569
Development 108,569-580(1990)
Printed in Great Britain © The Company of Biologists Limited 1990
Cell movements during epiboly and gastrulation in zebrafish
RACHEL M. WARGA and CHARLES B. KIMMEL
Institute of Neuroscience, University of Oregon, Eugene OR 97403, USA
Summary
Beginning during the late blastula stage in zebrafish,
cells located beneath a surface epithelial layer of the
blastoderm undergo rearrangements that accompany
major changes in shape of the embryo. We describe
three distinctive kinds of cell rearrangements. (1) Radial
cell intercalations during epiboly mix cells located deeply
in the blastoderm among more superficial ones. These
rearrangements thoroughly stir the positions of deep
cells, as the blastoderm thins and spreads across the yolk
cell. (2) Involution at or near the blastoderm margin
occurs during gastrulation. This movement folds the
blastoderm into two cellular layers, the epiblast and
hypoblast, within a ring (the germ ring) around its entire
circumference. Involuting cells move anteriorwards in
the hypoblast relative to cells that remain in the epiblast;
the movement shears the positions of cells that were
neighbors before gastrulation. Involuting cells eventually form endoderm and mesoderm, in an anteriorposterior sequence according to the time of involution.
The epiblast is equivalent to embryonic ectoderm.
(3) Mediolateral cell intercalations in both the epiblast
and hypoblast mediate convergence and extension movements towards the dorsal side of the gastrula. By this
rearrangement, cells that were initially neighboring one
another become dispersed along the anterior-posterior
axis of the embryo. Epiboly, involution and convergent
extension in zebrafish involve the same kinds of cellular
rearrangements as in amphibians, and they occur during comparable stages of embryogenesis.
Introduction
opposite direction: deep-lying blastoderm cells spread
outward towards the margin to form the hypoblast.
Ballard's view has been generally accepted, but very
recently, involution was observed directly in the small
embryo of a teleost, the rosy barb (Wood and Timmermans, 1988).
During the course of cell-lineage analyses, we have
followed cell movements during epiboly and gastrulation in zebrafish. We observed cell rearrangements
that seemed nonsensical if considered only in terms of
the eventual fates that the lineages produced. First, in
the late blastula, cells scatter chaotically (Kimmel and
Law, 1985b; Kimmel and Warga, 1986). Second, in the
gastrula, neighboring cells at the blastoderm margin
undergo anterior-posterior inversions in their positions
(Kimmel and Warga, 1987a). Finally, cells in either
ectodermal (Kimmel and Warga, 1986) or mesodermal
(Kimmel and Warga, 1987a) lineages disperse along the
anterior-posterior axis of the embryo.
We now show that each of these cellular rearrangements are understandable if they are considered in
relation to the changes in form of the blastoderm that
occur at the same time. Studies done mostly in Xenopus
suggest that cells undergo specific rearrangements to
mediate the changes in form (Keller, 1987). We find the
same rearrangements occur in zebrafish at the comparable stages of development.
In the zebrafish embryo, after an early developmental
period of rapid cleavages, morphogenetic movements
occur that rapidly produce major changes in the appearance and organization of the blastoderm. During epiboly (Trinkaus, 1984a; 19846), beginning at the late
blastula stage about 4h after fertilization, the blastoderm thins and spreads to completely cover the yolk cell
during the course of 6h. Gastrulation begins about an
hour after epiboly is underway. The blastoderm, a
single multilayer of cells, rearranges into a two-layered
structure consisting of a more superficial epiblast, and
an inner hypoblast (Wilson, 1891). Shortly after gastrulation begins, the embryonic axis appears and lengthens
along one side of the embryo (the dorsal side), as cells
accumulate and line up specifically at that location. The
rearrangements that occur among the cells of the
blastoderm during early morphogenesis, particularly
with respect to their lineal relationships and their future
fates, are not well understood.
For example, several early embryologists concluded
that during gastrulation the hypoblast originates by cell
involution, a streaming of cells lying at the blastoderm
margin inward and underneath their neighbors (Wilson,
1891; Morgan, 1895; Pasteels, 1936). Later, Ballard
(I966a,b,c) concluded that the movement was in the
Key words: blastula, gastrula, morphogenetic movements,
involution, clonal analysis, cell lineage.
570
R. M. Warga and C. B. Kimmel
Materials and methods
Embryos and stages
Zebrafish embryos were obtained from natural spawnings and
staged by cell number during early cleavage. They were
dechorionated with watchmaker's forceps and kept at 28.5°C
in an incubation medium of 14mM NaCl, 0.6mM KC1, 1.3mM
CaCl2, lmM MgSO4, and 0.07mM sodium-potassium phosphate buffer (pH7.2). In some experiments, we used embryos
homozygous for the gol-1 (golden) mutation (Streisinger et al.
1981), because they are lightly pigmented relative to the wild
type, and fluorescently labeled cells in their bodies can be
observed more clearly in whole-mount preparations after
pigment cells differentiate.
Developmental time usually was determined from the
morphological features of the embryo, and Table 1 gives a
staging series for the period of development of interest, from
midblastula period until somites begin to form. We use the
letter h to mean hours after fertilization at 28.5°C. A
previously published series, although less complete, includes
useful sets of photographs (Hisaoka and Battle, 1958;
Hisaoka and Firlit, 1960). In our series, names in common
usage in embryology denote major periods of development
(e.g. midblastula, gastrula), and the stages subdivide these
periods. We name rather than number the stages, which
seems to help one to remember them, and is more flexible.
