DEVELOPMENTAL DYNAMICS 19529-42 (1992)

Segregation and Early Dispersal of Neural Crest Cells in the Embryonic Zebrafish DAVID W. RAIBLE, ANDREW WOOD, WENDY HODSDON, PAUL D. HENION, JAMES A. WESTON, AND JUDITH S. EISEN Institute of Neuroscience, University of Oregon, Eugene, Oregon 97403

ABSTRACT We have exploited our ability to visualize and follow individual cells in situ, in the living embryo, to study the development of trunk neural crest in the embryonic zebrafish. In most respects, the development of zebrafish trunk neu- ral crest is similar to the development of trunk neural crest in other species: zebrafish trunk neu- ral crest cells segregate from the dorsal neural keel in a rostrocaudal sequence, migrate ventrally along two pathways, and give rise to neurons of the peripheral nervous system, Schwann cells, and pigment cells. However, some aspects of the development of zebrafish trunk neural crest differ from those of other vertebrates: zebrafish trunk neural crest cells are significantly larger and fewer in number than those in avian embryos and the locations of their migratory pathways are slightly different. This initial description of neural crest development in the zebrafish embryo pro- vides the foundation for future experimental studies. o 1992 Wiley-Liss, Inc.

Key words: Cell migration, Basal lamina, Teleost, Brachydanio rerio

INTRODUCTION The neural crest of vertebrate embryos provides a

useful system to learn how stable phenotypic differ- ences between cells arise during embryogenesis and how these differences are influenced by environmental and intrinsic cellular factors. Neural crest cells can be identified early and give rise to diverse cellular pheno- types after they disperse through interstitial spaces (Horstadius, 1950; Weston, 1970, 1982, 1991; Le Douarin, 1982; Newgreen and Erickson, 1986; Noden, 1987; Bronner-Fraser, 1987; Erickson, 1988; Bronner- Fraser and Fraser, 1991). Our present knowledge of neural crest development has come primarily from large numbers of studies in aves (Weston, 1970, 1982; Le Douarin, 1982; Noden, 1987; Bronner-Fraser, 1987; Erickson, 1988), amphibia (Lofberg et al., 1980; Spieth and Keller, 1984; Krotoski et al., 1988; Epperlein et al., 1988; Moury and Jacobson, 19901, and mammals (Erickson and Weston, 1983; Martins-Green and Erick- son, 1986; Serbedzija et al., 1990), and a few studies in teleosts (Lamers et al., 1981; Langille and Hall, 1987; Sadaghiani and Vielkind, 1989). These studies have shown that, in general, many of the processes of neural

0 1992 WILEY-LISS, INC

crest development are remarkably similar in these di- verse species.

Although most studies have emphasized the early pluripotentiality of neural crest cells, recent studies raise the possibility that individual crest cells may have different developmental potentials prior to or dur- ing their initial dispersal (Weston, 1991). Most studies on the neural crest have been carried out either on populations of crest cells or on individual cells whose initial properties were undefined. For these reasons, the relationship between the development of crest cell populations and the development of individual crest cells is not clear. Furthermore, for most studies carried out in vivo, embryos were fixed prior to analysis so that inferences were made about dynamic processes from static images.

In this paper, we describe the early development of trunk neural crest in the embryonic zebrafish, Bruchy- dunia rerio. We have chosen to study neural crest de- velopment in this species because its small size, optical clarity, and rapid development provide the opportunity for us to identify individual crest cells and follow their migration and differentiation in situ, in the living em- bryo. We show that, in general, the trunk neural crest of embryonic zebrafish develops in a way that is similar to what has been described in other vertebrates. We have also identified some differences in the develop- ment of zebrafish neural crest from what has been de- scribed in other vertebrates. Zebrafish neural crest cells are significantly larger and fewer in number than those in avian embryos. In addition, although zebrafish neural crest cells also migrate along a restricted path- way, that pathway has a different somitic location than the pathways in other species. Although these differ- ences may reflect important distinctions in the specific cellular interactions occurring in different species, they seem unlikely to represent major departures in underlying mechanisms.

The present paper provides the background for fu-

Received June 19, 1992; revision accepted September 30, 1992. Address reprint requestskorrespondence to Judith S. Eisen, Insti-

tute of Neuroscience, University of Oregon, Eugene, OR 97403. Andrew Woods present address is Department of Neurology, Uni-

versity of Cambridge, Addenbrooke’s Hospital, Hills Road, Cam- bridge, England.

30 RAIBLE ET AL.

ture studies. We hope to use the description of zebrafish neural crest development presented here, in combina- tion with our ability to manipulate embryos geneti- cally (Streisinger et al., 1981; Kimmel et al., 1991) and surgically (Eisen, 1991), to examine the relative influ- ences of intrinsic cellular properties and environmen- tal features in determination of the fate of individually identified neural crest cells.

