We have studied neural crest development in two teleost fish species, Xiphophorus maculatus (platyfish) and X. heUeri (swordtail), and found similarities to that in other vertebrates but also some important differences. Unlike in other vertebrates, segregation of neural crest cells occurs in masses or groups from the dorsal-lateral part of the neural keel (tube) except in the mesencephalon region, where neural crest cells segregate from the dorsal-midline and in the most anterior trunk region, where they segregate individually. However, the cells were found in the usual neural tube-somite and somite-ectoderm migration pathways. Notably numerous cells, presumed in part to be neural crest cells, were found in a third location, dorsally on the neural tube. These cells exhibit a series of morphological stages referred to as ‘covering’, ‘condensation’, and ‘differentiation’. A great amount of ECM was observed in these fish and can be temporally and regionally correlated with the appearance of the neural crest cells. No major differences could be detected between the two fish species with the exception that segregation and appearance of neural crest cells in various locations occur earlier in the platyfish. This time difference could lead to perturbations in neural crest cell development in certain platyfish-swordtail hybrids and may contribute to the formation of neural-crest-derived pigment cell tumours, melanomas, in these hybrids.

Certain platyfish, for example Xiphophorus maculatus, form a distinct pigment cell, macromelanophore, which after introgressive hybridization of the platyfish with the green swordtail, X. helleri, gives rise to hereditary melanomas in a Mendelian fashion (for review, see Vielkind & Vielkind, 1982; Vielkind et al. 1989). The genetics of this melanoma formation is well understood and a model has emerged for the normal functional role of identified genetic factors in the various steps in the developmental pathway of this pigment cell type. However, although it is clear that the macromelanophore, as for other pigment cells, originates from the neural crest (Humm & Young, 1956), to the best of our knowledge there is no study on neural crest formation and behaviour of neural crest cells in early embryogenesis in these fish. This is also true for fish in general and studies done so far all have been concerned with the fate of neural crest derivatives. Newth (1951, 1956) has shown in the lamprey using grafting and extirpation techniques that the head skeleton, dorsal root ganglia, melanophores and dorsal fin mesenchyme are derived from the neural crest, Lamers et al. (1981) have injected radioactively labelled crest cells into the trunk region of carp embryos and found similar pathways of migration in these fish as observed in other vertebrates, and recently Langille & Hall (1987, 1988) have mapped the contribution of the neural crest to the head skeleton in the Japanese medaka and lamprey.

Thus, there is a need to obtain basic information on neural crest development in fish. We have studied neural crest development in the two species X. maculatus and X. helleri with light and scanning electron microscopy from onset of neurulation with the aim to determine how it compares with that observed in other vertebrate species and whether or not differences in neural crest development can be found between the two species. These studies will form the basis for further studies on pigment cell developmental abnormalities. In addition, since studies on the neural crest are scarce in fish so that criteria for the identity of neural crest cells other than the association with the neural tube are not available, we have undertaken immunohistochemical staining using the HNK-1 antibody shown to be selective for neural crest cells in their early migratory phase in chicken (Bronner-Fraser, 1986) and also in newt (Tucker et al. 1984).

Fish and isolation of embryos from pregnant females

Two species of the viviparous cyprinodont fish, X. maculatus (platyfish) and Xiphophorus helleri (swordtail), were used in this study. From the platyfish two strains were studied, strain Jpl63B (kindly supplied by Dr Kaliman, New York Aquarium) and strain Sr’; they differ only in the extent of macromelanophore pattern formation (for a detailed description of the strains see Anders et al. 1973 and Kallman, 1975). Both species have an ovarian cycle of four weeks and give birth to 10–40 young, depending on the age of the female. A new set of oocytes matures and is fertilized after 7 days by sperm stored in the ovarian tract (Scrimshaw, 1945; Tavolga, 1949). This allows easy calculation of the theoretical age of the embryos

Pregnant females were sacrificed by decapitation between 11 and 13 days after the last brood, the ovaries were removed, washed in modified PBS and the embryos were separated from the ovarian tissue (Vielkind & Vielkind, 1983). Some of the embryos was cultured according to methods described earlier, which have been proven not to have any effect on embryonic development (Vielkind & Vielkind, 1983). Staging of the embryos was done using criteria described by Tavolga (1949). A description of criteria for embryonic development upon which staging was based is given in Table 1.

Table 1

Characteristic features of the various stages in embryogenesis of Xiphophorus maculatus and X. helleri (see Tavolga, 1949)

Characteristic features of the various stages in embryogenesis of Xiphophorus maculatus and X. helleri (see Tavolga, 1949)
Characteristic features of the various stages in embryogenesis of Xiphophorus maculatus and X. helleri (see Tavolga, 1949)

Scanning electron microscopy (SEM)

Embryos of stages 8–16 were fixed for 6 to 24 h in half-strength Kamovsky’s fixative (2·5% glutaraldehyde and 2% paraformaldehyde, 0-1 M-cacodylate (pH 7·3) (Kamovsky, 1965)). To preserve glycosaminoglycans (GAGs), 0·5% cetyl-pyridinium chloride (CPC) (Derby & Pintar, 1978) was added to the Kamovsky fixative for the fixation of some embryos.

Prior to fixation, an incision was made into the yolk sac, which later helped to separate the ectoderm. The embryos were then rinsed in 0·1 M-cacodylate buffer (pH 7·3) and postfixed in 1% osmium in the same buffer for 1 h, dehydrated through a graded series of ethanol, and critical-point dried using CO2 as transition fluid. Dried specimens were mounted on aluminium stubs using double-stick tape. The ectoderm was removed using a small piece of tape and mounted on the same stub for further examination of its inner surface. The specimens were viewed at 20 kV in a Cambridge 250T scanning electron microscope. In total, 73 platyfish and 77 swordtail embryos were examined.

Light microscopy (LM)

Embryos of stages 8·12 were fixed for 15 min in Kamovsky’s fixative with or without 0·5% CPC after removal of the yolk in most cases. The specimens were postfixed in 1% osmium, dehydrated in ethanol and propylene oxide, embedded in Epon 812, and serial semithin sections were cut on a Reichert ultramicrotome and stained in 0·1% toluidine blue in borax. The sections were viewed and photographed on a Zeiss photomicroscope.

Fluorescence microscopy

Embryos were fixed in 4% paraformaldehyde, embedded in paraffin, and serially sectioned (6 pm). Sections were deparaffinized, hydrated, washed in PBS, and incubated with 1:50 dilution HNK-1 monoclonal antibody (Becton-Dickinson) overnight at 4°C. After washing in PBS, sections were incubated for 1 h with rabbit anti-mouse IgM antibody, followed by 1 h incubation with FITC-conjugated goat anti-rabbit IgG antibody, rinsed in PBS and coverslipped. In control experiments, the HNK-1 antibody was replaced with mouse whole molecule IgM. Sections were analysed with a Zeiss epifluorescence photomicroscope.

Neurulation and indication of neural crest formation in the platyfish

It is generally known that neurulation in teleost fish differs from that in other vertebrates. Instead of the neural tube, a solid cord of cells called the neural keel is initially formed. This has been also observed for the teleosts studied here. In further embryonic development as early as late stage 9 (see Fig. 3A) a cavity, the neurocoel, forms in the neural keel in the head and progressively also in the trunk region; the neural keel is then referred to as the neural tube. Fig. 1A shows a cross-section of a stage-7 Xiphophorus maculatus (platyfish) embryo (for characterization of embryonic stages, see Table 1) in which a massive cell structure can be seen representing the neural keel. The cells that are located at the border of the ectoderm and the neural keel are presumed to represent the neural crest primordium, but the limit and extent of the neural crest primordium is not discernable from the surrounding tissues at this stage.

Fig. 1

Neural crest primordium and segregation of cells in Xiphophorus maculatus (platyfish) embryos at stages 7 and 8. (A–C) Light micrographs. (A) Platyfish embryo at stage 7: neural crest primordium at junction between ectoderm and neural keel. (B,C) Platyfish embryo at stage 8 (3 somites): putative neural crest cell masses appear

(B) laterally between the mesencephalon and the optic bud and (C) under the ectoderm in the more posterior regions. Ec, ectoderm; Ms, mesoderm; NCc, neural crest cells;

NCP, neural crest primordium; NK, neural keel; OB, optic buds; Y, yolk. Bar, 50 μm.

