Regulative interactions in zebrafish neural crest

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1 Development 122, (1996) Printed in Great Britain The Company of Biologists Limited 1996 DEV Regulative interactions in zebrafish neural crest David W. Raible* and Judith S. Eisen Institute of Neuroscience, University of Oregon, Eugene, Oregon , USA *Author for correspondence at present address: University of Washington, Box , Department of Biological Structure, Seattle, WA , USA ( 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. Key words: Danio rerio, neural crest, cell fate, lateral specification, dorsal root ganglia INTRODUCTION 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 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.

2 502 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 ( M r; 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 ( M r; 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 H 2O. 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% H 2O 2 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 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

3 Zebrafish crest cell interactions 503 Table 1. Cell transplantation reveals intrinsic biases Host stage Migration times Cells producing Cell type (h) of transplanted cells (h) DRG neurons (%) EMC (19.25) 4/21 (19) LMC (19.1) 0/30 (0)* EMC (22.1) 0/39 (0)* Native EMC /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 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 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. Table 2. Cell ablation reveals a hierarchy of fates Cells migrating Cells ablated Type of ablation before 18h DRG normal EMC Aspiration 9/9 15/15 EMC Laser 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 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

4 504 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.

5 Zebrafish crest cell interactions 505 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= 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. 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/lin- 12/glp-1 family of cell surface molecules (Greenwald, 1994; Artavanis-Tsakonas et al., 1995). Several Notch family

6 506 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. 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