MATERIALS AND METHODS Animals

Embryos were obtained from our laboratory colony. Adults were maintained on a 14:lO hr lightldark cycle a t 28.5"C (see Kimmel and Warga, 1987; Kimmel et al., 1989). At this temperature, embryos develop the first pair of somites a t about 10 h (hours postfertilization a t 28.5"C), and two pairs are added per hour until the embryo has the full complement of 30 somite pairs (Hanneman and Westerfield, 1989).

Chorions were removed with sharpened watchmak- ers forceps and the embryos maintained in embryo me- dium (Kimmel and Warga, 1987) at 28.5"C. Observa- tions on living embryos were made using Nomarski (DIC) microscopy of specimens embedded in agarose (Wood and Timmermans, 1988) or in agar (Eisen et al., 1989). Alternatively, embryos were placed between 24 x 60 mm coverslips held apart by 200 km spacers in a drop of embryo medium containing 2 mgiml agarose. When necessary, embryos were immobilized in a dilute solution of tricaine-methanesulfonate (MS222; Sigma Chemical Co., St. Louis, MO). The movement of indi- vidual neural crest cells was recorded by drawing cell outlines a t regular intervals using a camera lucida tube, photographing the cells a t regular intervals, or time-lapse video recording. All observations were made in segments 1-18 in embryos between 15.5 h (13 somite) and 3 days of development.

Cell Labeling To follow the fates of individual neural crest cells, we

labeled them by intracellular injection of rhodamine- dextran (Molecular Probes, Eugene, OR) using glass micropipettes that were pulled on a Brown-Flaming P80 PC puller (Sutter Instrument Co., San Rafael, CA). Micropipette tips were filled with 10% rhodamine-dex- tran in 0.2 M KC1 and the shanks backfilled with 0.5 M LiCl in distilled water (dH,O). Embryos were mounted as described in Eisen et al. (1989). Cells were impaled by overcompensating the capacitance of a high-input impedance amplifier (Biodyne Electrical Co., Santa Monica, CA) for 1-2 sec; typically enough dye was in- jected into the cell during impalement that the elec- trode could be withdrawn immediately. Labeled cells were monitored using low light level, video-enhanced, fluorescence microscopy; video images were stored on an optical disk recorder (Panasonic Industrial Co., Se- caucus, NJ) and image processed using NeuroVideo software (generous gift of Paul Z. Myers).

Scanning Electron Microscopy For SEM, embryos were fixed for 1 min in primary

fixative (2.0% glutaraldehyde, 0.5% acrolein, 2.0% formaldehyde, 0.5% dimethyl sulfoxide, 8 Mm CaC1,; in 0.1 M sodium cacodylate buffer pH 7.0-7.2) a t room temperature. They were then rinsed in embryo me- dium and the periderm and ectoderm were removed by dissection with watchmakers forceps. Specimens were returned to primary fixative for 2 hr, rinsed in three changes of 0.1 M sodium cacodylate buffer for 10 min each and post-fixed for 1 hr in 2.0% OsO, and 8 mM CaCl, in 0.1 M sodium cacodylate buffer at room tem- perature. The fixed embryos were rinsed again in so- dium cacodylate buffer, dehydrated through a graded series of ethanols, and dried from liquid COz in a Sor- vall critical point apparatus. Dried embryos were mounted on aluminum stubs with double-sided adhe- sive tape, sputter-coated with gold-palladium, and viewed in a Phillips AMR 1000 SEM.

Light Microscopy and Transmission Electron Microscopy

Embryos were dechorionated, washed in embryo me- dium, and placed in primary fixative (see above) a t room temperature; for TEM, 1.0% tannic acid was in- cluded in the primary fixative and embryos were transected to allow for greater penetration of fixative to enhance preservation of the extracellular matrix. After 30 min, the yolk sac was removed with tungsten needles and the embryos were returned to primary fix- ative for 60-90 min. All embryos were washed in three changes of 0.1 M sodium cacodylate buffer for 10 min at room temperature. Embryos to be processed for TEM were post-fixed either with 2.0% OsO, and 8.0 mM CaC1, in 0.1 M cacodylate buffer (pH 7.2) for 60 min, or with 0.5% OsO,, 0.8% K,Fe(CN)6 and 8.0 mM CaC1, in 0.1 M sodium cacodylate (pH 7.2) for 15 rnin at room temperature. After post-fixation, embryos were given three 10-min rinses in 0.1 M sodium cacodylate buffer (pH 7.2) followed by 60 min in 0.1% tannic acid in dH20 at room temperature to minimize extraction ar- tifacts produced during ethanol dehydration. Embryos were rinsed twice in sodium cacodylate buffer and once in dH20 (5 min each) prior to the ethanol dehydration series. The duration of the dehydration series was crit- ical in determining the degree of shrinkage. Thus, a rapid dehydration procedure consisting of 10 rnin in each of 30, 50, 70, 80, 95, and twice in 100% ethanol was adopted. Embryos were then transferred into 1:l ethano1:propylene oxide (PO) followed by two washes in 100% PO, a 1 : l P0:Epon mixture for 60 min and a 1:3 P0:Epon mixture overnight. All dehydration and infiltration stages were carried out with constant stir- ring. Embryos were oriented and embedded in fresh resin for 4 h r at room temperature and polymerized for 16 hr at 60°C. For light microscopy, blocks were seri- ally sectioned at 6 pm and 10 pm using an LKB ul- tratome. Individual sections were transferred to sili-