Fig. 1

Neural crest primordium and segregation of cells in Xiphophorus maculatus (platyfish) embryos at stages 7 and 8. (A–C) Light micrographs. (A) Platyfish embryo at stage 7: neural crest primordium at junction between ectoderm and neural keel. (B,C) Platyfish embryo at stage 8 (3 somites): putative neural crest cell masses appear

(B) laterally between the mesencephalon and the optic bud and (C) under the ectoderm in the more posterior regions. Ec, ectoderm; Ms, mesoderm; NCc, neural crest cells;

NCP, neural crest primordium; NK, neural keel; OB, optic buds; Y, yolk. Bar, 50 μm.

First appearance and segregation of neural crest cells

By stage 8, in the anterior part of the platyfish embryo, ridges of cells are visible on the dorsolateral sides of the neural keel. This position is occupied by the neural crest in other vertebrates, suggesting that this ridge of cells may represent the neural crest.

By advanced stage 8 (3 somites), the presumptive neural crest is also formed in the posterior part of the embryo. At the same time in the anterior cranial region (Fig. 1B), a layer of cells is located between the neural keel and the closely attached ectoderm and masses of cells have already segregated from the ridges and can be found laterally over the neural keel. On the left in Fig. 1B, the ridge can be seen to be arising from the dorsolateral aspect of the neural keel. In the posterior presumptive rhombencephalon region, segregation is not so intense (Fig. 1C) and in the trunk region no segregation is observed from the neural keel. Thus, it appears that the formation of the neural crest and the segregation of its cells follow an anterior-posterior gradient.

Segregation and location of neural crest cells in the cranial region

By stage 9, neural crest cell segregation intensifies in all cranial regions (Fig. 2A–D). In the anterior part of the embryo, a group of cells can be seen in the furrow between the prosencephalon and the optic vesicle (Fig. 2A). At higher magnification (Fig. 2B), it can be clearly seen that the long axes of these cells are oriented in an anterior-posterior direction and some of them are connected dorsally to the mesencephalon suggesting that they have originated at the dorsal midfine of the mesencephalon (see also Fig. 3B). They are the only ones to segregate dorsally whereas in the other head regions, the crest cells segregate dorsolaterally from the neural keel (Fig. 2A,C). It is important to note that the neural crest cells of the cranial region segregate in masses rather than as individual cells.

Fig. 2

Segregation and location of neural crest cells in the cranial regions of platyfish embryos at stage 9 and 11. (A-F) SEM micrographs. (A) Part of head of a platyfish embryo at stage 9 (6 somites), from which the ectoderm is removed: in mes- and rhombencephalon region presumptive crest cells are expanded over the mesenchymal cells. (B) Optic area (inset in A), at higher magnification: elongated crest cells appear in the furrow between the optic vesicle and the prosencephalon (arrow indicates midline). (C) Part of head of a platyfish embryo at stage 9 (7 somites): more crest cells appear in all regions. (D) Optic area (inset in C) at higher magnification: crest cells can be seen over the optic vesicle.

(E) Anterior cranial region of a platyfish embryo at stage 11 (19 somites): stellar and flat cells cover the eyes and the brain, respectively. (F) Lateral view of the rhombencephalon region of a platyfish embryo at stage 11 (19 somites): cell aggregation behind the otic vesicle is covered by a layer of cells and a cord-like structure is connected to it (arrow). M, mesencephalon; OpV, optic vesicle; OtV, otic vesicle; P, prosencephalon; Rh, rhombencephalon. Bar, 20 μm.

Fig. 2

Segregation and location of neural crest cells in the cranial regions of platyfish embryos at stage 9 and 11. (A-F) SEM micrographs. (A) Part of head of a platyfish embryo at stage 9 (6 somites), from which the ectoderm is removed: in mes- and rhombencephalon region presumptive crest cells are expanded over the mesenchymal cells. (B) Optic area (inset in A), at higher magnification: elongated crest cells appear in the furrow between the optic vesicle and the prosencephalon (arrow indicates midline). (C) Part of head of a platyfish embryo at stage 9 (7 somites): more crest cells appear in all regions. (D) Optic area (inset in C) at higher magnification: crest cells can be seen over the optic vesicle.

(E) Anterior cranial region of a platyfish embryo at stage 11 (19 somites): stellar and flat cells cover the eyes and the brain, respectively. (F) Lateral view of the rhombencephalon region of a platyfish embryo at stage 11 (19 somites): cell aggregation behind the otic vesicle is covered by a layer of cells and a cord-like structure is connected to it (arrow). M, mesencephalon; OpV, optic vesicle; OtV, otic vesicle; P, prosencephalon; Rh, rhombencephalon. Bar, 20 μm.

Fig. 3

Origin and appearance of neural crest cells in cranial regions of a platyfish embryo at late stage 9 (9 somites). (A-E) Light micrographs of cross-sections. (A,B) Optic region: crest cells (arrows) can be seen in the space (A) between the prosencephalon, the optic vesicle and the optic stalk and (B) between the mesencephalon and the optic vesicles. Note the site of origin of the crest cells on the mesencephalon (arrowheads). (C,D) Preotic and (F) postotic region: neural crest cell condensations can be seen rostrally and caudally of the otic vesicle (arrows). (E) Otic region: no neural crest cells are observed. Note: the planes of these sections are marked in Fig. 5A. En, endoderm; M, mesencephalon; Ms, mesoderm; OpV, optic vesicle; OtV, otic vesicle; P, prosencephalon; Pl, placodal thickening; Rh, rhombencephalon. Bar, 50 μm.

Fig. 3

Origin and appearance of neural crest cells in cranial regions of a platyfish embryo at late stage 9 (9 somites). (A-E) Light micrographs of cross-sections. (A,B) Optic region: crest cells (arrows) can be seen in the space (A) between the prosencephalon, the optic vesicle and the optic stalk and (B) between the mesencephalon and the optic vesicles. Note the site of origin of the crest cells on the mesencephalon (arrowheads). (C,D) Preotic and (F) postotic region: neural crest cell condensations can be seen rostrally and caudally of the otic vesicle (arrows). (E) Otic region: no neural crest cells are observed. Note: the planes of these sections are marked in Fig. 5A. En, endoderm; M, mesencephalon; Ms, mesoderm; OpV, optic vesicle; OtV, otic vesicle; P, prosencephalon; Pl, placodal thickening; Rh, rhombencephalon. Bar, 50 μm.

By late stage 9 in the anterior head region, cells are observed over the optic vesicle with their filopodia attached to the lateral wall of the prosencephalon and to the dorsal part of the optic vesicle (Fig. 2C,D). Progressively, some flat cells appear further laterally over the optic vesicle in the narrow space between the ectoderm and the optic vesicle which can be seen in the overall view of the embryo taken at a different angle (Fig. 5A). At this stage, the most distal part of the optic vesicle remains free of neural crest cells, presumably due to the close apposition of the ectoderm, which can be seen in the cross-sections of this region (Fig. 3A,B). The cells posterior to the optic vesicle appear as a network expanding laterally over the neural keel and the mesenchymal cells (Fig. 2A,C). Most of these cells appear elongated, are oriented perpendicular to the neural keel axis and show lamelli- and filopodia. With these structures, they are connected to the brain as well as to each other and it seems as if they might also have been connected to the ectoderm, which has been removed.

From stage 10 onwards, large numbers of cells are seen in the cranial region and one can observe that the morphology of these cells is now changing more drastically. Generally, two kinds of cells can be observed; flat cells occupy the dorsal and distal parts of brain and the optic vesicles, and stellar cells fill the space between the brain and the optic vesicles. In the anterior part of head, however, cells were observed to cover the eye, and have already spread to the anterior portion of the eye and to the prosencephalon (Fig. 2E). At higher magnification (data not shown), one can observe that the cells of the optic area show a stellar morphology and many of their cell processes are intermingled with the extracellular matrix (ECM) fibrils present in this region. The mesencephalon is now also covered with the flat cells. These cells are in close contact with each other so that their boundaries cannot be distinguished, and cover the mesencephalon as a thin layer of cells (Fig. 2E). A layer of cells (Fig. 2F) is also seen posteriorly to the otic vesicle covering an aggregation of cells, which can be first observed at late stage 9 and is best demonstrated in Fig. 5A in which also an anteriorly located condensation of cells of the otic vesicle can be observed. The cord-like structure seen in the later stages (Fig. 2F) seems to be connected to the posterior cell aggregation. Similar cell aggregations have been observed in zebrafish (Metcalf et al. 1985) and have been interpreted to represent the primordia of the anterior and posterior lateral line ganglia and the cord-like structure as representing the posterior lateral line primordium.