Blastomere injections
Single blastomeres were injected (Kimmel and Law, 1985a),
in mid- and late blastula embryos with the lineage tracer dye
tetramethylrhodamine-isothiocyanate dextran (Molecular
Probes, Eugene, OR; lOxlO3 Afr, diluted to 5 % (wt./vol.) in
0.2 M KC1). The second dye for double-label experiments was
fluorescein-dextran (Sigma), dissolved the same way. Injections were made by pressure, usually over the course of a few
seconds, either into a cell in the surface enveloping layer
(EVL), or, in other cases, into a cell in the deep layer (DEL)
of the blastoderm. To inject a DEL cell, the injection pipette
was advanced through the intact EVL. It was technically more
difficult to specifically inject single DEL cells than EVL cells,
even under visual control. As an aid, we monitored voltage
through the injection pipette. We observed that successful
passage of the pipette through the EVL was accompanied by a
rise in voltage of up to 40 mV; the extracellular space
surrounding DEL cells is at a positive potential relative to the
bath (Bennett and Trinkaus, 1970). Upon intracellular penetration of a DEL cell, we then observed the expected shift to
negative potential, reflecting the membrane potential of the
cell.
Observations of fluorescent cells in live embryos
For short-term viewing of labeled cells, embryos were usually
positioned as desired in a gel of 3 % methyl cellulose made in
the aqueous incubation medium described above and viewed
without a coverglass. Alternatively, embryos in incubation
medium were sandwiched between two micro cover glasses
that were spaced apart with three pairs of cover glasses (each
0.13-0.17mm thick). For longer term viewing and for timelapse recordings, the embryos were held stationary in such
chambers in a gel of 0.1 % agarose made in the same medium,
and the chamber was then sealed with Vaseline to prevent
evaporation. Observations were made using a Zeiss microscope with illumination from both a transmitted and an epilight source (Zeiss filter set 48-77-14), which permitted
simultaneous imaging of labeled and unlabeled cells. The
fluorescent image was amplified with a Silicon-IntensifiedTarget (SIT) video camera (Dage) to prevent light-induced
damage to the labeled cells. In some experiments, the depths
of fluorescent cells were determined with a digital shaft
encoder fitted to the fine-focus knob of the microscope.
For time-lapse recordings, single-frame images were taken
with a Gyre video recorder at 4 s intervals. The epi-light
source was controlled by a shutter that illuminated the
embryo for only 60 ms during each exposure, in order to
minimize light-induced damage to the labeled cells. Frequent
refocusing of the image was required during the recording
Table 1. Series of normal stages for 3-10.5 h of development
1
Stage
h
1 k-cell
3
2k-cell
3.2
High
3.5
Oblong
3.7
Sphere
HB b
10
11
12
Dome
30%-epiboly
4.3
4.7
13
14
50%-epiboly
Germ-ring
Shield
75%-epiboly
100%-epiboly
5.2
5.5
6
8
9.5
15
16
17
Bud
l-Somite
10
10.5
Description
Midblastula; yolk syncytial layer present; cell cycles of blastoderm cells fairly
synchronous, determined by presence or absence of interphase nuclei
Single row of yolk syncytial layer nuclei; cell cycles of blastoderm cells highly
asynchronous
Blastoderm perched high upon the yolk cell, giving the embryo a dumbbell shape; yolk
syncytial layer nuclei in two rows
Flattening of the blastoderm over the yolk cell produces a single smooth contoured
outline, elongated along the animal-vegetal axis; multiple rows of yolk syncytial layer
nuclei
Late blastula; embryo has assumed a spherical shape; at a deep plane of focus the yolk
cell-blastoderm interface is flat
Yolk cell bulging (doming) towards animal pole as blastoderm rapidly thins by epiboly
Blastoderm shaped as an inverted cup of uniform thickness and covers 30% of the yolk
cell
Gastrula; 50% of the yolk cell is covered by the blastoderm
Germ ring visible from animal pole; 50%-epiboly
Embryonic shield visible from animal pole, 50%-epiboly
The blastoderm continues to spread over the yolk cell at a rate of 15 %-epiboly per hour
Yolk plug closed. Gastrulation movements nearly complete in the anterior parts of the
embryo
Tail bud prominent at the posterior end of the axis
Segmentation; the first furrow appears in the paraxial (presomitic) mesoderm; about 2
somites are added per hour (Hanneman and Westerfield, 1989)
°h: hours after fertilization at 28.5°C.
HB: Approximate stage number in the zebrafish staging series described by Hisoaka and Battle (1958).
b
Eplboly and gastrulation In zebrafish
session, which began at sphere stage (4 h) and continued for at
least 6h, when epiboly is completed, and generally for a few
hours longer. Afterwards, the embryo was released from the
viewing chamber and reexamined at 24-30 h, when many cell
types have begun to differentiate and can be distinguished by
their morphologies and positions in identifiable tissues (Kimmel and Warga, 1987«). Labeled cells that were still undiffer-
AP
571
entiated were reexamined on the 2nd and/or the 3rd day of
development.
Histology
We also studied a set of sectioned embryos. They were fixed at
intervals between late blastula (4h) and midgastrula (7h)
periods by immersion in Bouin's solution (Humason, 1962),
B
D
v
D
V
D
Fig. 1. Morphogenesis during zebrafish epiboly and gastrulation. Side views of living embryos with the animal pole (AP) to
the top. An outline of stage descriptions is given in Table 1. (A) Oblong stage, at the end of the midblastula period, 3.7 h.
The blastoderm is a thick cap of cells occupying about a third of the volume of the blastula. The blastoderm margin (m)
separates the blastoderm and the uncleaved yolk cell. At this time blastoderm cells are motile (D. A. Kane, in preparation),
but major rearrangements among them have not yet occurred. (B) 50%-epiboly stage, onset of gastrula period, 5.2 h.
Epiboly is well underway, and the blastoderm has thinned to take the form of a cup inverted over the yolk cell. Involution
and convergence movements appear to begin at this stage. (C) Shield stage, early gastrula, 6.Oh. Involution and
convergence movements have produced the embryonic shield, a pronounced accumulation of cells along the margin at the
dorsal (D) side. Hence the blastoderm appears thicker here than ventrally (V), and the hypoblast has become prominent
(arrow). To aid visualizing the hypoblast this embryo was slightly flattened between coverslips. Hence it appears a little
larger than the others in this figure. (D) Bud stage, beginning of the tailbud period, 10h. Epiboly is completed; the yolk
plug (YP) has just been enveloped by the blastoderm near the site of the original vegetal pole of the egg. The blastoderm is
obviously thicker dorsally (D) than ventrally (V), due to the forming embryonic axis on the dorsal side. The tail bud (arrow)
is present at the posterior end of the embryonic axis. Scale bar: 200 ^m.