ZEBRAFISH NEURAL CREST 31

conized glass slides, stained using 0.1% toluidine blue in 1.0% sodium tetraborate, and photographed under a 100 x oil immersion objective. Selected sections were re-embedded (see Schabtach and Parkening, 1974) and thin-sectioned. Silver sections were mounted on parlo- dian-coated nickel grids and stained using 1.0% uranyl acetate in double dH,O and lead citrate according to Reynolds (1963). Subsequently, grids were carbon coated and viewed using a Phillips 300 TEM operated at 60-80 Kv.

Serial Reconstructions Serial 6 km and 10 pm sections of a 19.5 h (21

somite) embryo were stained with toluidine blue and photographed. Selected features were traced and digi- tized with a PC Vision Plus framegrabber on a Dell 3086 microcomputer (Dell Computer Corporation). Co- ordinate files were established using Image Measure (Microsciences Incorporated). Sequences of between 8 and 60 sections were reconstructed using Model Mate Plus 3D solid modeling software (Control Automation Incorporated). Compiled images were photographed di- rectly from a flat tension mask video monitor (Zenith Data Systems).

Immunocytochemistry Whole mounts. Two and three day embryos were

processed and stained for immunoreactivity of the zn-5 monoclonal antibody (mAb) which recognizes a cell surface antigen on several different cell types, includ- ing zebrafish dorsal root ganglion sensory neurons, fol- lowing the procedures described in Eisen et al. (1989).

Sections. Individual neural crest cells were labeled with lysinated rhodamine-dextran and their develop- ment followed as described above. At four or five days embryos were fixed and prepared for labeling with the zn-5 mAb or antiserum to tyrosine hydroxylase, respec- tively, using the following protocol. Embryos were fixed in 4% paraformaldehyde for 4 hr, rinsed in PBS, incubated in 30% sucrose in PBS for 12 hr, frozen in O.C.T. (Miles Laboratories), mounted on a chuck, and 12 pm cryostat sections cut. Sections were mounted on gelatin-coated slides, air-dried for 1 hr, and washed in three 15 min changes of PBS. Nonspecific labeling was blocked by preincubation in a dilution buffer contain- ing 0.5 M NaC1, 0.01 M phosphate buffer, 0.1% NaN,, 2.0 % BSA, and 0.3 % Triton X-100. Sections were incubated in primary antibody in dilution buffer con- taining 10% goat serum for 12 hr at room temperature and washed in three 15 min changes of PBS. For zn-5 labeling, sections were incubated for 2 hr a t room tem- perature in biotinylated goat antiserum made against the constant region of mouse IgG in dilution buffer that included 10% goat serum, washed in three 15 min changes of PBS, incubated in avidin-fluorescein for 1 hr a t room temperature, washed in three 15 min changes of PBS, and mounted in 1: l glycero1:PBS. For tyrosine hydroxylase labeling, sections were treated as for zn-5 labeling, except that the secondary antibody

was biotinylated goat antiserum made against the con- stant region of rabbit IgG.

RESULTS Neural Crest Identification

To learn whether the cells that we observed segre- gating from the trunk neural tube in embryonic ze- brafish formed derivatives characteristic of the neural crest of other vertebrate species, we labeled individual cells in living embryos and examined the location and cellular phenotypes of their progeny. Figure 1 shows an example of a cell that was labeled with rhodamine- dextran when i t was located on the dorsolateral aspect of the neural tube. This cell migrated ventrally, di- vided a t least twice, and gave rise to a dorsal root gan- glion sensory neuron and melanocytes. Using this tech- nique, we found many of the derivatives produced by the trunk neural crest of other species (Figs. 1, 2), for example, sensory neurons, pigment cells, Schwann cells, and sympathetic neurons. No labeled cells were observed in locations inconsistent with those described for neural crest derivatives in other vertebrate species. We conclude that the cells, which arise and migrate from the dorsal surface of the zebrafish trunk neural tube, are the teleost equivalents of trunk neural crest cells in other vertebrate species.