Origin and location of neural crest cells and relationship to other structures in cranial region as observed in cross-sections

In order to reveal the origins and locations of the crest cells in more detail, cross-sections were made of embryos of the same stages in the regions of the various brain divisions. In the prosencephalon region, the crest cells are located between the brain and the optic vesicles and some dorsally between the ectoderm and the optic vesicles (Fig. 3A). These cells appear not to have originated, however, from the prosencephalon, but rather from the dorsal midline of the mesencephalon as is also seen in the next figure (Fig. 3B) and as already observed in the SEM micrographs discussed above (Fig. 2A-D). In the mesencephalon region, neural crest cells appear lateral to the neural keel (Fig. 3B). Fig. 3C,D,E,F shows sections around the otic vesicle. The condensations of crest cells mentioned above are now more obvious and there seems to be a close association of these cells with the cephalic placodes suggesting a dual origin of the ganglia (Fig. 3D) as was also observed in the chick (Le Douarin, 1986) and in Xenopus (Sadaghiani & Thiebaud, 1987). In this figure, it can also be seen that crest cells are located over the mesoderm. In the preotic region, the crest cells are observed in close contact with the endoderm that develops into the pharyngeal pouches (Fig. 3C,D). In the area where there is a close association between the otic vesicle and the brain region, no crest cells are observed (Fig. 3E).

Segregation and location of neural crest cells in the trunk region

The first signs of neural crest cell segregation in the trunk region of the platyfish can be observed in late stage 9 in the area of the first 4 of the 9 somites (Fig. 5A). The behaviour and other characteristics of these early segregating cells can be seen in more detail in the SEM micrographs shown in Fig. 4A-F. Some neural crest cells anterior to the first somite appears to segregate individually from the neural keel in a lateral direction (Fig. 4A). After turning in a rostral or caudal direction, they become aligned with the longitudinal axis of the neural keel (Fig. 4B). They are connected to the neural keel and to each other with lamelli- and filopodia and intermingle with the ECM fibrils. In contrast, the cells between the first and the fourth somite, as the cells in the head region already described, segregate simultaneously in groups and extend towards the intersegmental grooves of consecutive somites (data not shown).

Fig. 4

Segregation, location and characteristics of neural crest cells in the postotic and trunk region of platyfish embryos at late stage 9 and stage 11. (A-F) SEM micrographs. (A,B) Postotic-trunk region of a platyfish embryo at late stage 9 (9 somites): neural crest cells (A) in the postotic region appear to aggregate posterior to the otic vesicle while those (B) in the area of the first somite are aligned parallel to the longitudinal axis of the neural keel. (C,D) Dorsal edge of the neural keel at the level of the somites 6 and 7 and of the unsegmented mesoderm of a platyfish embryo at late stage 9 (11 somites): mass of individual neural crest cells (C) presumably in the process of segregation whereas (D) individualization of cells has just begun. (E) Postotic and anterior trunk region of a platyfish embryo at late stage 9 (11 somites): postotic crest cells have aggregated to form the presumed posterior lateral line ganglion; cells in the anterior trunk region have increased in number and are aligned over the somites (arrowheads).

(F) Same region of a platyfish embryo as in E, but at stage 11 (19 somites): crest cells can be observed in the postotic-trunk region over the mesoderm; in the trunk region cells appear between the ectoderm and the somites (arrows). Note the cord-like structure (presumptive lateral line primordium) originated from rhombencephalon region (arrow). (G,H) Light micrographs. (G) Trunk region of a platyfish embryo at late stage 9 (10 somites): few crest cells are observed between the neural keel and the somites.

(H) Trunk region of a platyfish embryo at stage 11 (19 somites): presence of many crest cells between the neural tube and somites. The fine meshwork around the somite is here easily visible because of the CPC added during fixation of the embryo. Ec, ectoderm; NCc, neural crest cells; NK, neural keel; NT, neural tube; OtV, otic vesicle; PLL, posterior lateral line ganglion; Rh, rhombencephalon; S, somite; uM, unsegmented mesoderm. Bar, 10 μm.

Fig. 4

Segregation, location and characteristics of neural crest cells in the postotic and trunk region of platyfish embryos at late stage 9 and stage 11. (A-F) SEM micrographs. (A,B) Postotic-trunk region of a platyfish embryo at late stage 9 (9 somites): neural crest cells (A) in the postotic region appear to aggregate posterior to the otic vesicle while those (B) in the area of the first somite are aligned parallel to the longitudinal axis of the neural keel. (C,D) Dorsal edge of the neural keel at the level of the somites 6 and 7 and of the unsegmented mesoderm of a platyfish embryo at late stage 9 (11 somites): mass of individual neural crest cells (C) presumably in the process of segregation whereas (D) individualization of cells has just begun. (E) Postotic and anterior trunk region of a platyfish embryo at late stage 9 (11 somites): postotic crest cells have aggregated to form the presumed posterior lateral line ganglion; cells in the anterior trunk region have increased in number and are aligned over the somites (arrowheads).

(F) Same region of a platyfish embryo as in E, but at stage 11 (19 somites): crest cells can be observed in the postotic-trunk region over the mesoderm; in the trunk region cells appear between the ectoderm and the somites (arrows). Note the cord-like structure (presumptive lateral line primordium) originated from rhombencephalon region (arrow). (G,H) Light micrographs. (G) Trunk region of a platyfish embryo at late stage 9 (10 somites): few crest cells are observed between the neural keel and the somites.

(H) Trunk region of a platyfish embryo at stage 11 (19 somites): presence of many crest cells between the neural tube and somites. The fine meshwork around the somite is here easily visible because of the CPC added during fixation of the embryo. Ec, ectoderm; NCc, neural crest cells; NK, neural keel; NT, neural tube; OtV, otic vesicle; PLL, posterior lateral line ganglion; Rh, rhombencephalon; S, somite; uM, unsegmented mesoderm. Bar, 10 μm.

Fig. 5

Segregation and location of crest cells in platyfish and swordtail embryo at late stage 9. (A-F) SEM micrographs. (A) Platyfish embryo (9 somites), (B) swordtail embryo (9 somites): comparing the head and trunk regions of these embryos, it can be seen that the neural crest cells are in a more advanced stage in the platyfish, and in the optic region the crest cells already have appeared on the optic vesicle. Arrows indicate aggregations, and arrowheads the segregation, of crest cells from the neural keel, dorsal to the border of consecutive somites. (C) Mesencephalon region (inset in B) at higher magnification of swordtail embryo shown in B: the cells are connected to the neural keel and presumably to the ectoderm with long processes (arrowheads) and to each other by lamellipodia (small arrowheads). (D) Transverse fracture of swordtail embryo at late stage 9 (9 somites) in the preotic region (same level as cross-section shown in Fig. 3D): crest cells are found over the mesoderm and are in close association with the placodal thickening and the endoderm. (E) Postotic-trunk region of a swordtail embryo at late stage 9 (9 somites): neural crest cells in the postotic region appear laterally, perpendicular to the longitudinal axis of the embryo. Those in the area of the first somite are aligned parallel to the neural keel. (F) Part of the trunk region in the area of somites 1, 2 and 3 of a swordtail embryo of the same stage as in B: crest cells segregate from the edge of the neural keel and seem to be directed towards the space between two consecutive somites (arrows), or seem to curve rostrally to find the next available space (above the second somite). Ec, ectoderm; En, endoderm; M, mesencephalon; Ms, mesoderm; NCc, neural crest cells; NK, neural keel; OpV, optic vesicle; OtV, otic vesicle; P, prosencephalon; Pl, placodal thickening; Rh, rhombencephalon; S, somite. Bar, 30 μm. Note: dotted lines in Fig. 5A indicate planes of sections shown in Fig. 3A-F.

