Cell Movements During Epiboly and Gastrulation in Zebrafish

Development 108,569-580(1990) 569 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, neighbors before gastrulation. Involuting cells eventu- cells located beneath a surface epithelial layer of the ally form endoderm and mesoderm, in an anterior- blastoderm undergo rearrangements that accompany posterior sequence according to the time of involution. major changes in shape of the embryo. We describe The epiblast is equivalent to embryonic ectoderm. three distinctive kinds of cell rearrangements. (1) Radial (3) Mediolateral cell intercalations in both the epiblast cell intercalations during epiboly mix cells located deeply and hypoblast mediate convergence and extension move- in the blastoderm among more superficial ones. These ments towards the dorsal side of the gastrula. By this rearrangements thoroughly stir the positions of deep rearrangement, cells that were initially neighboring one cells, as the blastoderm thins and spreads across the yolk another become dispersed along the anterior-posterior cell. (2) Involution at or near the blastoderm margin axis of the embryo. Epiboly, involution and convergent occurs during gastrulation. This movement folds the extension in zebrafish involve the same kinds of cellular blastoderm into two cellular layers, the epiblast and rearrangements as in amphibians, and they occur dur- hypoblast, within a ring (the germ ring) around its entire ing comparable stages of embryogenesis. circumference. Involuting cells move anteriorwards in the hypoblast relative to cells that remain in the epiblast; Key words: blastula, gastrula, morphogenetic movements, the movement shears the positions of cells that were involution, clonal analysis, cell lineage.

Introduction opposite direction: deep-lying blastoderm cells spread outward towards the margin to form the hypoblast. In the zebrafish embryo, after an early developmental Ballard's view has been generally accepted, but very period of rapid cleavages, morphogenetic movements recently, involution was observed directly in the small occur that rapidly produce major changes in the appear- embryo of a teleost, the rosy barb (Wood and Timmer- ance and organization of the blastoderm. During epi- mans, 1988). boly (Trinkaus, 1984a; 19846), beginning at the late During the course of cell-lineage analyses, we have blastula stage about 4h after fertilization, the blasto- followed cell movements during epiboly and gastru- derm thins and spreads to completely cover the yolk cell lation in zebrafish. We observed cell rearrangements during the course of 6h. Gastrulation begins about an that seemed nonsensical if considered only in terms of hour after epiboly is underway. The blastoderm, a the eventual fates that the lineages produced. First, in single multilayer of cells, rearranges into a two-layered the late blastula, cells scatter chaotically (Kimmel and structure consisting of a more superficial epiblast, and Law, 1985b; Kimmel and Warga, 1986). Second, in the an inner hypoblast (Wilson, 1891). Shortly after gastru- gastrula, neighboring cells at the blastoderm margin lation begins, the embryonic axis appears and lengthens undergo anterior-posterior inversions in their positions along one side of the embryo (the dorsal side), as cells (Kimmel and Warga, 1987a). Finally, cells in either accumulate and line up specifically at that location. The ectodermal (Kimmel and Warga, 1986) or mesodermal rearrangements that occur among the cells of the (Kimmel and Warga, 1987a) lineages disperse along the blastoderm during early morphogenesis, particularly anterior-posterior axis of the embryo. with respect to their lineal relationships and their future We now show that each of these cellular rearrange- fates, are not well understood. ments are understandable if they are considered in For example, several early embryologists concluded relation to the changes in form of the blastoderm that that during gastrulation the hypoblast originates by cell occur at the same time. Studies done mostly in Xenopus involution, a streaming of cells lying at the blastoderm suggest that cells undergo specific rearrangements to margin inward and underneath their neighbors (Wilson, mediate the changes in form (Keller, 1987). We find the 1891; Morgan, 1895; Pasteels, 1936). Later, Ballard same rearrangements occur in zebrafish at the compar- (I966a,b,c) concluded that the movement was in the able stages of development. 570 R. M. Warga and C. B. Kimmel