Neural Crest Segregation Neural crest segregation from the neuroepithelium

follows separation of the neuroepithelium from the non-neural ectoderm. During this process, the neuroep- ithelium undergoes a series of morphogenetic changes to become a neural tube. As in other vertebrates, these processes occur in a rostrocaudal sequence. Figure 3 illustrates formation of the neural tube and segrega- tion of neural crest cells in segment 8. At 14 h (10 somites), the neuroepitheiium is continuous with the basal surface of the overlying non-neural ectoderm, and the boundary between the two tissues is indistinct. At this stage, the neuroepithelium is considered a neu- ral keel (see Lamers et al., 1981) because it does not yet have a central canal. Whereas the neural tube in the trunk of avian, amphibian, and murine embryos forms by folding of the neural plate, the neural keel of tele- osts forms by a ventral thickening of the ectoderm (Lamers et al., 1981). Thus, there are no neural folds in teleost embryos; the neural crest cells segregate di- rectly from the dorsal region of the neural keel. How- ever, in the posterior region of avian embryos, forma- tion of the neural tube is similar to formation of the teleost neural keel (Hamilton, 1952; Schoenwolf, 1991).

By 15 h (12 somites), the neuroepithelium begins to separate from the overlying ectoderm (Fig. 3). At this stage, a continuous basal lamina is observed on the basal surface of the non-neural ectoderm. However, on its dorsomedial and dorsolateral surfaces, where neu- ral crest cells are beginning to segregate, the neural keel basal lamina is discontinuous. In slightly older embryos, fragments of basal lamina are associated

Fig. 1. Individual zebrafish neural crest cells can be labeled and fol- lowed in living embryos. A-C show image processed photomicrographs of a single neural crest cell in segment 10 that was labeled by intracellular injection of rhodamine-dextran just as it began to migrate along the me- dial pathway. In this and all subsequent figures, dorsal is to the top and rostra1 is to the left, unless indicated. A shows the labeled cell at 18 h (18 somites); the main cell body is located at the level of the dorsal aspect of the somite and the cell has extended a ventral process between the somite and neural tube. B shows the same embryo at 31 h; the cell divided to produce four daughters. The two most ventral cells have dif- ferentiated as melanocytes, although the pigment granules are not obvi-

ous in this photomicrograph. The most dorsal cell later differentiated as the dorsal root ganglion (DRG) sensory neuron shown in C. The fate of the remaining cell is unknown. C shows the DRG sensory neuron at 52 h; the cell has a dorsal process extending toward the central nervous sys- tem and a ventral process extending toward the segmental nerve. D shows the same embryo at 5 days, after it was fixed and processed for immunoreactivity with the 21-1-5 mAb that recognizes DRG neurons and secondary motoneurons. The DRG containing the cell shown in C is indicated with an arrow; DRGs in adjacent segments are indicated with arrowheads. Scale bar: A-C, 20 pm; D, 37 pm.

ZEBRAFISH NEURAL CREST 33

Fig. 2. Neural crest cells in the zebrafish trunk form typical neural crest derivatives. Zebrafish sensory neurons of the dorsal root ganglia were identified by their morphology and location and by their labeling with the zn-5 mAb (as shown in Fig. 1). A,B: Some pigment cells were rec- ognized by the melanin granules they contained. A shows a pigment cell in a living embryo whose progenitor was labeled with rhodamine dextran. B shows a bright field view of the same cell revealing the pigment gran- ules it contains. C,D: Non-melanogenic pigment cells (iridiphores and/or xanthophores) were recognized by their stellate morphology and auto- fluorescence. C shows a pigment cell in a living embryo whose progenitor was labeled with rhodamine dextran. D shows a fluorescence image of the same cell. E,F: Schwann cells were identified by their morphology and location along peripheral nerve fibers. E shows a Schwann cell (ar-

row) in a fixed, sectioned embryo whose progenitor was labeled with lysinated rhodamine dextran. F shows the same section labeled with the zn-5 mAb to reveal the ventral roots (arrowheads). The cell shown in E is aligned along the root designated with an arrow. G,H: Sympathetic neu- rons were recognized by their location and expression of tyrosine hydrox- ylase. G shows a section of an embryo containing three sympathetic neurons (arrowheads) and one Schwann cell (arrow) whose progenitor was labeled with lysinated rhodamine dextran. H shows a chain of sym- pathetic neurons in the same section following labeling with an antiserum to tyrosine hydroxylase. The three sympathetic neurons shown in G are designated with arrowheads. The sympathetic neurons are positioned among melanin-containing pigment cells. Scale bar: A-D, 30 pm; E-F, 60 pm; G-H, 50 pm.