Fig. 5

Segregation and location of crest cells in platyfish and swordtail embryo at late stage 9. (A-F) SEM micrographs. (A) Platyfish embryo (9 somites), (B) swordtail embryo (9 somites): comparing the head and trunk regions of these embryos, it can be seen that the neural crest cells are in a more advanced stage in the platyfish, and in the optic region the crest cells already have appeared on the optic vesicle. Arrows indicate aggregations, and arrowheads the segregation, of crest cells from the neural keel, dorsal to the border of consecutive somites. (C) Mesencephalon region (inset in B) at higher magnification of swordtail embryo shown in B: the cells are connected to the neural keel and presumably to the ectoderm with long processes (arrowheads) and to each other by lamellipodia (small arrowheads). (D) Transverse fracture of swordtail embryo at late stage 9 (9 somites) in the preotic region (same level as cross-section shown in Fig. 3D): crest cells are found over the mesoderm and are in close association with the placodal thickening and the endoderm. (E) Postotic-trunk region of a swordtail embryo at late stage 9 (9 somites): neural crest cells in the postotic region appear laterally, perpendicular to the longitudinal axis of the embryo. Those in the area of the first somite are aligned parallel to the neural keel. (F) Part of the trunk region in the area of somites 1, 2 and 3 of a swordtail embryo of the same stage as in B: crest cells segregate from the edge of the neural keel and seem to be directed towards the space between two consecutive somites (arrows), or seem to curve rostrally to find the next available space (above the second somite). Ec, ectoderm; En, endoderm; M, mesencephalon; Ms, mesoderm; NCc, neural crest cells; NK, neural keel; OpV, optic vesicle; OtV, otic vesicle; P, prosencephalon; Pl, placodal thickening; Rh, rhombencephalon; S, somite. Bar, 30 μm. Note: dotted lines in Fig. 5A indicate planes of sections shown in Fig. 3A-F.

By the end of stage 9, segregation of neural crest cells proceeds caudally towards the level of the 6-7th somite. Unlike the cells in the anterior regions, the crest cells here appear as a mass of individualized cells while still attached to the neural keel, before they segregate (Fig. 4C). In the most posterior region, this phenomenon cannot yet be observed (Fig. 4D).

While segregation of crest cells is progressing posteriorly, the elongated cells anterior to the first somite now appear as stellar cells and can be seen laterally over the tightly packed mesenchymal cells. They also appear to be coated with ECM fibrils (Fig. 4E). Those cells that were presumably the first to segregate in the somite region are observed between the somites and the neural keel (Fig. 4G), while more cells have segregated and are aligned with the apex of the somites (Fig. 4E).

During stages 10 and 11, in the area between the otic vesicle and the first somite, the neural crest cells cover the mesenchymal cells like a sheet, while other cells still segregate from the neural tube (Fig. 4F). In the trunk region, in addition to cells located between the neural tube and the somites (Fig. 4H), crest cells can be observed in a new location, laterally over the somites (Fig. 4F). The appearance of cells in both locations is presumably due to the widening of the spaces between the neural tube, the somites and the ectoderm and the appearance of ECM material. These events occur to the level of the 13-14th somite; posterior to this level, the neural crest cells are still in the phases of segregation and individualization.

During stage 12, crest cell segregation continues in all areas with the exception of the region of the unsegmented mesoderm and some of the cells appear in groups in the space between the neural tube and the somites (data not shown). We assume that these separated groups represent the primordia of the spinal ganglia as judged by their location which is similar to that found in other vertebrates studied (see Le Douarin, 1982; Erickson, 1986).

Neural crest development in platyfish and swordtail

The results described so far are concerned with neural crest development in the platyfish strain Sr’. We did not observe differences in any feature of neural crest development in the other platyfish strain. The two strains are virtually identical and differ only with respect to a pigment cell locus determining different extents of pigment patterns in the two strains. In order to obtain further basic information on neural crest development in the teleost fish we included in our studies also the swordtail since introduction of the pigment cell locus into this species through genetic crosses results in melanomas.

The SEM observations of the swordtail embryos at different stages reveal, in general, that neural crest development and the sequence of events is very similar if not identical to that of the platyfish. The only exception is that segregation, and as a consequence appearance, of neural crest cells occurs earlier in the platyfish. This feature can be seen in Fig. 5A and B which represent SEM micrographs of overall views of late stage-9 platyfish and swordtail embryos, respectively. For example, in the platyfish, cells have already covered part of the optic vesicle while, in the swordtail, the optic vesicle is still free of cells. Similarly, in the anterior trunk region of the platyfish, a greater number of cells have segregated than in the swordtail. In early stages, this is a general feature of the platyfish with respect to all the other regions. However, after stage 11, this difference between the two species is no longer apparent.

As indicated, the other features of neural crest development appear to be the same. For example, neural crest appearance and segregation follow the same anterior-posterior gradient as already shown for the platyfish. In addition, in the anterior head region of embryos of both species, elongated crest cells can be observed in the narrow space between the optic vesicle and the neural keel, some of them are still connected to the mesencephalon; these cells have already been described earlier for the platyfish (compare Fig. 5A and B with Fig. 2B which shows the optic region of a younger platyfish embryo). Other examples of similarities are shown in Fig. 5C,D. The network of cells discussed above (see Fig. 2) which spans the mes- and rhombencephalon region can also be seen in the overall picture and at higher magnification these cells appear also as elongated cells and are oriented perpendicular to the embryonic axis (Fig. 5C). That these cells in the swordtail appear to cover the mesenchymal cells can be seen in a transverse fracture of this embryonic region in the SEM (Fig. 5D). In the anterior trunk area, the crest cells, as already described in the platyfish embryo, were seen to be aligned with the longitudinal axis of the neural keel, while those of the vagal region rostrally to them are elongated perpendicular to the neural keel axis (Fig. 5E, compare with Fig. 4B). In the area of the first three somites, the segregating crest cells were found on the dorsolateral edge of the neural keel. Their migrating heads are usually oriented towards the inter-segmental grooves of the consecutive somites, while their bodies are still part of the neural keel (Fig. 5F).

HNK-1 immunostaining of sections

Neural crest cells are generally identified by their association with the neural tube and their ability to distance themselves from their point of origin. Our studies have identified cells that fulfill these criteria, and we have therefore classified these cells as neural crest cells. To further characterize these cells, we have immunostained serial cross-sections of embryos of stages 9-12 with the HNK-1 monoclonal antibody; this antibody recognized neural crest cells in chicken and newt in their early migratory phase (Tucker et al. 1984).

Examples of immunostained cross-sections of embryos of both species are shown in Fig. 6. Fig. 6A,B,C represent micrographs of immunostained cross-sections of a stage-9 swordtail embryo in the optic region, rhombencephalon and anterior trunk region. In the optic region (Fig. 6A), a heavy immunostaining can be observed on the cells that are located between the optic vesicles and the mesencephalon and are dorsally associated with the mesencephalon (compare with Fig. 3B). In the rhombencephalon region (Fig. 6B), the immuno-positive cells were found laterally over the mesenchymal cells and under the ectoderm, and are associated dorsally with the rhombencephalon (compare with Fig. 5D). Staining, however, was also found on the lateral walls of the neural keel (Fig. 6A,B). In the anterior trunk region (Fig. 6C), immunostaining was only observed in the cells which are associated with the dorsolateral edge of the neural keel (compare with Fig. 4G). Examples of the immunostaining of cross sections of an advanced stage, i.e. stage 12 of a platyfish embryo are shown in Fig. 6D,E,F. In the head regions, there were no positive cells dorsolaterally on the neural tube; the staining seemed to be restricted to the cells of the peripheral nervous system (Fig. 6D,E). Positive cells could only be detected on the dorsolateral aspect of the neural tube in the regions of the midtrunk, under the ectoderm, and between the somites and the neural tube (Fig. 6F, compare with Fig. 4F,H). In all these regions, we observed also staining of the ventral walls of the neural tube (Fig. 6D,E,F). In control sections that were immunoreacted with mouse IgM instead of HNK-1, no fluorescence has been observed at all.