572
R. M. Warga and C. B. Kimmel
dehydrated and embedded in Epon A12. Serial 5 fm\ sections
were cut and stained with azure A, methylene blue and basic
fuchsin (Humphrey and Pittman, 1974).
Results
Deep and shallow DEL blastomeres intercalate during
epiboly
In zebrafish, cleavages generate two populations of
distinctive blastoderm cells; flattened epithelial cells in
a surface enveloping layer (EVL), and rounded, more
loosely associated deep layer (DEL) cells lying beneath
the EVL. The EVL is a monolayer and the DEL a
multilayer of cells. All of the movements we describe in
this paper pertain to the DEL: the EVL cells behave
relatively passively. Neighb6r exchanges occur within
the EVL (Keller and Trinkaus, 1987), but they are
infrequent. We have not observed neighbor exchanges
between the EVL and DEL.
B
Fig. 2. The double-label method used to distinguish the
morphogenesis and fates of sibling clones located at
different depths in the blastoderm. (A) At the 32-cell stage,
the blastomeres are often arranged in a single-layered 4x8
array. One of the central rows of 8 cells is shown, and a cell
in this row, adjacent to the animal pole, is injected with
rhodamine-dextran (coarse stippling). (B) Following the
next (sixth) cleavage, which is horizontal, the upper of the
two labeled sister cells is reinjected, now with
fluorescein-dextran (fine stippling). The lower daughter,
containing only rhodamine-dextran is adjacent to the yolk
cell. (C) Following several more cleavage divisions the two
sister clones are expected to remain coherent in the
midblastula, since cell mixing is very limited before late
blastula stage. The rhodamine-labeled clone is expected to
lie deep in the blastoderm, immediately beneath the doubly
labeled clone, which extends to the blastoderm surface.
DEL cells become motile in the midblastula, after the
tenth cleavage at 3h (D. A. Kane, unpublished observations). The embryo flattens to take on a spherical
shape by 4h (late blastula; Fig. 1A), and during the
next hour of development, a rapid thinning of the
blastoderm becomes evident, signifying epiboly is
underway. The first change observable is very deep in
the embryo, where the yolk cell begins to bulge or
'dome' towards the animal pole (Fig. IB). The blastoderm then rapidly takes on a cup-shaped appearance,
and spreads to cover the yolk cell (Fig. 1C).
At the beginning of the late blastula stage, single
clones descended from a progenitor cell labeled earlier
are coherent groups of cells. Later, during epiboly, the
DEL cells in such clones rapidly spread apart, interspersing with unlabeled cells (Kimmel and Law, 1985b).
In Xenopus, epiboly is known to occur by radial cell
intercalations, in which cells at different depths in the
blastoderm intercalate, thus producing its thinning
(Keller, 1980). Such a rearrangement could produce the
cell scattering we observed in zebrafish, and we have
examined whether radial intercalations occur in this
species.
We took advantage of the pattern of cell division
during early cleavages to label, with two different
colored dyes, two sibling blastomeres; one underlying
the other at the 64-cell stage (Fig. 2). The deeper cell
generated a clone located deep in the DEL of the
midblastula, and immediately underlying the clone
originating from its superficial sib, as confirmed by
direct inspection (Fig. 3A). DEL cells of the two clones
became thoroughly intermixed by early gastrula stage
(Fig. 3B; note that the intermixing does not extend into
the EVL). Subsequently, both sets of DEL cells gave
rise to very similar sets of derivatives in the later
embryo. In this example, both clones developed head
ectodermal cell types (Fig. 3C). These results establish
that blastoderm cells intercalate along radii during early
epiboly, and that such movements are confined to the
DEL.
The hypoblast arises by involution
At the time when the blastoderm half covers the yolk
cell (5.2h; referred to as 50%-epiboly; Fig. 1C) new
cell movements begin, including involution movements
that form the hypoblast. These new movements mark
the onset of gastrulation (see Discussion). Within about
15 minutes, the blastoderm becomes noticeably thicker
in a circumferential band at its margin. The band, or
germ ring, at first appears uniform in structure, and
using time-lapse video microscopy, we observed in
views from the animal pole (3 embryos) that it forms
more-or-less simultaneously, within about 15min,
around the entire circumference of the blastoderm.
An analysis of involution is shown in Fig. 4, a case
where we kept track of the depths of cells in the
blastoderm as their rearrangements occurred. Here,
minutes after the onset of gastrulation, a clone of 5
labeled cells was located near the margin of the blastoderm. The cells initially occupied a shallow position
within the DEL, indicated by blue color-coding in
Fig. 3. Deep cells in the DEL intercalate with superficial DEL cells, and then both populations exhibit similar fates. The double-labeling
experiment is explained in Fig. 2. The images shown here are computer-enhanced, with cells containing rhodamine alone shown as red,
and cells doubly labeled with rhodamine and fluorescein shown as yellow. (A) A side view (as in Fig. 1A) at a deep plane of focus of the
midblastula. As expected (Fig. 2C), the red clone lies underneath, deep to, the yellow one. The deepest cells in the red clone are adjacent
to the yolk cell. The most superficial cells in the yellow clone are EVL cells. (B) Surface view (at a shallow plane of focus) of the early
gastrula. Red and yellow cells are intermingled within the DEL, with red cells now occupying very superficial positions in the DEL. Red
cells are not present in the EVL. The only labeled cells within the EVL (arrow) are yellow, as expected from the labeling regime, and
because DEL cells do not intercalate into the EVL. (C) view of clusters of labeled neuro-epithelial cells within the brain of the embryo at
24 h (side view, with dorsal to the top). Both red and yellow cells have contributed to the CNS. The experiment was repeated 5 times, and
the mixing among DEL cells was always observed. Scale bars: 100,um (A & B), 50f*m (C).