34 RAIBLE ET AL.

Fig 3 Rostrocaudal gradient of segregation of neural crest cells in the trunk The SEM montage in A shows a dorsal view (rostral to the left) of a 19 h (20 somite) embryo and illustrates almost every stage of neural crest development from segregation of cells from the neuroepithelium in the most caudal regions to penetration of the medial pathway in the rostral segments The outlines of neural crest cells and somites have been redrawn in 6, and the areas shown in C-E indicated by boxes C: In rostral areas of the trunk (somite pairs 1-5) flattened mesenchymal cells are associated with the neuroepithelium and many of these cells appear oriented perpendicular to the rostrocaudal axis of the embryo Laterally, mesenchymal cells overlie the entrance to the medial pathway Dorsally, in several places the surface of the neuroepithelium is relatively

with segregating crest cells and these cells interrupt the basal lamina on the dorsolateral aspect of the neu- ral keel. By 17 h (16 somites), the neuroepithelium has formed a central canal, so that it is now referred to as a neural tube. At 19 h (20 somites), the basal lamina surrounding the neural tube appears to be complete at this axial level, and neural crest cells are more closely associated with the basal lamina of the neural tube than with the basal lamina underlying the non-neural ectoderm.

smooth and free of neural crest cells D: Between segments 6-12 nu- merous neural crest cells overlie the neural tube and almost obscure its dorsal surface from view Between somites 13-17 many neural crest cells appear on the surface of the neuroepithelium However, in some areas, it is difficult to determine whether complete segregation has taken place Contacts between adjacent cells appear extensive and some cell overlap is visible in this micrograph E: Caudal of somite 17, neural crest cells have not segregated from the neuroepithelium The rough surface may partially result from the pulling away of part of the dorsal neural tube during removal of the nonneural ectoderm in preparation for SEM Scale bar A-B, 125 pm, C-E, 62 pn

As in other vertebrates, zebrafish neural crest cells segregate from the neuroepithelium of the trunk neu- ral tube in a rostrocaudal progression. Figure 4 shows a scanning electron micrograph of a 19 h (20 somite) embryo, from which the epithelium was removed to reveal the underlying neural crest, neural tube, and somites. In this embryo, neural crest cells in segments 1-5 have dispersed, so that the dorsal surface of the neural tube appears relatively smooth and largely de- void of neural crest cells. At the level of segments 6-12,

Fig. 4. Basal lamina formation during neural crest segregation. A shows transverse sections at the level of segment 8 in (left to right) embryos of 14 h (10 somite), 17 h (16 somite), and 19 h (20 somite). At 14 h the neural keel has not separated from the overlying ectoderm. By 17 h, the neural keel has formed a cavity; thus, it is now considered a neural tube. At this stage, separation has begun on the dorsolateral as- pect of the neural tube. During separation, two basal laminae form, one underlying the epidermal ectoderm and one on the dorsal surface of the neural keel. As neural crest cells segregate, they come to lie between these two laminae. By 19 h, separation is complete. Complete basal laminae are present underlying the epidermal ectoderm and on the dorsal surface of the neural tube; neural crest cells reside between these two

laminae. Individual cells from the most lateral region of the crest have begun to disperse along the medial pathway. At this time, sclerotome cells have begun to appear at the ventromedial aspect of the somite. B shows a TEM of the region of separation of the epidermis (E) and neural keel (NK) of a 15 h (12 somite) embryo at the level of segment 8. The arrow points to the incompletely formed basal lamina. C shows parts of 2 neural crest cells (NC) between the complete basal laminae of the epi- dermis (E) and neural tube (NT) at the level of segment 8 of a 19 h embryo. The neural crest cells are in close apposition to the complete basal lamina on the dorsal surface of the neural tube (arrow). Scale bar: A, 60 pm; E C , 1 pm.

36 RAIBLE ET AL.

Fig. 5. The beginning of neural crest migration is concomitant with somite elevation. The lower panel shows a Nomarski photomicrograph of segments 5-18 of a 18 h (18 somite) embryo; the same embryo has been redrawn in the upper panel. The box in the upper panel indicates the region of the inset in the lower panel. In segments 1-8, the somites have elevated and neural crest cells have entered the medial pathway. In

segments 9-13, neural crest cells are contacting the dorsal aspect of the somites (arrows). Caudal to segment 13, there is a space between the dorsal aspect of the somites and the segregating neural crest. At this level, cells on the dorsal aspect of the somites show extensive protrusive activity (arrowheads). NT, neural tube; N, notochord; Y , yolk; B, blood cells. Scale bar: 50 pm; inset, 130 pm,

