos examined,
cells had migrated ventrally in ablated segments and were indistinguishable
from unablated segments. Embryos were then raised to 3 days, fixed and
stained with zn-5 or anti-Hu antibodies to examine DRG development. In all
cases, DRGs were present with a normal number of cells.
Although transplanted EMC cells can produce DRG neurons
even when they migrate late, they do not behave the same
under all environmental conditions. EMC cells transplanted
into 18-19h hosts (‘late’ hosts) migrated 3-5.5 hours after host
EMC cells. Under these conditions, transplanted EMC cells no
longer generated DRG neurons (Table 1). Because we cannot
specifically recognize neuronal precursors among the EMC
cell population, we do not know in these experiments the fates,
after transplantation, of EMC cells normally destined to make
neurons; these cells may instead generate another type of crest
derivative or they may die. Our results suggest that EMC cells
destined to produce neurons require proper environmental
signals to carry out a neurogenic developmental program; these
environmental signals change with time, and are present ‘early’
but not ‘late’.
Early-migrating neural crest cells can be
functionally replaced
To test whether EMC cells influence LMC cell fate, we ablated
EMC cells before they began to migrate and asked whether the
DRG formed normally (Table 2). An example of such an
ablation experiment is shown in Fig. 2. Panel A shows a lateral
view of a living zebrafish embryo at 16.5h, just as EMC cells
began to enter the migration pathway. The same embryo is
shown in panel B directly after EMC cells were removed by
aspiration. Presumptive LMC cells immediately moved ventrolaterally to fill the position of the ablated EMC cells, so that
after 20 minutes, embryos that had undergone EMC ablation
were nearly indistinguishable from unoperated embryos (not
shown). LMC cells precociously entered the migration
pathway and migrated ventrally to the level of the notochord
(Fig. 2C), reaching it at about the same time that EMC cells
would in unablated segments. After ablation of EMC cells, the
DRG formed normally (Fig. 2D), suggesting that EMC cells
have been functionally replaced. These results suggest that
LMC cells have the potential to produce DRG neurons and that
interactions between EMC cells and LMC cells prevent LMC
cells from migrating early and adopting neural fates.
To test whether continued interactions between EMC and
LMC cells are necessary to maintain cell fate choices, we
ablated EMC cells after they had all entered the migration
pathway and LMC cells remained associated with the dorsolateral neural tube (Fig. 3). Under these conditions, LMC cells
migrated at their normal times. Fig. 3A shows EMC cells
migrating ventrally on the medial pathway in a living zebrafish
embryo at 18h, and the same segment is seen in Fig. 3B immediately after laser irradiation. All visible EMC cells have been
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D. W. Raible and J. S. Eisen
Fig. 1. After transplantation, EMC cells retain the ability to produce
DRG neurons. The embryo has been fixed and sectioned for
immunohistochemistry. A shows two progeny of an EMC cell
transplanted from a host labeled with rhodamine dextran. B shows
the same cells are positive for the anti-Hu antibody, shown in green.
The section has grazed the developing spinal cord (sc), which is also
Hu-positive. Scale bar, 60 µm.
killed, and the remaining cellular debris is readily observed.
After such an ablation, however, the DRG still formed (Fig.
3C). To confirm that cells contributing to the DRG were
derived from LMC cells from the same segment, EMC cells
were first ablated and LMC cells were labeled immediately
afterward with fluorescent dextran. After EMC ablation, LMC
cells contributed cells to the DRG (Fig. 3D). These results
suggest that interactions between EMC cells and LMC cells
continue even after the onset of migration.
DISCUSSION
Several models can be proposed to account for the fate differences between EMC and LMC cells. LMC cells may be the
same as EMC cells, but normally not produce DRG neurons
because environmental factors necessary for neurogenesis are
transient and thus absent by the time LMC cells migrate.
However, ablating EMC cells after they begin to migrate
allows some LMC cells to generate neurons even when they
migrate at their normal times, suggesting that if appropriate
environmental cues are necessary, they remain present. Alternatively, EMC cells may preferentially compete for limiting
amounts of survival/differentiation factors so that not enough
of the factor is available for the LMC cells that follow them.
However, some transplanted EMC cells that migrate with host
LMC cells are still able to produce neurons even after such a
factor would presumably have been removed by host EMC
cells. Thus, we favor the idea that interactions between EMC
cells and LMC cells maintain intrinsic differences in the ability
to generate neurons. Interactions may be direct, through cell
contact, or indirect, by modification of environmental signals.
Although we do not think that EMC and LMC cells compete
for factors, we cannot rule out the possibility that EMC cells
modify factors that are unnecessary for their own development
but that still regulate LMC cell development.
