Download Neural crest development in Xiphophorus fishes

487
Development 105, 487-5W (1989)
Printed in Great Britain © The Company of Biologists Limited 1989
Neural crest development in Xiphophorus fishes: scanning electron and
light microscopic studies
BAHRAM SADAGHIANI and JUERGEN R. VIELKIND*
BC Cancer Research Centre and Department of Pathology, University of British Columbia, Vancouver, BC, Canada
•Address for correspondence: BC Cancer Research Centre, 601W 10th Ave., Vancouver, BC, V5Z 1L3, Canada
Summary
We have studied neural crest development in two teleost
fish species, Xiphophorus maculatus (platyfish) and X.
helleri (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 somiteectoderm 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 tune 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.
Introduction
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
infishso 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).
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
Key words: neural crest, Xiphophorus, fish, teleost.
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Table 1. Characteristic features of the various stages in
embryogenesis of Xiphophorus maculatus and
X. helleri (see Tavolga, 1949)
Theor. age
Develop. (days after No. of
stages
fertiliz.) somites
1
1-9
2-3
0
0-3
P
3
5-7
7
3-4
12-13
3-9
19-20
11
4-2
21-22
11
#1
23
14
5-0
5-5
24-25
6-9
26
10
13
25
Developmental events
Neural keel formation
Optic bud and otic placode
formation
Neurocoel formation;
formation of pros-, mes-, and
rhombencephalon and tail bud
Optic cup and otic vesicle
formation; appearance of first
three gills
Widening of mes- and
rhombencephalon; complete
invagination of otic vesicle and
appearance of pectoral fin buds
First movements of body; closure
of the blastopore; formation of 5
pairs of caudal nerves, the
intestine, and the genital ridge;
heart beating
Appearance of pigment cells in
outer layer of retina; formation
of 4 pairs of gill pouches
Formation of liver primordium
Appearance of melanophores on
the rhombencephalon; appearance
of the swim bladder
First indication of the dorsal fin
Materials and methods
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 Kallman, 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
Pregi.ant 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.
Scanning electron microscopy (SEM)
Embryos of stages 8-16 were fixed for 6 to 24 h in halfstrength Karnovsky's fixative (2-5 % glutaraldehyde and 2 %
paraformaldehyde, 0-1 M-cacodylate (pH7-3) (Karnovsky,
1965)). To preserve glycosaminoglycans (GAGs), 0-5 % cetylpyridinium chloride (CPC) (Derby & Pintar, 1978) was added
to the Karnovsky fixative for the fixation of some embryos.
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 fan.
Neural crest in Xiphophorus fishes 489
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/an.
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En
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; PI, placodal thickening; Rh, rhombencephalon. Bar, 50/mi.
Neural crest in Xiphophorus fishes 491
Prior tofixation,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 Karnovsky'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 jcn). 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 antirabbit 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.
Results
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.
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. IB), 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. IB, 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 midline 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.
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
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Neural crest in Xiphophorus
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 lateraj 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^m.
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.
fishes
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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).
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
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¥
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 in Xiphophorus fishes 495
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 swordtaO
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; PI, placodal thickening; Rh,
rhombencephalon; S, somite. Bar, 30 (xm. Note: dotted lines
in Fig. 5A indicate planes of sections shown in Fig. 3A-F.
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 intersegmental 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
496
B. Sadaghiani and J. R. Vielkind
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 fan.
Neural crest in Xiphophorus fishes
497
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 fan.
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 immunopositive 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 HNKl t no fluorescence has been observed at all.
498
B. Sadaghiani and J. R. Vielkind
covering
condensation
differentiation
#"•:
'/' .*%fy-<*A
Neural crest in Xiphophorus fishes
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. DorsaOy 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/an.
499
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).
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.
Taken in sum, staining was found in those cells that
While the above mentioned cells advance in their
we had identified in the SEM and LM studies described
development through the three phases, round cells
above as neural crest cells that were in progress of early
bearing a few filopodia can be observed to appear
segregation and migration and we did observe identical
among and over those covering the neural tube in
staining patterns in both species. As in the newt and
almost all trunk regions. In addition, some cells could
chicken, some staining was found on other structures
be observed on the apex of the somites, between the
such as the neurones (Tucker et al. 1984) and, as was
somites and the ectoderm, and in the lateral grooves
found in chicken (Bronner-Fraser, 1986), on the lateral
between consecutive somites. The latter cells are round
and ventral walls of the neural tube.
in shape, while a few, which seem to remain on the
Characteristics of cells that appear on the dorsal aspect lateral part of the somites, usually exhibit a flat or
of the trunk neural tube in advanced embryogenesis
elongated shape (Figs 8C,D and 9B). It should be
mentioned that at these advanced embryonic stages,
While in the head region, neural crest segregation
many cells remain attached to the inner surface of the
seems to be terminated by stage 10, segregation in the
ectoderm when it is removed (Fig. 9C).
trunk region seems to continue even beyond stage 13.
Up to this stage, we have observed on the dorsolateral
Temporal and regional appearance of the extracellular
aspect of the neural tube in the trunk region only cells
matrix (ECM)
that exhibit a round or elongated shape and possess a
few filopodia. From stage 13 (23 somites) onwards,
Since it has been proposed that the structure of the
however, the picture becomes more complex as adECM may have some influence on the neural crest cell
ditional flat cells begin to accumulate on the dorsolatmigration and behaviour (Loefberg etal. 1980), we also
eral sides of the neural tube. The boundaries of these
describe observations on the ECM structure of embryos
flat cells are hardly recognizable and they bear very fine made in our SEM studies.
processes which extend across adjacent cells (Fig. 7B)
A few strands of ECM fibrils can be seen on the
and the dorsal midline. During further development,
neural keel and under the ectoderm in regions where
stages 13 to 15 (Figs 7A; 8A), it appears that mainly the the neural crest cells segregate. The fibrils increase in
flat cells cover the dorsal part of the neural tube in
number during segregation of the crest cells
characteristic phases which we describe as covering,
(Fig. 4A,B). Similarly, ECM material appears in other
condensation and differentiation phases. They progress
locations seemingly preceding the appearance of crest
cells. For example, ECM fibrils can be observed on the
in an anterior-posterior fashion; as a result all three
somites at the onset of crest cell segregation in the trunk
phases can be observed in a stage-15 embryo in an
region (Fig. 4E,F). The ECM increases when crest cells
anterior-posterior sequence.
appear on the somites (compare Fig. 4A,B with
The covering of the neural tube is intense in the
Fig. 4E,F). More ECM fibrils are present at the lateral
anterior trunk region and diminishes gradually towards
500
B. Sadaghiani and J. R. Vielkind
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/OTI.
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).
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
Neural crest in Xiphophorus fishes
501
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 lateral 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 /an.
502
B. Sadaghiani and J. R. Vielkind
more abundant in the lateral parts than in the middorsal 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 Karnovsky's
fixative supplemented with cetylperidinium chloride
(CPC). This compound presumably retains glycosaminoglycans (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
0-5-1-0 jim) (Fig. 10F) whereas the ECM of a CPCtreated 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 swordtail 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.
Discussion
Using scanning electron and light 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; BronnerFraser, 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 & MorrissKay, 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
(Lamers et al. 1981), the ectoderm-associated cells gave
rise to pigment cells.
We have also observed many cells on the dorsal part
Neural crest in Xiphophorus fishes 503
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. Theflatmorphology 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 etal. 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 areflatand
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 thefibrilsand 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.
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(Accepted 12 December 1988)