Materials and methods was advanced through the intact EVL. It was technically more difficult to specifically inject single DEL cells than EVL cells, Embryos and stages even under visual control. As an aid, we monitored voltage through the injection pipette. We observed that successful Zebrafish embryos were obtained from natural spawnings and passage of the pipette through the EVL was accompanied by a staged by cell number during early cleavage. They were rise in voltage of up to 40 mV; the extracellular space dechorionated with watchmaker's forceps and kept at 28.5°C surrounding DEL cells is at a positive potential relative to the in an incubation medium of 14mM NaCl, 0.6mM KC1, 1.3mM bath (Bennett and Trinkaus, 1970). Upon intracellular pen- CaCl2, lmM MgSO4, and 0.07mM sodium-potassium phos- etration of a DEL cell, we then observed the expected shift to phate buffer (pH7.2). In some experiments, we used embryos negative potential, reflecting the membrane potential of the homozygous for the gol-1 (golden) mutation (Streisinger et al. cell. 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 Observations of fluorescent cells in live embryos pigment cells differentiate. For short-term viewing of labeled cells, embryos were usually Developmental time usually was determined from the positioned as desired in a gel of 3 % methyl cellulose made in morphological features of the embryo, and Table 1 gives a the aqueous incubation medium described above and viewed staging series for the period of development of interest, from without a coverglass. Alternatively, embryos in incubation midblastula period until somites begin to form. We use the medium were sandwiched between two micro cover glasses letter h to mean hours after fertilization at 28.5°C. A that were spaced apart with three pairs of cover glasses (each previously published series, although less complete, includes 0.13-0.17mm thick). For longer term viewing and for time- useful sets of photographs (Hisaoka and Battle, 1958; lapse recordings, the embryos were held stationary in such Hisaoka and Firlit, 1960). In our series, names in common chambers in a gel of 0.1 % agarose made in the same medium, usage in embryology denote major periods of development and the chamber was then sealed with Vaseline to prevent (e.g. midblastula, gastrula), and the stages subdivide these evaporation. Observations were made using a Zeiss micro- periods. We name rather than number the stages, which scope with illumination from both a transmitted and an epi- seems to help one to remember them, and is more flexible. light source (Zeiss filter set 48-77-14), which permitted simultaneous imaging of labeled and unlabeled cells. The Blastomere injections fluorescent image was amplified with a Silicon-Intensified- Single blastomeres were injected (Kimmel and Law, 1985a), Target (SIT) video camera (Dage) to prevent light-induced in mid- and late blastula embryos with the lineage tracer dye damage to the labeled cells. In some experiments, the depths tetramethylrhodamine-isothiocyanate dextran (Molecular of fluorescent cells were determined with a digital shaft 3 encoder fitted to the fine-focus knob of the microscope. Probes, Eugene, OR; lOxlO Afr, diluted to 5 % (wt./vol.) in 0.2 M KC1). The second dye for double-label experiments was For time-lapse recordings, single-frame images were taken fluorescein-dextran (Sigma), dissolved the same way. Injec- with a Gyre video recorder at 4 s intervals. The epi-light tions were made by pressure, usually over the course of a few source was controlled by a shutter that illuminated the seconds, either into a cell in the surface enveloping layer embryo for only 60 ms during each exposure, in order to (EVL), or, in other cases, into a cell in the deep layer (DEL) minimize light-induced damage to the labeled cells. Frequent of the blastoderm. To inject a DEL cell, the injection pipette refocusing of the image was required during the recording

Table 1. Series of normal stages for 3-10.5 h of development Stage h1 HBb Description 1 k-cell 3 10 Midblastula; yolk syncytial layer present; cell cycles of blastoderm cells fairly synchronous, determined by presence or absence of interphase nuclei 2k-cell 3.2 Single row of yolk syncytial layer nuclei; cell cycles of blastoderm cells highly asynchronous High 3.5 Blastoderm perched high upon the yolk cell, giving the embryo a dumbbell shape; yolk syncytial layer nuclei in two rows Oblong 3.7 11 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 Sphere 12 Late blastula; embryo has assumed a spherical shape; at a deep plane of focus the yolk cell-blastoderm interface is flat Dome 4.3 13 Yolk cell bulging (doming) towards animal pole as blastoderm rapidly thins by epiboly 30%-epiboly 4.7 14 Blastoderm shaped as an inverted cup of uniform thickness and covers 30% of the yolk cell 50%-epiboly 5.2 Gastrula; 50% of the yolk cell is covered by the blastoderm Germ-ring 5.5 Germ ring visible from animal pole; 50%-epiboly Shield 6 15 Embryonic shield visible from animal pole, 50%-epiboly 75%-epiboly 8 16 The blastoderm continues to spread over the yolk cell at a rate of 15 %-epiboly per hour 100%-epiboly 9.5 17 Yolk plug closed. Gastrulation movements nearly complete in the anterior parts of the embryo Bud 10 Tail bud prominent at the posterior end of the axis l-Somite 10.5 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. bHB: Approximate stage number in the zebrafish staging series described by Hisoaka and Battle (1958). Eplboly and gastrulation In zebrafish 571 session, which began at sphere stage (4 h) and continued for at entiated were reexamined on the 2nd and/or the 3rd day of least 6h, when epiboly is completed, and generally for a few development. hours longer. Afterwards, the embryo was released from the viewing chamber and reexamined at 24-30 h, when many cell Histology types have begun to differentiate and can be distinguished by We also studied a set of sectioned embryos. They were fixed at their morphologies and positions in identifiable tissues (Kim- intervals between late blastula (4h) and midgastrula (7h) mel and Warga, 1987«). Labeled cells that were still undiffer- periods by immersion in Bouin's solution (Humason, 1962), AP B