Fig. 4. Changes in the
positions of cells in a clone
of DEL cells during
involution in the gastrula.
Depth in the blastoderm is
color-coded in these
computer-enhanced images:
blue, superficially lying
labeled DEL cells; red, most
deeply positioned cells;
green, intermediate. The
blastoderm margin is
indicated with arrows, and
the orientation is
approximately the same in
all the panels. A - D show
successive times during
gastrulation, at 6.6h (60%epiboly),7.6h(70%epiboly), 8h (75%-epiboly),
and 9h (90%-epiboly)
respectively. Eventually the
progeny of the involuting
cells in this clone formed
somitic muscle. (A) All the
labeled cells occupy shallow
positions in the DEL. The
cells are moving towards the
blastoderm margin. (B) Cell
number in the clone has
increased by cell division,
and the cells nearest the
margin (green) have begun
to involute. (C) Twenty
minutes later the involuting
cells form a stack at the
margin. The cells that had
begun involution earliest (in
B) are at the bottom of the
stack. Those involuting last
are at the top. (D) An hour
later the first involuting cells
(red and green pair) have
moved away from the
margin, and are now in a
deep location (in the
hypoblast) beneath their
superficial relatives, which are still in the epiblast (blue). The second pair of involuting cells have moved to the deepest location at the
margin by this time, and shortly later will also move away from the margin within the hypoblast. Scale bar: 25 ,um.
Fig. 5. The germ ring forms during involution. The panels (A-D) show selected frames from a time-lapse video tape
following cells during the earliest gastrulation movements. At the beginning of the experiment a single DEL cell,
located beneath the EVL at the blastoderm margin in the blastula (3.1 h), was injected with rhodamine-dextran. (A) By
4.5 h (30%-epiboly), before the onset of gastrulation, the labeled cell had divided, and its daughters are close neighbors
at the margin (arrow). The color for this figure was computer-generated, and codes cell lineage (i.e. a red lineage and a
green lineage), so that the reader can keep track of the fate of these two daughter cells. (B) An hour later (5.5 h, 50%epiboly), involution movements are just beginning. The two labeled cells have become separated by a single unlabeled
one. (C) Six minutes later both cells are dividing, and are in the process of involution. (D) Forty minutes later (6.3 h,
shield stage) the germ ring has formed. All of the cells of the clone have now moved well away from the margin and are
present in the hypoblast within the confines of the germ ring. One cell in each of the two lineages is very close to the
upper boundary of the germ ring (upper arrow). The lower boundary of the germ ring is the margin (lower arrow). As
in this example, we invariably observed that only DEL cells located near the blastoderm margin involute during
gastrulation. In all, in this study, involution was followed by time-lapse analysis in labeled clones in six embryos that
were oriented such that we could clearly distinguish the borders of the hypoblast, and in each of these cases the labeled
involuting DEL cells (31 in all) entered, and then remained in the hypoblast. Conversely, in three embryos with clones
located farther from the margin, 18 DEL cells were followed that never involuted and were later present in derivatives
of the epiblast. A clone in one additional embryo was located about 80% of the way between the animal pole and the
margin, and in this case 5 of the DEL cells involuted, and 6 of the DEL cells (initially farther from the margin) did not
involute. In eight of the embryos the clones included labeled EVL cells as well as labeled DEL cells, and in none of
these cases was an EVL cell observed to involute. Scale bar: 25 ^m.
Epiboly and gastrulation in zebrafish
Fig. 4A. The labeled cells moved towards the margin,
apparently actively, since we observed blebbing and
formation of filopodia, and two of them divided. Upon
reaching the margin, each cell protruded processes and
moved deeper within the DEL (Fig. 4B; green), away
from the EVL and towards the surface of the yolk cell.
Each marked cell then reversed its direction relative to
the margin, and proceeded away from the margin, now
located deeply in the blastoderm (Fig. 4C and D; red),
within its new hypoblast layer.
These findings show that involuting DEL cells form
the hypoblast. The first ones to involute are those
located just at the blastoderm margin at the beginning
of gastrulation (Fig. 5A), and as they migrate inwards
the germ ring forms behind them (Fig. 5B and C).
Analysis of sectioned embryos revealed that before
germ ring formation there is no layering of cells within
the DEL itself (Fig. 6A). However, after the germ ring
forms, the DEL appears folded inwards at the margin,
and is split, within the germ ring specifically, into the
epiblast and hypoblast (Fig. 6B). As epiboly and gastrulation continue, cells that initially were located
distantly from the margin move towards it within the
573
epiblast, and involute. Consequently the hypoblast
increases in area and extent beneath the epiblast,
eventually spreading all the way from the margin to the
animal pole.
Fates of epiblast and hypoblast cells
Single cells present during gastrulation generate clones
restricted to single types of tissues (Kimmel and Warga,
1986). Now we have shown that some of these cells, but
not others, involute during gastrulation, and we can ask
whether involuting and noninvoluting cells have different fates. We kept track of cells in labeled clones during
gastrulation and determined the fates of their descendants at a later stage, as illustrated in Fig. 7A. From such
records, we reconstructed the cell lineage (Fig. 7B) and
the pathways of movement of the cells during gastrulation (Fig. 7C). In this example, all of the labeled cells
involuted, inverting their relative positions as they did
so. The progeny of one of the cells originally present
(Fig. 7, black lineage) all formed somitic mesoderm,
including differentiated muscle fibers. The progeny of
the other cell (stippled) formed derivatives of two
B
V.
a-
f
Fig. 6. Involution produces layers of DEL cells within the blastoderm. Stained plastic 5;<m sections that show the
blastoderm margin. (A) The onset of gastrulation (50%-epiboly; 5.2 h); (B) the midgastrula (75%-epiboly, 8 h). Both
micrographs are oriented the same way with the blastoderm margin near the bottom, the EVL present as a very thin cell
layer to the left, and the yolk to the right. In A the DEL is a homogeneous multilayer of cells. In B, except just at the
margin where cells are involuting, the DEL is layered into the epiblast (underlying the EVL) and the hypoblast (overlying
the YSL of the yolk cell). This interpretation exactly follows Wilson (1891), who did not have the benefit of following
labeled cells in live embryos, but relied exclusively on studying fixed material such as shown in this figure. Scale bar: 50fan.