Fig. 6

Identification of neural crest cells by HNK-1 antibody in swordtail and platyfish embryos at late stage 9 and late stage 12, respectively. (A-F) Fluorescence micrographs of cross-sections. (A-C) Swordtail embryo at late stage 9 (8 somites): immunopositive cells are (A) in the optic region associated with the mesencephalon and some appear between the mesencephalon and the optic vesicle, and within the walls of the neural tube and the optic vesicles, (B) in the rhombencephalon region located over the mesenchymal cells and under the ectoderm (arrows), and (C) in the anterior trunk region located only dorsolaterally to the neural keel. (D-F) Platyfish embryo at late stage 12 (20 somites): immunopositive staining can be seen (D) in the preotic and (E) postotic region only over the ganglia and in the ventral wall of the neural tube, and (F) in the midtrunk region, dorsolaterally of the neural tube, under the ectoderm (arrows) and between the somites and the neural tube (arrowheads). NK, neural keel; NT, neural tube; OpV, optic vesicle; M, mesencephalon; Ms, mesenchymal cells; Rh, rhombencephalon; S, somite. Bar, 50 μm.

Fig. 6

Identification of neural crest cells by HNK-1 antibody in swordtail and platyfish embryos at late stage 9 and late stage 12, respectively. (A-F) Fluorescence micrographs of cross-sections. (A-C) Swordtail embryo at late stage 9 (8 somites): immunopositive cells are (A) in the optic region associated with the mesencephalon and some appear between the mesencephalon and the optic vesicle, and within the walls of the neural tube and the optic vesicles, (B) in the rhombencephalon region located over the mesenchymal cells and under the ectoderm (arrows), and (C) in the anterior trunk region located only dorsolaterally to the neural keel. (D-F) Platyfish embryo at late stage 12 (20 somites): immunopositive staining can be seen (D) in the preotic and (E) postotic region only over the ganglia and in the ventral wall of the neural tube, and (F) in the midtrunk region, dorsolaterally of the neural tube, under the ectoderm (arrows) and between the somites and the neural tube (arrowheads). NK, neural keel; NT, neural tube; OpV, optic vesicle; M, mesencephalon; Ms, mesenchymal cells; Rh, rhombencephalon; S, somite. Bar, 50 μm.

Taken in sum, staining was found in those cells that we had identified in the SEM and LM studies described above as neural crest cells that were in progress of early segregation and migration and we did observe identical staining patterns in both species. As in the newt and chicken, some staining was found on other structures such as the neurones (Tucker et al. 1984) and, as was found in chicken (Bronner-Fraser, 1986), on the lateral and ventral walls of the neural tube.

Characteristics of cells that appear on the dorsal aspect of the trunk neural tube in advanced embryogenesis

While in the head region, neural crest segregation seems to be terminated by stage 10, segregation in the trunk region seems to continue even beyond stage 13. Up to this stage, we have observed on the dorsolateral aspect of the neural tube in the trunk region only cells that exhibit a round or elongated shape and possess a few filopodia. From stage 13 (23 somites) onwards, however, the picture becomes more complex as additional flat cells begin to accumulate on the dorsolateral sides of the neural tube. The boundaries of these flat cells are hardly recognizable and they bear very fine processes which extend across adjacent cells (Fig. 7B) and the dorsal midfine. During further development, stages 13 to 15 (Figs 7A; 8A), it appears that mainly the flat cells cover the dorsal part of the neural tube in characteristic phases which we describe as covering, condensation and differentiation phases. They progress in an anterior-posterior fashion; as a result all three phases can be observed in a stage-15 embryo in an anterior-posterior sequence.

Fig. 7

Onset of the covering phase in platyfish embryo at stage 13. (A-D) SEM micrographs. (A) Embryo at stage 13 (23 somites) at low magnification. Arrows indicate the enlarged areas in B,C,D. (B) Somite level 2-4: fiat cells possessing fine processes cover the dorsolateral part of the neural tube. (C) Somite level 8-10: flat and some round (arrows) cells cover only the lateral margins of the neural tube. (D) Somite level 12-16: covering has not yet progressed into this region, most of the cells are round in shape. A, anterior. Bar, 40 μm.

Fig. 7

Onset of the covering phase in platyfish embryo at stage 13. (A-D) SEM micrographs. (A) Embryo at stage 13 (23 somites) at low magnification. Arrows indicate the enlarged areas in B,C,D. (B) Somite level 2-4: fiat cells possessing fine processes cover the dorsolateral part of the neural tube. (C) Somite level 8-10: flat and some round (arrows) cells cover only the lateral margins of the neural tube. (D) Somite level 12-16: covering has not yet progressed into this region, most of the cells are round in shape. A, anterior. Bar, 40 μm.

The covering of the neural tube is intense in the anterior trunk region and diminishes gradually towards the posterior regions (compare Fig. 7B with 7C,D). With further embryonic development, covering proceeds and reaches the most posterior part of the neural tube by stage 15. As the cells appear dorsally, they exhibit thicker processes which are intermingled with the fibrils of the ECM that are aligned transversely to the neural tube (Fig. 8B). In addition, the dorsal midline is now also covered by these cells (Fig. 8C).

Fig. 8

Covering, condensation and differentiation phases of cells in a swordtail embryo at stage 15. (A-E) SEM micrographs. (A) Trunk of a swordtail embryo at stage 15 (25 somites) at low magnification: the characteristics of the phases can be observed even at this low magnification. The lines indicate the limits of the regions showing the various phases and the numbers correspond to the somite level. They are shown at higher magnification in B-E. Arrows in these micrographs mark the dorsal midline. (B,C) Posterior trunk region: covering phase has reached the posterior region. In B flat cells have almost completely covered the neural tube and some cells have already met each other (arrowheads). The ECM fibrils and the cell processes are aligned with each other and extend across the neural tube. Some round cells can also be seen (white arrowheads). In C flat cells have covered entirely the neural tube. Round cells exhibit one or two filopodia and can be seen over the flat cells (white arrowheads). (D) Mid-trunk region: covering phase is now followed by the condensation phase: cells are packed closely together. Round cells can be observed on the somites (arrowheads). (E) Anterior trunk region: condensation phase is followed by the final phase, the differentiation phase: extracellular spaces have developed between the cells. Dorsally projecting cell processes can be observed, presumably connected to the ectoderm, but have been disrupted during the removal of the ectoderm. A, anterior. Bar, 20 μm.

Fig. 8

Covering, condensation and differentiation phases of cells in a swordtail embryo at stage 15. (A-E) SEM micrographs. (A) Trunk of a swordtail embryo at stage 15 (25 somites) at low magnification: the characteristics of the phases can be observed even at this low magnification. The lines indicate the limits of the regions showing the various phases and the numbers correspond to the somite level. They are shown at higher magnification in B-E. Arrows in these micrographs mark the dorsal midline. (B,C) Posterior trunk region: covering phase has reached the posterior region. In B flat cells have almost completely covered the neural tube and some cells have already met each other (arrowheads). The ECM fibrils and the cell processes are aligned with each other and extend across the neural tube. Some round cells can also be seen (white arrowheads). In C flat cells have covered entirely the neural tube. Round cells exhibit one or two filopodia and can be seen over the flat cells (white arrowheads). (D) Mid-trunk region: covering phase is now followed by the condensation phase: cells are packed closely together. Round cells can be observed on the somites (arrowheads). (E) Anterior trunk region: condensation phase is followed by the final phase, the differentiation phase: extracellular spaces have developed between the cells. Dorsally projecting cell processes can be observed, presumably connected to the ectoderm, but have been disrupted during the removal of the ectoderm. A, anterior. Bar, 20 μm.

The condensation phase can be observed in the area of somites 8-16 of an embryo at stage 15. It is characterized by an increase of cells which are densely packed and in intimate contact with each other. The cells in this phase generally possess short cell processes. Those cells on the midline form a band of elevated cells possessing dorsally projected cell processes (Fig. 8D).

The differentiation phase can be observed in the most anterior part of the trunk region, between somites 1 and 7 of the same stage-15 embryo. Two important changes have occurred that distinguish this phase from the condensation phase. Extracellular spaces can be found between cells and it is possible to identify individual cells. The morphology of the cells has changed from a flat shape to an irregular stellar one. At the midline, the cells are projected dorsally and are more closely packed; thus less extracellular space can be seen between them (Fig. 8E). The cells are arranged in several layers which can be seen in the fracture of an embryo shown in Fig. 9A.