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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 DEL cells become motile in the midblastula, after the were cut and stained with azure A, methylene blue and basic tenth cleavage at 3h (D. A. Kane, unpublished obser- fuchsin (Humphrey and Pittman, 1974). vations). 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 Results blastoderm becomes evident, signifying epiboly is underway. The first change observable is very deep in Deep and shallow DEL blastomeres intercalate during the embryo, where the yolk cell begins to bulge or epiboly 'dome' towards the animal pole (Fig. IB). The blasto- In zebrafish, cleavages generate two populations of derm then rapidly takes on a cup-shaped appearance, distinctive blastoderm cells; flattened epithelial cells in and spreads to cover the yolk cell (Fig. 1C). a surface enveloping layer (EVL), and rounded, more At the beginning of the late blastula stage, single loosely associated deep layer (DEL) cells lying beneath clones descended from a progenitor cell labeled earlier the EVL. The EVL is a monolayer and the DEL a are coherent groups of cells. Later, during epiboly, the multilayer of cells. All of the movements we describe in DEL cells in such clones rapidly spread apart, inter- this paper pertain to the DEL: the EVL cells behave spersing with unlabeled cells (Kimmel and Law, 1985b). relatively passively. Neighb6r exchanges occur within In Xenopus, epiboly is known to occur by radial cell the EVL (Keller and Trinkaus, 1987), but they are intercalations, in which cells at different depths in the infrequent. We have not observed neighbor exchanges blastoderm intercalate, thus producing its thinning between the EVL and DEL. (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 B 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 Fig. 2. The double-label method used to distinguish the the onset of gastrulation (see Discussion). Within about morphogenesis and fates of sibling clones located at different depths in the blastoderm. (A) At the 32-cell stage, 15 minutes, the blastoderm becomes noticeably thicker the blastomeres are often arranged in a single-layered 4x8 in a circumferential band at its margin. The band, or array. One of the central rows of 8 cells is shown, and a cell germ ring, at first appears uniform in structure, and in this row, adjacent to the animal pole, is injected with using time-lapse video microscopy, we observed in rhodamine-dextran (coarse stippling). (B) Following the views from the animal pole (3 embryos) that it forms next (sixth) cleavage, which is horizontal, the upper of the more-or-less simultaneously, within about 15min, two labeled sister cells is reinjected, now with around the entire circumference of the blastoderm. fluorescein-dextran (fine stippling). The lower daughter, containing only rhodamine-dextran is adjacent to the yolk An analysis of involution is shown in Fig. 4, a case cell. (C) Following several more cleavage divisions the two where we kept track of the depths of cells in the sister clones are expected to remain coherent in the blastoderm as their rearrangements occurred. Here, midblastula, since cell mixing is very limited before late minutes after the onset of gastrulation, a clone of 5 blastula stage. The rhodamine-labeled clone is expected to labeled cells was located near the margin of the blasto- lie deep in the blastoderm, immediately beneath the doubly derm. The cells initially occupied a shallow position labeled clone, which extends to the blastoderm surface. 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 573