ZEBRAFISH NEURAL CREST 37

tig. 6. Neural crest cells migrate along two distinct pathways. A: 6 km transverse section of a 19 h (20 somite) embryo stained with toluidine blue. B: Schematic representation of neural crest cell pathways, drawn from the transverse section shown in A. The neural crest cell on the right is located on the dorsolateral aspect of the neural tube (NT); it is shown

beginning to migrate along the medial pathway between the neural tube and somite (S). The neural crest cell on the left is located on the dorso- medial aspect of the neural tube; it will migrate either on the lateral pathway between the somite and epidermis (E) or on the medial pathway. NC, notochord. Scale bar: 15 km,

neural crest cells are present on the dorsomedial and dorsolateral surfaces of the neural tube. These cells possess broad lamellapodia and filopodia and appear to overlap one another. In segments 13-17, neural crest cells have just begun to separate from the neural tube. In these segments, it is difficult to distinguish individ- ual crest cells from the other cells of the neuroepithe- lium. Neural Crest Dispersal

contrast, examination of SEMs from avian (Tosney, 1978) and mammalian (Erickson and Weston, 1983) embryos reveal that, prior to dispersal, there are 3-5 fold the number of neural crest cells associated with the neural tube at each segmental level than in ze- brafish embryos.

Zebrafish neural crest cells are larger and less nu- merous than their counterparts in avian embryos. For example, the short and long axes of individual ze- brafish cells, measured from the SEM shown above, were 11.7 & 4.3 pm by 22.8 * 3.8 pm (average ? s.d.; n = 10). In comparison, the corresponding measure- ments of avian neural crest cells (from SEMs in Tosney, 1978; Fig. 2B,C; with permission of author) were 5.0 * 1.0 pm by 8.2 5 0.8 pm (n = 7). We found that in counts of neural crest cells on the dorsal aspect of the neural tube from 4 separate zebrafish embryos, there were 11.3 ? 3.5 neural crest cells per segment. In

The onset of neural crest cell migration coincides with changes in the shape of the somites. Before a crest cell begins its ventral migration, it may move either rostrally or caudally along the neural tube for up t o a segment length, although not all crest cells appear to move in this way. Prior to the onset of dispersal, crest cells extend long pseudopodial processes which appear, in time-lapse recordings, to probe the dorsal surface of the somite. During this time, the cells of the dorsal surface of the somite also exhibit protrusive activity (Fig. 5 ) . In zebrafish, each somite elongates dorsoven- trally as it differentiates (see van Raamsdonk et al.,

38 RAIBLE ET AL.

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0 o e

o e 0

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age of embryo (h)

Fig. 7. Timing of entry of neural crest cells on the medial and lateral pathways. Embryos were mounted and neural crest migration observed on one side with Nomarski DIC optics; 2-7 embryos were observed for each time point. A cell was defined as migratory when it had extended a substantial pseudopodial process medial to the dorsal margin of the somite (medial pathway) or lateral to the dorsal margin of the somite (lateral pathway). The solid line shows the rate of somite formation (mod- ified from Hannenman and Westerfield, 1989, with permission of the authors). The squares show the onset of migration on the medial pathway and the circles show the onset of migration on the lateral pathway. For both pathways, the open symbols represent the earliest time at which a cell was observed migrating at a given axial level in any embryo and the solid symbols represent the earliest time at which cells were migrating at that axial level in all embryos.

1974). As the dorsal margin of a somite elevates, stable contacts are established between i t and neural crest cell processes; concomitantly, protrusive activity on the surface of the somite ceases, and crest cells begin to migrate.

Zebrafish trunk crest cells normally migrate along two distinct pathways (Fig. 6). Both pathways corre- spond to pathways taken by crest cells in other verte- brates. However, in contrast to avian embryos, in ze- brafish embryos both of these pathways extend ventrally. Thus, we have renamed them based on their anatomical location relative to the somites. Crest cells initially located on the lateral aspect of the dorsal neu- ral tube are the first to migrate; they enter a pathway between the neural tube and somite. The correspond- ing pathway in avian embryos has been called the ven- tral pathway; we have designated the zebrafish path- way as the “medial pathway.” Crest cells initially located on the dorsal-most surface of the neural tube migrate later. Many, but not all of these cells, extend ventrally along a path between the somite and the overlying ectoderm. The corresponding pathway in avian embryos has been called the dorsolateral path- way; we have designated this as the “lateral pathway.”

The medial pathway. Neural crest cells entering

the medial pathway have distinctive shapes. As they leave the dorsolateral neural tube, neural crest cells typically extend a substantial process. This process of- ten extends 15-25 pm before the body of the cell begins to translocate, so that cells just beginning to migrate are often extremely elongated. Cells remain extended while they are between the somite and the underlying neural tube or notochord, often measuring as much as 2 9 . 1 ~ 1 . 9 p m b y 1 0 . 6 ~ 1 . 4 p m ( a v e r a g e r t s . d . , n = 7).

To establish when crest cells first enter the medial pathway, we examined living embryos and plotted the onset of migration against axial level (Fig. 7). Crest cells begin to enter the medial pathway in a rostrocau- dal sequence that does not precisely parallel the ros- trocaudal sequence of somite formation. Moreover, in some cases individual crest cells begin to migrate ear- lier in a caudal segment than in more rostral segments.