Fig. 9

Appearance of neural crest cells in the anterior trunk region and inner ectoderm of swordtail embryo stage 15. (A-C) SEM micrographs. (A) Transverse fracture of the anterior trunk region at the first somite: many cells are observed between the neural tube and the ectoderm (arrowheads). (B) Anterior trunk region at somites 13 and 14: many cells are seen in the grooves between consecutive somites. (C) The inner surface of the ectoderm: many cells (arrowheads) seem to be attached to the ectoderm and some of them may be part of lateral line primordium (arrow). The ridges of the ectoderm mark the intersomitic grooves. A, anterior; Ao, aorta; D, dorsal; I, intestine; Mn, mesentry; N, notochord; NT, neural tube; Pn, pronephric duct; S, somite. Bar, 20 μm.

Fig. 9

Appearance of neural crest cells in the anterior trunk region and inner ectoderm of swordtail embryo stage 15. (A-C) SEM micrographs. (A) Transverse fracture of the anterior trunk region at the first somite: many cells are observed between the neural tube and the ectoderm (arrowheads). (B) Anterior trunk region at somites 13 and 14: many cells are seen in the grooves between consecutive somites. (C) The inner surface of the ectoderm: many cells (arrowheads) seem to be attached to the ectoderm and some of them may be part of lateral line primordium (arrow). The ridges of the ectoderm mark the intersomitic grooves. A, anterior; Ao, aorta; D, dorsal; I, intestine; Mn, mesentry; N, notochord; NT, neural tube; Pn, pronephric duct; S, somite. Bar, 20 μm.

While the above mentioned cells advance in their development through the three phases, round cells bearing a few filopodia can be observed to appear among and over those covering the neural tube in almost all trunk regions. In addition, some cells could be observed on the apex of the somites, between the somites and the ectoderm, and in the lateral grooves between consecutive somites. The latter cells are round in shape, while a few, which seem to remain on the lateral part of the somites, usually exhibit a flat or elongated shape (Figs 8C,D and 9B). It should be mentioned that at these advanced embryonic stages, many cells remain attached to the inner surface of the ectoderm when it is removed (Fig. 9C).

Temporal and regional appearance of the extracellular matrix (ECM)

Since it has been proposed that the structure of the ECM may have some influence on the neural crest cell migration and behaviour (Loefberg et al. 1980), we also describe observations on the ECM structure of embryos made in our SEM studies.

A few strands of ECM fibrils can be seen on the neural keel and under the ectoderm in regions where the neural crest cells segregate. The fibrils increase in number during segregation of the crest cells (Fig. 4A,B). Similarly, ECM material appears in other locations seemingly preceding the appearance of crest cells. For example, ECM fibrils can be observed on the somites at the onset of crest cell segregation in the trunk region (Fig. 4E,F). The ECM increases when crest cells appear on the somites (compare Fig. 4A,B with Fig. 4E,F). More ECM fibrils are present at the lateral sides of the somites and, in particular, in the lateral grooves where they bridge consecutive somites (Fig. 10A). At stage 11, when the crest cells appear in this area, some cells can be observed to be oriented towards these grooves (Fig. 10A). These cells increase in number in later stages (13-15) while the amount of fibrils seems to have declined (Fig. 7B). By stage 16, the surface of somites is free of ECM material again (data not shown).

Fig. 10

Appearance of ECM and association of ECM with neural crest cells in swordtail and platyfish embryos.

(A-G) SEM micrographs. (A) Platyfish embryo at stage 11: ECM fibrils cover the somites and fill the laterai grooves between the somites and crest cells seem to be oriented towards these grooves. (B,C) Platyfish embryo at stage 9: ECM fibrils are less abundant on the inner surface of B the head ectoderm than C the trunk ectoderm. (D) Swordtail embryo at stage 13: a thick layer of ECM fibrils is observed at the onset of the covering phase covering the cells which appeared dorsally on the neural tube. (E) Swordtail embryo at stage 16: a dense ECM is seen on the inner surface of the dorsal ectoderm intermingled with the cells. (F) Platyfish embryo at stage 11: neural cells are intermingled with the ECM fibrils and spherical bodies (arrowheads). (G) Platyfish embryo at stage 11: CPC-treated specimens show a heavy fibre-associated precipitation. Ec, ectoderm; ECM, extracellular matrix; NCc, neural crest cells; NT, neural tube. Bar, 10 μm.

Fig. 10

Appearance of ECM and association of ECM with neural crest cells in swordtail and platyfish embryos.

(A-G) SEM micrographs. (A) Platyfish embryo at stage 11: ECM fibrils cover the somites and fill the laterai grooves between the somites and crest cells seem to be oriented towards these grooves. (B,C) Platyfish embryo at stage 9: ECM fibrils are less abundant on the inner surface of B the head ectoderm than C the trunk ectoderm. (D) Swordtail embryo at stage 13: a thick layer of ECM fibrils is observed at the onset of the covering phase covering the cells which appeared dorsally on the neural tube. (E) Swordtail embryo at stage 16: a dense ECM is seen on the inner surface of the dorsal ectoderm intermingled with the cells. (F) Platyfish embryo at stage 11: neural cells are intermingled with the ECM fibrils and spherical bodies (arrowheads). (G) Platyfish embryo at stage 11: CPC-treated specimens show a heavy fibre-associated precipitation. Ec, ectoderm; ECM, extracellular matrix; NCc, neural crest cells; NT, neural tube. Bar, 10 μm.

The inner surface of the ectoderm in all regions is decorated by a fine meshwork of ECM fibrils as early as stage 9. This seems to increase gradually with the age of the embryo and appears to be more dense in the trunk (Fig. IOC) than in the head region (Fig. 10B). It is also more abundant in the lateral parts than in the mid-dorsal region. From stage 13 onwards, concurrently with the appearance of crest cells on the dorsal part of the neural tube (covering phase), a thick layer of densely interwoven ECM fibrils can be seen under the ectoderm (Fig. 10D,E). At the same time, the amount of subectodermal ECM in the lateral regions begins to decrease.

In order to obtain additional information about the ECM, some specimens were fixed in Kamovsky’s fixative supplemented with cetylperidinium chloride (CPC). This compound presumably retains glycos-aminoglycans (GAGs) resulting in a better image of the ECM (Pratt et al. 1975). The ECM of an embryo fixed without CPC appears as a network of thin fibrous material which carries tiny spherical bodies (diameter 0·5𠄷1·0 μm) (Fig. 10F) whereas the ECM of a CPC-treated embryo appears as a heavy fibre-associated precipitation, suggesting a very high content of GAGs in the ECM (Fig. 10G). We assume that is was this precipitate that did not allow the removal of the ectoderm from CPC-treated embryos of other stages suggesting that also in these stages of ECM has a high content of GAGs.

The overall observations in both platyfish and sword-tail embryos suggest that the ECM is temporally and regionally correlated with the appearance of the crest cells. In addition, in numerous cases, the processes of the crest cells seem to be connected to, and aligned with, the ECM fibrils. However, we did not observe a consistent orientation of neural crest cells in the direction of the ECM fibrils.

Using scanning electron and fight microscopy we have studied the development of neural crest in Xiphophorus fish (platyfish and swordtails). In similar studies of other vertebrates, cells have been recognized as neural crest cells by their association with the neural tube and subsequent segregation from this structure (Tosney, 1978, 1982; Loefberg et al. 1980; Anderson & Meier, 1981; Erickson & Weston, 1983; Spieth & Keller, 1984; Tan & Morriss-Kay, 1985; Sadaghiani & Thiebaud, 1987); we have made similar observations in these fish. Furthermore, radioactively labelled grafts (Weston, 1963) and nuclear markers (Le Douarin, 1982; Sadaghiani & Thiebaud, 1987) have been used to prove the identity of these cells. More recently, antibodies such as NC-1 and HNK-1 were recognized to be selective for neural crest cells in their migratory phase (Tucker et al. 1984; Vincent & Thiery, 1984; Bronner-Fraser, 1986). Because of the lack of nuclear markers in these fish and because grafting experiments as well as the use of tracer dyes are limited due to the small size of the embryos as well as their cells, we have decided to use the HNK-1 antibody to further document the identity of cells which we classified as neural crest cells by light and scanning electron microscopy.