574
R. M. Warga and C. B. Kimmel
4
B
-
5
-
6
-
7
-
8
-
9 -
as h
to 5.6h
60h
6.5h
11
-
12
mus.und
mus
gut
mes
mes
end
ZOh
78h
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*PPP ©aaa
30h
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different germ layers; gut epithelium (endoderm) and
somitic muscle (mesoderm).
Summary lineage analyses for cells in other embryos
analyzed the same way are shown in Fig. 8. Without
exception (and also for 5 additional clones not illustrated), cells that involuted (arrows) later formed
mesoderm and endoderm, and cells in lineages where
involution did not occur (no arrows) formed ectoderm.
The lineage shown in Fig. 8B is noteworthy, for in this
case two sibling subclones, separated at the first division
shown in the diagram, developed differently from one
another but still followed the general rule: None of the
cells in one of the subclones involuted and they subsequently developed as ectoderm. The cells in the
second subclone all involuted and then formed mesoderm. We conclude from these results that the epiblast
is the rudiment of ectoderm and the hypoblast is the
rudiment of both mesoderm and endoderm.
The time of involution is related to fate
Cells that involute early during gastrulation usually
form endoderm, and cells that involute later formed
mesoderm. This can be seen most clearly from single
clones that contributed to both germ layers (e.g. arrows
in Figs. 7B and 8D). Except for the early involution of a
cell that later formed heart tissue, a mesodermal
derivative (Fig. 8D), this rule was generally followed
(including those clones that contributed cells to only a
single germ layer; compare the times of involution of
cells in Fig. 8C and E).
Cells involuting at different times during gastrulation
also had different pathways of movement within the
hypoblast, and later occupied different positions in the
embryo. Cells that involuted soon after the beginning of
gastrulation sharply reversed their direction of movement as they involuted, turning towards the animal pole
(Fig. 7C; stippled cell ppp). Cells that involuted somewhat later turned less sharply (stippled cell aaa), and
cells that involuted much later during epiboly (black
cell aaa) did not turn at all, but continued to move
towards the vegetal pole after entering the hypoblast.
No matter whether a turn occurred or not, all involuting
Epiboly and gastrulation in zebrafish
Fig. 7. Analysis of movements, lineages and fates of
clonally related cells that involute. A single DEL cell
located near the margin of a blastula embryo was labeled
with rhodamine-dextran (2.3 h). Its subsequent
development was recorded by time-lapse video during
epiboly and early tailbud stages (4-13 h). The fates of the
cells in this clone were then determined by comparing
positions of the labeled cells at 13 h (after gastrulation
movements were over), and later, when they had begun
differentiation in the embryo at 30 h. (A) A series of
tracings from selected frames during the first 4h from the
time-lapse recording (3.8-7.8 h), and, in the last drawing,
the fates of the cells in the 1-day embryo. Anterior is to the
top, the blastoderm margin is to the bottom (except for the
last drawing, where there is no margin). The founding
labeled DEL cell had divided a single time by the time the
recording session began. Cells of the subclone generated by
its more anterior daughter are coded black in these
drawings, and cells of the subclone generated by its more
posterior daughter are stippled. Cell divisions are indicated
by v's, and arrowheads point to newly involuting cells.
Initially the stippled cell is nearest the margin. Its progeny
involute earliest, and some of them eventually differentiate
as epithelial cells of the foregut (gut). Other cells in this
subclone, and cells of the black subclone differentiate as
somitic muscle (mus). A few cells in the black subclone that
are present within one somite remain undifferentiated
(und). Notice that the stippled cells are initially closer to
the margin, involute earlier, and then form more anterior
fates, including endoderm. (B) Cell lineage diagram for the
same two subclones for the period 3.8-13 h. Hours of
development are shown to the left. Horizontal lines in the
diagram indicate divisions and vertical lines indicate cells;
the more anterior daughter cells (and farther from the
blastoderm margin) arising at each divisions are shown as
left-side branches in the diagram. Arrowheads indicate
when individual cells involuted; in general cells towards the
left involute later because they are farther from margin.
The endodermal (end) and mesodermal (mes) cell fates are
indicated below the diagram. Notice the symmetrical
(generative) form of the lineage: sibling cells tend to divide
at the same time, as is also the case in the examples shown
in Fig. 8. (C) Pathways of movements of selected cells
present in the same two subclones. aaa refers to the most
anterior, and ppp to the most posterior cells in the lineages
(i.e. the left-most and right-most branches in B). During
the recording session, the embryo itself was held stationary
in agarose, and serves as a fixed reference for the
movements of these individual cells within it. The pathways
were reconstructed directly from the video tapes. The star
shows the starting position of the cell, and the arrowhead
shows the time of involution. The orientation is as in A.
Individual points along the pathways represent approximate
one-half hour intervals. Notice that the cells that involute
earlier (in the stippled subclone in this case) turn anterior
as they involute. Whether or not a turn occurs, cells recede
from the margin after involution. Scale bars (in A & C):
100 ^m.
cells recede from the margin of the blastoderm, which
continues to rapidly advance by epiboly towards the
vegetal pole. We measured the rates that the involuting
DEL cells moved (18 cells from 3 embryos), but no
significant differences were found in the speed of
575
movement that correlated with whether a turn occurred, or with later fate (data not shown).