The intrinsic differences between EMC and LMC cells
revealed by transplantation represent a stage in cell fate
decisions not easily described by conventional terminology
concerning cell fate determination. When EMC and LMC cells
are tested under the same conditions, they behave differently.
Taken alone, these results suggest that EMC and LMC cells
have different developmental potentials. However, under the
appropriate conditions, LMC cells generate the same full complement of neural crest derivatives as EMC cells, suggesting
that they have the same developmental potential. Cells thus
seem to exhibit intrinsic conditional biases in developmental
potential that reflect the different probabilities with which they
will generate neurons under different conditions. These conditional biases are present before neural crest cells can be recognized as different from one another by other criteria (Raible
and Eisen, 1994), and biases are expressed before cells become
restricted to produce a single type of derivative. The state in
which cells display conditional biases is not adequately
portrayed by terms normally used to describe cell potential
such as specification, determination, or commitment. When
cells are specified they may display characteristics that allow
them to be recognized as distinct from other cells. Cells that
are specified may change their fates upon transplantation to
new environments (Davidson, 1990; Kimmel et al., 1991), yet
may continue to display their original fates when isolated in a
‘neutral’ environment such as tissue culture (Slack, 1991).
When cells are committed (Stent, 1985; Kimmel et al., 1991)
or determined (Slack, 1991), they undergo irreversible restrictions in potential, and thus display a default fate under all conditions. The conditional biases we have observed seem intermediate, so that developmental potential is restricted under
some conditions but not others.
The regulative interactions we have observed among
zebrafish neural crest cells have some parallels with lateral
Fig. 2. Presumptive LMC cells replace presumptive EMC cells
after ablation. (A) Lateral view of a zebrafish embryo at 16.5h.
EMC cells (arrows) sit poised to enter the medial migration
pathway between somite and neural tube. At this time, all EMC
cells are positioned dorsolaterally, adjacent to the dorsal somite. In
our original cell fate study, all EMC cells were located in this
position (Raible and Eisen, 1994), and in time lapse study of living
embryos all cells that migrate before 18h are located in this position
(D. W. R., unpublished observation). Occasionally a cell in this
position migrates as an LMC cell; however, most presumptive
LMC cells are located dorsomedially to EMC cells. (B) Same
embryo after EMC cells have been aspirated (arrows). (C) Embryo
at 18h showing cells migrating ventrally (arrows). Some cells have
reached the level of the notochord. (D) Same embryo at 3 days
fixed and stained with anti-Hu antibodies to reveal DRG neurons.
The ablated segment is on the left. Scale bar, 18 µm.
Zebrafish crest cell interactions
Fig. 3. Feedback interactions regulate LMC fate. (A) embryo at
18h showing EMC cells on the medial pathway (arrows). (B) The
same embryo after laser irradiation of EMC cells, showing cellular
debris (arrows). (C) Ablated segments are indistinguishable from
control in 9/9 embryos. Representative embryo shown at 4 days
stained with zn-5. Rostral (left) DRG is in ablated segment. (D)
After ablation, LMC cells produce DRG neurons while migrating at
their normal times. Progeny of a fluorescently labeled LMC cell is
shown in the position of the DRG with a characteristic process
extending to the dorsal spinal cord. 2 of 18 LMC cells formed DRG
neurons after EMC ablation, while none of 54 LMC cells did in
unablated segments (Raible and Eisen, 1994), significantly
different at P=0.007. In comparison, about 1/3 of EMC cells give
rise to DRG neurons (see Table 1; Raible and Eisen, 1994). Scale
bar, 20 µm, A, B, D; 30 µm, C.
specification in invertebrate equivalence groups (Greenwald
and Rubin, 1992). Cells of an equivalence group make hierarchical choices between two fates: primary or secondary.
Ablation of primary-fate cells results in their replacement by
secondary-fate cells. However, the converse is not the case.
Equivalence can be broken by lateral specification interactions,
in which a cell inhibits its neighbors from adopting its fate. In
zebrafish neural crest, fates may be represented by cells that
produce DRG neurons (some EMC cells), and cells that do not
(remaining EMC cells and LMC cells). Removal of DRG
neuron-producing cells allows some LMC cells to take their
place. In contrast, ablation of LMC cells has no effect on DRG
neuron formation (DWR, unpublished observations). DRG
neuron production could, therefore, be thought of as a primary
fate, and production of other derivatives as a secondary fate.
Interactions between the two cell populations could then
establish or maintain the different intrinsic biases revealed by
transplantation of EMC and LMC cells. Although these
systems have some parallels, several important characteristics
of zebrafish neural crest cells remain unknown. For example,
we have no evidence that the two cell types are initially equivalent. Observing the types of derivatives produced by EMC
and LMC cells in isolation may answer this question.