After neural keel formation is complete, ridges of cells appeared on the lateral sides of the keel from which cells segregate; the formation of these ridges as well as the segregation followed an anterior-posterior gradient. These observations are similar to those made in other vertebrates (see Erickson, 1986) and we have interpreted therefore this cell population as neural crest cells. In support of this interpretation is the staining of these cell groups with the HNK-1 antibody as well as studies of Newth (1951, 1956) and Langille & Hall (1987) in the lamprey and by Langille & Hall (1988) in the Japanese medaka. These researchers ablated the dorsal structure of the neural keel and observed alterations in the appearance of neural-crest-derived structures such as head skeleton, cephalic nerves, head mesenchyme, and melanocytes.

Segregation of the neural crest cells occurred with a few exceptions from the dorsolateral aspect of the neural keel. This is in marked contrast to observations made in other vertebrates where segregation takes place from the dorsal midline. Only in the head region of a few organisms, Xenopus (Sadaghiani & Thiebaud, 1987), mouse (Nichols, 1981), and rat (Tan & Morriss-Kay, 1985) do neural crest cells segregate laterally from the neural tube. The lateral segregation behaviour in these fish might be the consequence of the neurulation process during which the ectoderm remains closely attached to the neural keel except in the mesencephalon region. As a result, neural crest cells find only enough space for segregation on the lateral sides and in the dorsal mesencephalon region in which we observed dorsally segregating cells.

In the head region, the neural crest cells can be distinguished from the mesenchymal cells for a short period of time, from stage 8 to 9. From stage 10 onwards many cells, which seem to cover the forming brain and the optic vesicles, appear in the head. These seem to be crest cells intermingled with the mesenchyme cells and they fill the spaces that are created by development of the brain. Therefore, in the head region the crest cells can no longer be recognized or followed from this stage based on their morphology. In the trunk region, as the wave of crest cell segregation progresses posteriorly, newly segregating crest cells can be observed as late as stage 16. The segregated cells appear between the neural tube and the somites and between the somites and the ectoderm. These locations have been observed in other vertebrates studied and were described as neural tube-somite and somite-ectoderm migration pathways (see Hoerstadius, 1950; Le Douarin, 1982). The use of HNK-1 antibody on the cross-sections of embryos, also confirm the migration of crest cells in these two pathways. Of major interest to us are the numerous cells found associated with the ectoderm particularly between stages 14 and 16. It is interesting to note that the appearance of melanocytes in both platyfish and swordtail in the trunk region coincide with these stages. In newt (Epperlein, 1982; Tucker & Erickson, 1986), and also presumably in carp (Larners et al. 1981), the ectoderm-associated cells gave rise to pigment cells.

We have also observed many cells on the dorsal part of the neural tube in advanced embryogenesis. The observation that they appear on the dorsolateral aspect of the neural tube may suggest that the cells are of neural crest origin. The flat morphology of the majority of cells is, however, inconsistent with our observation that most segregated cells are round or elongated. However, shortly after segregation these cells migrate into the narrow space dorsally between the neural tube and the ectoderm which could cause their flat morphology. Alternatively, these cells could represent somitic sclerotome cells which migrate relatively late in embryogenesis dorsally onto the neural tube (see Loefberg et al. 1980). Ablation studies (see Hoerstadius, 1950) as well as grafting experiments in amphibia (Chibon, 1964; Sadaghiani & Thiebaud, 1987) and carp (Lamers et al. 1981) revealed that neural crest cells yield mesenchymal and pigment cells of the dorsal fin. Therefore, we assume that at least part of the cells on the dorsal aspect of the neural tube are of neural crest origin and represent the late-segregating cells. Additional experiments will have to clarify the origin of these dorsal cell populations. Similar observations, in particular with regard to the distinct morphological changes of the dorsal cells observed here, have not been yet described for the other organisms, perhaps because studies of such advanced stages have not been done and these features may appear only in late embryogenesis.

The appearance of crest cells in locations distant from the neural crest is considered to be the result of migration. Many aspects of this process have not yet been deciphered. An important question is whether or not the cells use existing spaces or create their own space. At the onset of segregation, crest cells are found in spaces created by the progressive development of the organ primordia. For example, in the trunk region, the somites provide space dorsally and laterally at their borders with each other and with the ectoderm. It is in these spaces that the newly segregated cells appear in large numbers. These organ primordia also seem to act as barriers. The close apposition of the otic placodes to the neural keel seems to allow segregation only rostrally and caudally. Conversely, cells appearing over the eye as well as those dorsally on the neural tube are flat and seem to push themselves beneath the ectoderm which is closely apposed to the underlying structures. Thus, it appears that in these fish the crest cells preferentially appear in prexisting spaces but also appear in quite narrow spaces suggesting that they may create their own space.

Another important component which has often been connected to neural crest cell migration and orientation is the surrounding environment, for example the ECM (see Loefberg et al. 1980). Our observations of the ECM do not reveal any overall orientation of its fibrils with the presumptive movement of the neural crest cells. Only dorsally on the neural tube was a parallel alignment of the fibrils and the cells seen, some of which we believe are of neural crest origin. Interestingly, in the lateral grooves the fibrils span adjacent somites and cell movement seemed to be directed across the fibrils. Thus, if the spatial organization of the fibrils influences neural crest cell migration, it does so in different ways in different areas of the embryo. We clearly observed an increase of ECM material before segregation in areas where cells could be found soon thereafter, suggesting an important temporal and regional association of the ECM and the neural crest cells. In many areas, we also observed an intermingling of ECM fibrils and crest cells which might indicate an interrelationship between the ECM and these cells.

Although we detected some previously undescribed characteristics of neural crest formation and subsequent crest cell migration, the overall picture is the same as that described for other vertebrates. One of the important questions for our study was whether or not differences in neural crest features could be found between the two fish species. Clearly, our studies did not reveal any obvious or major differences. However, the finding that the formation of the neural crest, the onset of segregation as well as the migration of crest cells, occurs earlier in the platyfish than in the swordtail is important with respect to our major interest. Melanoma formation occurs in segregants of introgressive hybrids of these two species in a Mendelian fashion (Vielkind & Vielkind, 1982; Vielkind et al. 1988). The melanoma cell is derived from a pigment cell produced only by the platyfish; this cell presumably escapes regulation in the backcross hybrids. Many studies (see Le Douarin, 1982) suggest that the fate of neural crest cells is influenced by their surroundings, i.e. ECM or the available space. Mistiming of neural crest cell segregation and migration in melanoma hybrids could lead to the appearance of cells in an inappropriate microenvironment where incorrect environmental cues could result in further unordered neural crest cell behaviour and, finally, abnormal pigment cell development. It is interesting to note that murine embryonic skin can ‘regulate’, i.e. inhibit growth of B16 melanoma cells, but this regulation is correlated with the time of arrival of normally migrating premelanocytes into the skin (Gerschenson et al. 1986). Thus abnormal timing of appearance of pigment cell precursors could contribute to melanoma formation. Having established normal development in Xiphophorus, future studies should concentrate on the development of the neural crest in the hybrids.

The authors wish to thank Dr J. A. Weston for helpful discussion, Dr B. Crawford and Bruce Woolcock for critical reading, and Barbara Schmidt for preparation of the manuscript. This work was supported by grants to JRV from the Medical Research Council of Canada and the National Institutes of Health (USA). JRV is a Scholar of the Medical Research Council of Canada.