The animal pole of the gastrula develops into the
anterior-most structures of the later embryo (Kimmel
and Warga, 1987b), and accordingly, cells that turn
towards the animal pole during gastrulation develop
more anterior structures. It follows that the order in
which cells of a clone involute corresponds to their
subsequent order along the anterior-posterior axis of
the embryo, as can be seen from the positions of the
black and stippled cells in Fig. 7. This relationship was
consistent, as shown in Fig. 9 where data is collected
from a set of 5 embryos in which the labeled clones were
positioned at approximately the same lateral blastoderm location at the beginning of gastrulation.
Convergent extension movements are mediated by
mediolateral cell intercalations
Convergence is a third morphogenetic cell movement
occurring simultaneously with epiboly and involution.
DEL cells move towards the dorsal side of the gastrula
(Ballard, 1973; Kimmel and Warga 1987b), 'converging'
there from their original locations in the blastoderm.
The formation of the embryonic shield, a local dorsal
thickening of the germ ring (Oppenheimer, 1937;
Hisaoka and Battle, 1958), is a prominent effect of early
convergence movements. In Xenopus, convergence
cannot be separated from extension, the elongation of
the embryonic axis (Keller and Danilchik, 1988). Cells
move from lateral positions dorsalwards by intercalating between neighboring cells that lie more medially
(i.e. towards the axis). Such 'mediolateral intercalations' (Keller and Tibbetts, 1989) produce both narrowing and elongation of the axis.
Convergent extension occurs both in the epiblast
(Kimmel and Warga, 1986; and see below) and in the
hypoblast. Convergence of hypoblast cells is illustrated
in Fig. 7C as a drift in the pathways of the cells towards
the right side of the figure, the direction towards the
embryonic shield in this example. That intercalations
occur during this movement is revealed by the separation and spreading apart of the clonally-related cells
along the anterior-posterior axis (Fig. 7A). During this
dispersion, the labeled cells are intercalating among
more medial unlabeled neighbors.
The intercalations are more completely illustrated in
Fig. 10, an example following cells that remained in the
epiblast. This case is instructive because intercalations
occurred not only between labeled and unlabeled cells,
but also between different labeled cells, and it is clear
when they occurred. Four DEL cells (from a single
clone) were present at the beginning of gastrulation.
Their descendants eventually formed a dispersed series
of clusters in the hindbrain, distributed along the axis,
and on both sides of the midline. The cells are shaded to
indicate their lineal relationships. Eventually black and
hatched cells were positioned between different white
cells. The intercalations effecting this intermixing occurred in the gastrula, beginning at about 6.8 h
(Fig. 10). Shortly after both the black and hatched cells
576
R. M. Warga and C. B. Kimmel
gastrulation, although mixing within each of these
layers is extensive.
divided, one of the two daughter cells from each of the
divisions inserted between the pair of white sister cells.
In contrast to the radial intercalations that we considered above, the intercalations occurring during convergent extension do not scatter cells indiscriminately.
For example, we have not observed DEL cells in the
hypoblast and epiblast to mix with one another during
4
-
5
"
6
-
7
-
8
-
9
-
10
-
Discussion
This work has revealed three distinctive cell movements
:f1
nnlln:
N/
6oto. 12 pip
18 nor
23ner
ect
ect
eel
B
—
4 rbn,4 ncr.l 1 ner
ect
3 mus, 4 und
6 mus
9 mus, 8 und
mes
D
4
-
5
-
6
-
7
-
h
8
-
9
-
10
-
10 gut
30 gut
11
-
end
end
12
-
13
-
nnnJ,
6 mus-
23 gut
mes
end
3hrt
mes
Fig. 8. DEL cell lineage diagrams for the period of epiboly in clones from eight embryos. The presentation is as described
for Fig. 7B, and the data were obtained the same way. The clones are grouped (A-E) according to whether involution
occurred (arrowheads) or not, and whether the cells gave rise to ectodermal (ect), mesodermal (mes) or endodermal (end)
derivatives. Thus for example, in A none of the DEL cells in clones in two embryos involuted (there are no arrowheads),
and all of the cells formed ectoderm. Individual cell fates are abbreviated as follows: gut, gut epithelium; mus, somitic
muscle; ncr, neural crest; ner, nervous tissue; oto, otocyst (ear vesicle); pip, posterior lateral line placode; pnd, pronephric
duct; rbn, Rohon-Beard (sensory) neuron; und, undifferentiated-appearing mesenchyme.
Epiboly and gastrulation in zebrafish
23
.
20
.
15
.
10
.
5
.
Fig. 9. The time during gastrulation when a cell involutes
correlates directly with the later anterior-posterior position
of its progeny. Thefivedifferent symbols represent cells in
clones from five separate embryos, from the set shown in
Figs 7 & 8. These five were selected because in the early
gastrula they were all located in the same lateral position,
as measured along the margin relative to the position of the
embryonic shield (at the dorsal side; see Kimmel el al.
1990). The horizontal axis indicates when involution
occurred, relative to the onset of gastrulation at 50%epiboly. The vertical axis indicates position along the
anterior-posterior axis of the embryo at 24 h. Vertical lines
above the individual symbols indicate the extent of
anterior-posterior spread within the subclone derived from
the involuting cell, in those cases where the spread was
extensive.
C
o
2
1
•L «
1
<
0
aa
1
.
3
.
1
.
1
.
•
577
[1
i "i
•
•
«n .
o
0
1
1 —t—
1
H
2
1
1
1
1
3
Hours after 50%-eplboly
that accompany, and appear to produce, the early
changes in shape of the zebrafish embryo. These
movements are epiboly, involution and convergent
extension. They are diagrammed respectively in
Figs 11-13.