505
We envision that regulative interactions between EMC and
LMC cells are only one of several mechanisms that determine
zebrafish neural crest cell fate. Our transplantation results
suggest that the presence of appropriate environmental cues are
necessary for EMC cells to produce DRG neurons. Thus biases
that may be established by regulative interactions are further
affected by environmental factors. Shah et al. (1994) have
recently suggested that regulative interactions may also play a
role later in neural crest development, during cell fate determination within mammalian DRGs. In zebrafish, cells do not
begin to differentiate as DRG neurons until almost a day later
than the interactions we have described. Regulative interactions may thus play a role in cell fate decisions at several stages
of neural crest development, just as lateral specification does
at successive stages of sensory bristle development in
Drosophila (Jan and Jan, 1995).
Our model of regulative interactions among neural crest
cells shares some features with other models of neural crest
development. Current ideas about neural crest development
differ on the relative importance of lineage and environmental
factors in determining cell fate. At one extreme, the neural
crest population may be heterogeneous, with cells predetermined to produce specific derivatives. At the other extreme,
neural crest cells may be homogeneous, and only directed to
generate specific derivatives by local cues encountered after
migration. The idea that neural crest cells have different developmental potentials has been proposed based on several lines
of evidence. Neural crest populations from different stages
behave differently when tested under the same environmental
conditions in vivo (Artinger and Bronner-Fraser, 1992;
Erickson and Goins, 1995; Vogel and Weston, personal communication) and in vitro (Morrison-Graham and Weston, 1993;
Lahav et al., 1994; Henion and Weston, 1994; Henion et al.,
1995; Reid et al., 1995). Subsets of avian and murine neural
crest cells express different genes, such as those for adhesion
molecules (Nakagawa and Takeichi, 1995; Wehrle-Haller and
Weston, 1995; Henion et al., 1995), before entering the
migration pathway. Evidence also supports the idea that neural
crest cells have equivalent developmental potentials. Clonal
analysis in vitro (Sieber-Blum et al., 1993; Le Douarin and
Dupin, 1993) and in vivo (Bronner-Fraser and Fraser, 1991)
has shown that individual neural crest cells can generate
multiple derivative types in all combinations. Some neural
crest cells in culture have stem-cell properties that can be
regulated by a variety of environmental cues (Stemple and
Anderson, 1992; Shah et al., 1994).
A regulative model of neural crest cell fate determination
accommodates both sets of results. In our model, neural crest
cells are initially equivalent. At this stage, they would be likely
to generate several different derivatives, respond to multiple
growth factors and may act as stem cells. As neural crest cells
undergo regulative interactions, they would become intrinsically different from one another. At this stage they would
behave differently when transplanted into the same environment, would be likely to express different genes, and might
have different growth factor requirements.
What molecular mechanisms might support regulative interactions in the neural crest? In invertebrates, lateral specification is mediated through cell-cell contact by the Notch/lin12/glp-1 family of cell surface molecules (Greenwald, 1994;
Artavanis-Tsakonas et al., 1995). Several Notch family
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D. W. Raible and J. S. Eisen
members have been isolated from vertebrates (Coffman et al.,
1990; Reaume et al., 1992; Weinmaster et al., 1992; Del Amo
et al., 1992; Lardelli and Lendhal, 1993; Lardelli et al., 1994;
Lindsell et al., 1995, Chitnis et al., 1995; Henrique et al., 1995)
including zebrafish (Bierkamp and Campos-Ortega, 1993).
Although some family members are expressed in DRGs, no
earlier expression has been reported in the neural crest.
Zebrafish trunk neural crest cells have the opportunity for regulation by cell-cell contact since they extend filopodia before
migrating (D. W. R., unpublished observations), and migrate
in close proximity to one another (Fig. 2; Raible et al., 1992;
see also Krull et al., 1995). Whether contact-mediated interactions are involved in neural crest cell fate determination will
require further investigation. We and others have recently identified mutations disrupting the development of specific
zebrafish neural crest derivatives (Henion et al., 1996; Kelsh
et al., personal communication), that may reveal mechanisms
underlying neural crest regulative interactions.
We thank Bruce Bowerman, Marianne Bronner-Fraser, Chuck
Kimmel, Jim Weston and members of the Eisen lab for critical
comments on the manuscript, Kirsten Stoesser for technical assistance, Jerry Gleason for photographic help, and the staff of the University of Oregon zebrafish facility for providing embryos. Research
was supported by NIH grant HD22486 and the Dysautonomia Foundation.
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(Accepted 20 November 1995)