In avian embryos, neural crest migration between the neural tube and the myotome extends only through the rostral half somite (Rickmann et al., 1985; Bron- ner-Fraser, 1986; Teillet et al., 1987; Loring and Erick- son, 1987). To learn whether zebrafish neural crest cells were similarly restricted in their migration, we observed migrating cells in living embryos and recon- structed serial sections of a 19.5 h (21 somite) embryo. We found that crest cells could enter the medial path- way a t any location along the length of the somite. Once they enter the medial pathway, however, most cells converge toward the middle of the somite, so that by the time they reach the junction of the neural tube and the notochord, their ventral migration appears to be restricted to the region midway between adjacent segmental boundaries (Fig. 8).

The lateral pathway. At any axial level, neural crest cells begin to enter the lateral pathway approxi- mately 4 hr later than they first enter the medial path- way (Fig. 7). However, crest cells continue to enter the medial pathway for some time after migration has be- gun on the lateral pathway (Fig. 9). As with the medial pathway, entry into the lateral pathway follows a ros- trocaudal sequence that is not precisely tied to visible events in somitogenesis. Crest cells appear to enter the lateral pathway anywhere along the rostrocaudal length of the somite and then to migrate along a re- stricted region of the path. Figure 9 illustrates crest cells migrating on the lateral path of segments 7-9 of a 24 h (30 somite) embryo. Such cells are very large, measuring 50.7 * 6.1 pm by 8.0 -+ 1.0 pm and are oriented obliquely. The cells shown in Figure 9A ex- hibit a repeated pattern of two cells per segment; how- ever, this regularity is not observed in all embryos.

DISCUSSION We have described the segregation and early dis-

persal of neural crest cells in the embryonic zebrafish. Zebrafish neural crest cells are larger and fewer in number than their counterparts in avian embryos. These characteristics, together with the optical clarity and rapid development of the zebrafish embryo, have

ZEBRAFISH NEURAL CREST 39

allowed us to follow the segregation and migration of individual neural crest cells in living embryos. Al- though the development of zebrafish neural crest dif- fers in some details from that of other vertebrate spe- cies, most aspects appear to be similar.

Fig. 9. Crest cells migrating on the lateral pathway have an elongate morphology. A: This Nomarski photomicrograph shows the rostrocaudal orientation of individual cells (arrows) on the lateral pathway of segments 7-9 of a 24 h (30 somite) living embryo. B: This scanning EM also shows the rostrocaudal orientation of individual neural crest cells (arrows) on the lateral pathway. At this stage, some cells are still entering the medial pathway (stars). Scale bar: A, 25 pm; B, 12 pm.

Fig. 8. Crest cells migrating on the medial pathway show a restricted pattern. Over 80 transverse 6 pm sections through segments 7-10 of a 19.5 h (21 somite) embryo stained with toluidine blue (see Fig. 5) were manually traced and the outlines digitized to make these computer re- constructions. In the reconstructions illustrated here, dorsal is to the top and rostra1 is to the left. These reconstructions reveal that the medial pathway extends dorsoventrally halfway between adjacent segment boundaries. A: The ectoderm has been digitally eroded from this recon- struction revealing the dorsal portions of the somites and mesenchymal cell types (yellow) overlying the neural tube (blue). Although a lumen is present in the neural tube at this stage, it has not been represented. The notochord is represented in red. The reconstruction has been surface rendered and shadowed to enhance natural contours. Individual yellow blocks do not represent individual cells, but are constructed from render- ing, in the 2 axis, the two-dimensional outlines of cross-sectional profiles of adjacent tracings. The most ventral part of the notochord and somite outlines was completed by drawing a transverse line perpendicular to the dorsoventral axis of these structures. B: The somites have been removed to reveal mesenchymal cells on the surfaces of the neural tube and notochord. Some of the ventrally located mesenchymal cells are likely to be cells of the sclerotome, rather than neural crest cells. C: The neural tube and notochord have been removed to reveal the relationship be- tween mesenchymal cells and the medial surface of the somites. Scale bar: 50 wrn.

40 RAIBLE ET AL.

Comparison of Neural Crest in Zebrafish and Other Vertebrates

Segregation of the neural crest from the m u - roepithelium. Neural crest segregation from the neu- ral keel precedes formation of the overlying basal lam- ina. In zebrafish, as in avian and mammalian embryos (Tosney, 1978, 1982; Newgreen and Gibbins, 1982; Martins-Green and Erickson, 1986, 19871, the basal lamina overlying the nascent neural tube is discontin- uous before neural crest segregation and only becomes continuous after neural crest cells have begun to emi- grate. In avian embryos, neural crest cells will not in- vade an intact basal lamina (Erickson, 1987). Thus, i t has been suggested that the discontinuity of the na- scent basal lamina is necessary for crest cells to dis- perse. It seems likely that neural crest dispersal in zebrafish may be regulated in a manner similar to that of other species.