Epiboly
Radial intercalations (Keller, 1980) among DEL blastomeres occur first, in the late blastula (Fig. 11), and
along with an expansion and change in shape of the yolk
cell that occur simultaneously, these cell movements
appear to mediate the thinning of the blastoderm that
occurs rapidly during this period of development.
Intercalations thoroughly scatter DEL cells and are
responsible for marked dispersion of clonally related
cells that we have described elsewhere (Kimmel and
Law, 1985b). It is interesting, however, that DEL cells
do not intercalate outward into the EVL. The EVL, by
this stage, has acquired the form of a highly flattened
squamous epithelium. In Fundulus at a comparable
stage of development, junctional complexes are present
between cells of the EVL (Betachaku and Trinkaus,
1978), and it may be that such junctions mediate high
adhesivity among the EVL cell, such that the underlying DEL cells are unable to penetrate this layer.
25.0h
12.7h
9.2 h
8.7 h
5.0 h
6.5h
6.8h
Fig. 10. DEL cells intercalate during convergent extension in the gastrula. The drawings are selected from a larger series,
taken from video taped records, and show the positions of labeled DEL cells that were descended from a blastomere
injected in the midblastula with rhodamine-dextran. Methods are as in Kimmel and Warga (1986). The cell lineage is shown
in the summary diagram, and the shading of the cells codes their lineal relationships. EVL cells were present in this clone,
but are ignored after the first drawing (5.Oh), where 2 are shown by dotted outlines. None of the cells in this clone
involuted, but remained in the epiblast and by 25 h had formed a periodic series of bilateral clusters in the hindbrain (see
Kimmel and Warga, 1986). mid: brain midline. pia, brain pial or outermost surface; oto, otocyst. As individual cells move
apart from one another they are intercalating among invisible (i.e. unlabeled) cells. Intercalations among the labeled cells
also occur, (e.g. at 6.8h) such that subclonal groups often do not remain together. Scale bar: 100^m.
578
R. M. Warga and C. B. Kimmel
AP
blastoderm
>• DEL
M
\
yolk
cell
Fig. 11. Cut-away diagram of radial intercalations
beginning in the. midblastula (A), and through the onset of
gastrulation (B). Deep DEL cells move outward (radially),
intercalating among more superficial DEL cells but not
among EVL cells (See Fig. 3). This movement contributes
to epiboly, thinning and spreading the blastoderm*
epiblast
hypoblast
Fig. 12. Cut-away diagram of involution. (A) Onset of
gastrulation. (B) Germ-ring stage, 20min later. Side views.
A DEL cell first at the blastoderm margin (black) is at the
front of the wave of involuting cells (See Fig. 5). This
movement generates the hypoblast. EVL cells do not
involute.
Fig. 13. Diagram of mediolateral intercalations. (A) shield
stage, early gastrula. (B) 80%-epiboly, late gastrula. Dorsal
views. DEL cells converge towards the dorsal midline,
moving from lateral to medial positions. This movement
(convergent extension) lengthens the embryonic axis
(dashed line).
Involution
Involution movements of DEL cells located near the
blastoderm margin produce the hypoblast, an inner
layer of the blastoderm (Fig. 12). Involution (or in
some animals its counterpart invagination; see Trinkaus, 19846) is the singular morphogenetic movement
that characterizes gastrulation in many different types
of animals; hence we consider the beginning of gastrulation in zebrafish as the time when involution begins.
This is the stage when the blastoderm has advanced, by
epiboly, to cover just one-half of the yolk cell. The cells
move first towards the blastoderm margin, in the
general direction of the vegetal pole. When they reach
the margin they involute to take up a new, deeper,
position. Afterwards they either reverse their direction
of movement and move towards the animal pole, or, in
the case of cells that involute later during gastrulation,
they continue moving towards the vegetal pole. In
either case, once cells are in the hypoblast, they are left
behind the leading edge of the blastoderm, which
continues to advance across the yolk cell by epiboly
during the gastrula period. We have obtained no
evidence that cells can enter the hypoblast by any other
movement than involution, although we have not yet
carefully examined cell movements within the embryonic shield (at the dorsal side of the embryo).
Wood and Timmermans (1988) recently also observed involution in the rosy barb, another teleost in the
same family as zebrafish. However, Ballard could not
find involution in his careful and extensive studies of a
variety of other (and larger) teleost embryos
(1966a,b,c; 1973; 1981; 1982). It may be that gastru-
Epiboly and gastrulation in zebrafish
lation is dramatically different in large and small teleost
embryos, but we think it more likely that all teleosts
gastrulate as the zebrafish and rosy barb do, and that
the cell marking procedures available to Ballard were
inadequate to reveal all the cell movements that occur
in the DEL.
Involution is special in teleost fish, as compared to
other types of vertebrates, in that the EVL is not
involved (see below). Moreover, involution doesn't
seem to be initiated first at the dorsal side of the embryo
(as it does for example in amphibians). As judged from
the time-course of appearance of the germ ring, involution in the zebrafish begins more-or-less simultaneously around the circumference of the blastoderm.
Convergent extension
During gastrulation DEL cells also undergo mediolateral intercalations (Fig. 13), producing a general dorsalwards drift of the cells that has been described previously in other teleosts (e.g. Ballard, 1973; 1982). The
cells accumulate dorsally to form the embryonic shield,
and the subsequent narrowing (convergence) and
lengthening (extension) of the shield produces a welldefined embryonic axis within about two hours after the
shield first forms.
We have shown that cells in both the hypoblast and
epiblast undergo convergent extension. The intercalations appear to be regulated such that extensive mixing
occurs among the cells within both of these layers, but
not between the layers. Moreover, the fact that most
gastrula lineages are tissue-restricted (Kimmel and
Warga, 1986) shows that mixing among cells must occur
within, but not between, the primordia of different
tissues. However, the boundaries of the primordia are
invisible in the gastrula, such that we could not hope to
observe distinctive cellular behaviors in their vicinities.