Anders
,
A.
,
Anders
,
F.
&
Klinke
,
K.
(
1973
).
Regulation of gene expression in the Gordon-Kosswing melanoma system. I. The distribution of the controlling genes in the genome of the xiphophorin fish, Platypoecilus maculatus and Platypoecilus vanatus
.
In Genetics and Mutagenesis of Fish
(ed.
J. H.
Schroeder
), pp.
33
52
.
Berlin, Heidelberg, NY
:
Springer-Verlag
.
Anderson
,
C. B.
&
Meier
,
S.
(
1981
).
The influence of the metameric pattern in the mesoderm on migration of the cranial neural crest cells in the chick embryo
.
Devi Biol
.
85
,
385
402
.
Bronner-Fraser
.
R. M.
(
1986
).
Analysis of the early stages of trunk neural crest migration in avian embryos using monoclonal antibody HNK-1
.
Devi Biol
.
115
,
44
55
.
Chibon
,
P.
(
1964
).
Analyse par la méthode de marquage nucléaire à la thymidine tritiée des dérivés de la crête neurale céphalique chez l’Urodèles Pleurodeles waltlii
.
C. r. hebd Séanc Acad. Sci
.
259
,
3624
7
.
Derby
,
M. A.
&
Pintar
,
J. E.
(
1978
).
The histochemical specificity of Streptomyces hyaluronidase and chondroitinase ABC
.
Histochem. J
.
10
,
529
537
.
Epperlein
,
H. H.
(
1982
).
Different distribution of melanophores and xantophores in early tailbud and larval stages of Triturus alpestris
.
Wilhelm Roux’s Arch, devl Biol
.
191
,
19
27
.
Erickson
,
C. A.
(
1986
).
Morphogenesis of the neural crest
.
In: Developmental Biology: A Comprehensive Synthesis
(ed.
L.
Browder
), pp.
481
542
.
New York
:
Plenum Publishing Company
.
Erickson
,
C. A.
&
Weston
,
J. A.
(
1983
).
An SEM analysis of neural crest migration in the mouse
.
J. Embryol. exp. Morph
.
74
,
97
118
.
Gerschenson
,
M.
,
Graves
,
K.
,
Carson
,
S. D.
,
Wells
,
R. S.
&
Pierce
,
G. B.
(
1986
).
Regulation of melanoma by the embryonic skin
.
Proc. natn. Acad. Sci. U.S.A
.
83
,
7307
7310
.
Hoerstadius
,
S.
(
1950
).
The Neural Crest, Its Properties and Derivatives in the Light of Experimental Research
.
London
:
Oxford University Press
.
Humm
,
D. G.
&
Young
,
R.
(
1956
).
The embryological origin of pigment cells in platyfish-swordtail hybrids
.
Zoológica
41
,
1
10
.
Kallman
,
K. D.
(
1975
).
The platyfish, Xiphophorus maculatus
.
In Handbook of genetics. Vol. 4
(ed.
R. C.
King
), pp.
81
132
.
New York
:
Plenum Press
.
Karnovsky
,
M. J.
(
1965
).
A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy
.
J. Cell Biol
.
27
,
137A
138A
.
Lamers
,
C. H. J.
,
Rombout
,
J. W. H. M.
&
Timmermans
,
L. P. M.
(
1981
).
An experimental study on neural crest migration in Barbus conchoius (Cyprinidae, Teleostei), with special reference to the origin of the enteroendocrine cells
.
J. Embryol. exp. Morph
.
62
,
309
323
.
Langille
,
R. M.
&
Hall
,
B. K.
(
1987
).
Role of the neural crest in development of the trabeculae and branchial arches in embryonic sea lamprey, Petromyzon marinus (L
.).
Development
102
,
301
310
.
Langille
,
R. M.
&
Hall
,
B. K.
(
1988
).
Role of the neural crest in development of the cartilagous cranial and visceral skeleton of the medaka, Oryzias latipes (Teleostei)
.
Anat. Embryol
.
177
,
297
305
.
Le Douarin
,
N. M.
(
1982
).
The Neural Crest
.
Cambridge
:
Cambridge University Press
.
Le Douarin
,
N. M.
(
1986
).
Ontogeny of the peripheral nervous system from the neural crest and the placodes. A developmental model studied on the basis of the quail-chick chimaera system
.
In The Harvey Lectures Series
80
,
137
186
. (Alan R. Liss, Inc.).
Loefberg
,
J.
,
Ahlfors
,
K.
&
Faellstroem
,
C.
(
1980
).
Neural crest migration in relation to extracellular matrix organization in the embryonic axolotl trunk
.
Devi Biol
.
15
,
148
167
.
Metcalfe
,
W. K.
,
Kimmel
,
CH. B.
&
Schabtach
,
E.
(
1985
).
Anatomy of the posterior lateral line system in young larvae of the zabrafish
.
J. comp. Neurol
.
233
,
377
389
.
Newth
,
D. R.
(
1951
).
Experiments on the neural crest of the lamprey embryo
.
J. exp. Morph
.
28
,
247
260
.
Newth
,
D. R.
(
1956
).
On the neural crest of the lamprey embryo
.
J. Embryol. exp. Morph
.
4
,
358
375
.
Nichols
,
D. H.
(
1981
).
Neural crest formation in the head of the mouse embryo as observed using a new histological technique
.
J. Embryol. exp. Morph
.
64
,
105
120
.
Pratt
,
R. M.
,
Larsen
,
M. A.
&
Johnston
,
M. C.
(
1975
).
Migration of cranial neural crest cells in a cell-free hyaluronate-rich matrix
.
Devi Biol
.
44
,
298
305
.
Sadaghiani
,
B.
&
Thiebaud
,
C. H.
(
1987
).
Neural crest development in the Xenopus laevis embryo, studied by interspecific transplantation and scanning electron microscopy
.
Devi Biol
.
124
,
91
110
.
Scrimshaw
,
N. S.
(
1945
).
Embryonic development in poeciliid fishes
.
Biol. Bull. mar. biol. Lab. Woods Hole
88
,
233
246
.
Spieth
,
J.
&
Keller
,
R. E.
(
1984
).
Neural crest cell behavior in white and dark larvae of the Ambystoma mexicanum: Differences in cell morphology, arrangement, and extracellular matrix as related to migration
.
J. exp. Zool
.
229
,
91
107
.
Tan
,
S. S.
&
Morriss-Kay
,
G.
(
1985
).
The development and distribution of the cranial neural crest in the rat embryo
.
Cell Tissue Res
.
240
,
403
416
.
Tavolga
,
W. N.
(
1949
).
Embryonic development of the platyfish (platypoecilus), the swordtail (xiphophorus) and their hybrids
.
Bull. Amer. Mus. Nat. Hist
.
94
,
165
229
.
Tosney
,
K. W.
(
1978
).
The early migration of neural crest cells in the trunk region of the avian embryo: an electron microscopic study
.
Devi Biol
.
62
,
317
333
.
Tosney
,
K. W.
(
1982
).
The segregation and early migration of cranial neural crest cells in the avian embryo
.
Devi Biol
.
89
,
13
24
.
Tucker
,
G. C.
,
Aoyama
,
H.
,
Lipinski
,
M.
,
Tursz
,
T.
&
Thiery
,
J. P.
(
1984
).
Identical reactivity of monoclonal antibodies HNK-1 and NC-1: conservation in vertebrates on cells derived from the neural primordium and on some leukocytes
.
Cell Differentiation
,
14
,
223
230
.
Tucker
,
R. P.
&
Erickson
,
C. A.
(
1986
).
The control of pigment cell pattern formation in the california newt, Taricha torosa
.
J. Embryol. exp. Morph
.
97
,
141
168
.
Vielkind
,
J. R.
,
Kallman
,
K. D.
&
Morizot
,
D. C.
(
1989
).
Genetics of melanomas in Xiphophorus fishes
.
J. of Aquatic Animal Health (in press)
.
Vielkind
,
J.
&
Vielkind
,
U.
(
1982
).
Melanoma formation in fish of the genus Xiphophorus: A genetically-based disorder in the determination and differentiation of a specific pigment cell
.
Can. J. Genet. Cytol
.
24
,
133
149
.
Vielkind
,
U.
&
Vielkind
,
J.
(
1983
).
Culture of embryos from viviparous fishes of the genus Xiphophorus
.
J. Tissue Culture Methods
. Vol.
8
,
2
, 73-78.
Vincent
,
M.
&
Thiery
,
J. P.
(
1984
).
A cell surface marker for neural crest and placodal cells: Further evolution in peripheral and centeral nervous system
.
Devi Biol
.
103
,
468
181
.
Weston
,
J. A.
(
1963
).
A radioautographic analysis of the migration and localization of trunk neural crest cells in the chick
.
Devi Biol
.
6
,
279
310
.