Onset of migration along the embryonic axis. Neural crest cell migration begins earlier in rostral trunk segments than it does in caudal trunk segments. As has previously been shown in other teleosts (Sad- aghiani and Vielkind, 1989) and in avians (Newgreen and Erickson, 1986), the initiation of zebrafish neural crest cell migration follows a rostrocaudal sequence. However, in both zebrafish and avian embryos (New- green and Erickson, 19861, this sequence is not pre- cisely parallel to visible events in the process of somi- togenesis. This suggests that the beginning of neural crest migration is not directly related to somite forma- tion. However, i t may be related to other aspects of somite differentiation, for example, the formation of an intact basal lamina, which in other systems is corre- lated with loss of epithelial cell protrusive activity (Hay, 1984).

The choice of medial versus lateral pathway. Pathway selection by migrating neural crest cells changes over time. As in other vertebrate embryos (see Weston, 1991), neural crest cells in the zebrafish mi- grate along two distinct pathways: a medial pathway between the neural tube and somite and a lateral path- way between the somites and the overlying ectoderm. In avian (Weston and Butler, 1966; Serbedzija et al., 1989; Erickson et al., 19921, mammalian (Derby, 1978; Erickson and Weston, 1983; but see Serbedzija e t al., 1990), and zebrafish embryos, crest cells enter the me- dial pathway substantially before they enter the lat- eral pathway. However, this is not the case in all ver- tebrates, since in axolotl crest cells appear to enter the lateral pathway first (Lofberg et al., 1980). It is impor- tant to note, however, that zebrafish neural crest cells continue to enter the medial pathway even after mi- gration has begun on the lateral pathway. Thus, a t later stages the decision of an individual cell to select a particular pathway may involve factors other than simply when it begins to migrate. This idea is sup- ported by our observations that a few cells appear to extend processes into both pathways before selecting one to follow.

Toole e t al. (1984) have suggested that onset of mi- gration may be related to the dimensions of the inter- stitial space. Thus, if more space were available in the medial pathway than in the lateral pathway a t early developmental stages, neural crest cells might select the medial pathway preferentially. However, more re- cently, Newgreen (1989) has questioned the relation- ship between the dimensions of the interstitial space and the onset of neural crest migration. Erickson et al. (1992) have suggested that migration on the lateral pathway of avian embryos is transiently inhibited by the dermatome. Whether such a mechanism applies in the zebrafish remains to be determined. The availabil- ity of mutants, such as spt (Kimmel et al., 1989), that affect somitogenesis may provide an opportunity to test the role of somite-derived factors in neural crest cell pathway choice.

Spatial restrictions along the medial pathway. Neural crest cell migration along the medial pathway is spatially restricted. This restriction is distinctly dif- ferent from that in avian and murine embryos, how- ever, in that zebrafish neural crest cells are not re- stricted to the rostral half somite (Rickmann et al., 1985; Bronner-Fraser, 1986; Teillet et al., 1987; Loring and Erickson, 1987). Rather, zebrafish neural crest cells appear unrestricted until they reach the ventral edge of the neural tube, where they become restricted to migrate halfway between the segmental boundaries. In avian embryos, sclerotome is thought to be respon- sible for restricting the pathway of neural crest cell migration. In zebrafish, neural crest cells do not appear to encounter sclerotome as they migrate along the dor- sal portion of the medial pathway (A. Wood and Eliza- beth Morin-Kensicki, personal communication). There- fore, i t is not yet clear, in zebrafish, what role sclerotome plays in patterning crest migration.

We conclude that, in most respects, the development of the zebrafish neural crest is very similar to that described in other species. Our studies of zebrafish trunk neural crest have revealed the precise timing of neural crest segregation, the onset of migration, and pathway choice. With this information, we can take advantage of our ability to label and to transplant in- dividual neural crest cells, and to screen for neural crest mutants, to learn how the phenotypic differences between different neural crest derivatives arise and how they are influenced by environmental and intrin- sic cellular factors.

ACKNOWLEDGMENTS We thank Steve Allison and Greg Bobrowicz for pre-

liminary data, Paul Z. Myers for NeuroVideo software, Terry Takahashi for help with serial reconstructions, Ellie Melancon, Victoria A. Larson, Ed Sullivan, Steve Russell, Harrison Howard, Sean Poston, and Jerry Gleason for technical assistance, Pat Edwards for typ- ing, and Christine Gatchalian, Charles Kimmel, Ellie Melancon, Elizabeth Morin-Kensicki, Peter O’Day, and Kristine Vogel for critical reading of the manuscript.

ZEBRAFISH NEURAL CREST 41

Supported by HD22486, NS01476. D.W.R. is a postdoc- toral fellow of the Muscular Dystrophy Association.

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