Later in embryogenesis the boundaries become recognizable, and no mixing occurs across at least one of
them - the boundary separating the axial (prospective
notochord) and paraxial (prospective somite) mesoderm - as recently shown for Xenopus (Wilson et al.
1989) and rosy barb (Thorogood and Wood, 1987).
The enveloping layer
All of the movements we have described appear to
closely resemble their counterparts that have been
thoroughly described in Xenopus (Keller, 1986). Radial
intercalations, involution movements and mediolateral
intercalations occur at the equivalent stages, relative to
gastrulation onset, in zebrafish and Xenopus and they
produce equivalent changes in shape and organization
of the embryo. There is a single important difference,
however; the outside layer of cells in the teleost
blastoderm does not participate in any of them. DEL
cells do not enter the EVL during their radial intercalations, as we have shown in this study. We also
confirmed that EVL cells do not undergo involution, as
was first convincingly shown by Ballard for the trout
(1966a). Thisfindingwas expected in zebrafish since the
exclusive fate of the EVL is the periderm - an outermost epithelial cell layer covering the embryo (Kimmel
579
and Warga, 1986; Kimmel et al. 1990). We showed
earlier (Kimmel and Warga, 19876) that the EVL cells
do not undergo convergence, at least in the sense used
here to mean a specific dorsalwards movement.
The EVL may be a relatively passive participant in
blastoderm epiboly; as revealed by studies in Fundulus,
it seems to be pulled and stretched across the yolk cell
by the yolk syncytial layer of the yolk cell itself
(Betchaku and Trinkaus, 1978; Trinkaus, 1984a). However, perhaps active rearrangements among EVL cells
have recently been shown to occur both in Fundulus
(Keller and Trinkaus, 1987) and the medaka
(Kageyama, 1982), where they serve to continuously
decrease the diameter of the EVL as epiboly is completed and the marginal ring of EVL cells closes at the
vegetal pole of the yolk cell. It is likely that this
rearrangement also occurs in the zebrafish, for EVL
cells in single clones do sometimes become dispersed
from one another, rather than being present in a single
coherent patch (e.g. Kimmel and Warga, 1987b). The
dispersion is, however, markedly less than that occurring in the DEL.
Control and patterning of cell movements
Our studies are descriptive, and do not reveal the
mechanisms that underlie these morphogenetic movements. However, the rearrangements appear to be
active ones, for DEL cells constantly change in shape
and they move relative both to neighboring cells and to
a fixed point on the yolk cell. The yolk cell and EVL
cells both participate in epiboly and, as we have shown
here, so do DEL cells. The gastrulation movements of
involution and convergence may also depend upon
interactions among cells of all three classes. Recently
Symes and Smith (1987) suggested that activation of
gastrulation movements in amphibians is an early
consequence of mesodermal induction. This might
involve the yolk cell; Long (1983) obtained evidence
from transplantation experiments in the trout that the
yolk cell can induce dorsoventral polarity of the blastoderm.
Progress in understanding how such specific movements are produced may come through mutational
analysis in zebrafish. We have recently described a
mutation, spt-1, that appears to selectively disrupt
convergence of laterally positioned mesodermal cells
during gastrulation (Kimmel et al. 1989). Convergence
of ectoderm is not disturbed, suggesting that different
genes control dorsalwards movements of cells that
occupy different germ layers. Furthermore, mosaic
analysis suggests that the wild-type gene is required in
the mesoderm specifically (Ho et al. 1989). The gene
could code for, or regulate the expression of, a receptor
or adhesion molecule required for the convergence
movements of a subset of mesodermal cells.
An important finding from our study is that cells that
involute to enter the hypoblast then give rise to endoderm or mesoderm and, conversely, that the epiblast is
the equivalent of ectoderm in other vertebrates. This
observation is in accord with the interpretations of early
investigators of teleost embryology (Wilson, 1891; Mor-
580
R. M. Warga and C. B. Kimmel
gan, 1895; Pasteels, 1936). We also show that whether a
cell in the hypoblast will form endoderm or mesoderm,
and where its clonal descendants will come to lie along
the anterior-posterior axis of the embryo is directly
correlated with when it entered the hypoblast. Furthermore, we detected no differences in direction or rate of
movements of DEL cells as they approached the
margin, before involution, that correlated with their
future fates. Together, these observations lead to the
suggestion that whether and when a cell involutes is a
direct function of how far from the margin it was
positioned prior to the onset of gastrulation. We address this issue in the accompanying paper (Kimmel et
al. 1990), examining in more detail how cell fate is
related to cell position in the early gastrula.
KELLER, R. E. (1980). The cellular basis of epiboly: an SEM study
of deep cell rearrangement during gastrulation in Xenopus laevis.
J. Embryol. exp. Morph. 60, 201-234.
KELLER, R. E. (1986). The cellular basis of amphibian gastrulation.
In Developmental Biology: A Comprehensive Synthesis. Vol. 2.
The Cellular Basis of Morphogenesis (ed. L. Browder), pp.
241-327.
KELLER, R. E. (1987). Cell rearrangement in morphogenesis. Zool.
Sci. 4, 763-779.
KELLER, R. E. AND DANILCHIK, M. (1988). Regional expression,
pattern and timing of convergence and extension during
gastrulation of Xenopus laevis. Development 103, 193-209.
KELLER, R. E. AND TIBBETS, P. (1989). Mediolateral cell
intercalation in the dorsal, axial mesoderm of Xenopus laevis.
Devi Biol. 131, 539-549.
We thank D. A. Kane, A. Felsenfeld and D. Frost for their
critical comments on early versions of this paper, and for
stimulating discussion throughout the course of the study. C.
Cogswell, P. Myers, H. Howard, and R. Kimmel provided
technical assistance. The research was supported by NSF
grant BNS-8708638, NIH grant HD22486, and a grant from
the Murdock Foundation.
KIMMEL, C. B., KANE, D. A., WALKER, C , WARGA, R. M. AND
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{Accepted 28 January 1990)