Fig. 4A. The labeled cells moved towards the margin, epiblast, and involute. Consequently the hypoblast apparently actively, since we observed blebbing and increases in area and extent beneath the epiblast, formation of filopodia, and two of them divided. Upon eventually spreading all the way from the margin to the reaching the margin, each cell protruded processes and animal pole. 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 Fates of epiblast and hypoblast cells the margin, and proceeded away from the margin, now Single cells present during gastrulation generate clones located deeply in the blastoderm (Fig. 4C and D; red), restricted to single types of tissues (Kimmel and Warga, within its new hypoblast layer. 1986). Now we have shown that some of these cells, but These findings show that involuting DEL cells form not others, involute during gastrulation, and we can ask the hypoblast. The first ones to involute are those whether involuting and noninvoluting cells have differ- located just at the blastoderm margin at the beginning ent fates. We kept track of cells in labeled clones during of gastrulation (Fig. 5A), and as they migrate inwards gastrulation and determined the fates of their descend- the germ ring forms behind them (Fig. 5B and C). ants at a later stage, as illustrated in Fig. 7A. From such Analysis of sectioned embryos revealed that before records, we reconstructed the cell lineage (Fig. 7B) and germ ring formation there is no layering of cells within the pathways of movement of the cells during gastru- the DEL itself (Fig. 6A). However, after the germ ring lation (Fig. 7C). In this example, all of the labeled cells forms, the DEL appears folded inwards at the margin, involuted, inverting their relative positions as they did and is split, within the germ ring specifically, into the so. The progeny of one of the cells originally present epiblast and hypoblast (Fig. 6B). As epiboly and gas- (Fig. 7, black lineage) all formed somitic mesoderm, trulation continue, cells that initially were located including differentiated muscle fibers. The progeny of distantly from the margin move towards it within the the other cell (stippled) formed derivatives of two B

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different germ layers; gut epithelium (endoderm) and form endoderm, and cells that involute later formed somitic muscle (mesoderm). mesoderm. This can be seen most clearly from single Summary lineage analyses for cells in other embryos clones that contributed to both germ layers (e.g. arrows analyzed the same way are shown in Fig. 8. Without in Figs. 7B and 8D). Except for the early involution of a exception (and also for 5 additional clones not illus- cell that later formed heart tissue, a mesodermal trated), cells that involuted (arrows) later formed derivative (Fig. 8D), this rule was generally followed mesoderm and endoderm, and cells in lineages where (including those clones that contributed cells to only a involution did not occur (no arrows) formed ectoderm. single germ layer; compare the times of involution of The lineage shown in Fig. 8B is noteworthy, for in this cells in Fig. 8C and E). case two sibling subclones, separated at the first division Cells involuting at different times during gastrulation shown in the diagram, developed differently from one also had different pathways of movement within the another but still followed the general rule: None of the hypoblast, and later occupied different positions in the cells in one of the subclones involuted and they sub- embryo. Cells that involuted soon after the beginning of sequently developed as ectoderm. The cells in the gastrulation sharply reversed their direction of move- second subclone all involuted and then formed meso- ment as they involuted, turning towards the animal pole derm. We conclude from these results that the epiblast (Fig. 7C; stippled cell ppp). Cells that involuted some- is the rudiment of ectoderm and the hypoblast is the what later turned less sharply (stippled cell aaa), and rudiment of both mesoderm and endoderm. cells that involuted much later during epiboly (black cell aaa) did not turn at all, but continued to move The time of involution is related to fate towards the vegetal pole after entering the hypoblast. Cells that involute early during gastrulation usually No matter whether a turn occurred or not, all involuting Epiboly and gastrulation in zebrafish 575

Fig. 7. Analysis of movements, lineages and fates of movement that correlated with whether a turn oc- clonally related cells that involute. A single DEL cell curred, or with later fate (data not shown). located near the margin of a blastula embryo was labeled The animal pole of the gastrula develops into the with rhodamine-dextran (2.3 h). Its subsequent development was recorded by time-lapse video during anterior-most structures of the later embryo (Kimmel epiboly and early tailbud stages (4-13 h). The fates of the and Warga, 1987b), and accordingly, cells that turn cells in this clone were then determined by comparing towards the animal pole during gastrulation develop positions of the labeled cells at 13 h (after gastrulation more anterior structures. It follows that the order in movements were over), and later, when they had begun which cells of a clone involute corresponds to their differentiation in the embryo at 30 h. (A) A series of subsequent order along the anterior-posterior axis of tracings from selected frames during the first 4h from the the embryo, as can be seen from the positions of the time-lapse recording (3.8-7.8 h), and, in the last drawing, black and stippled cells in Fig. 7. This relationship was the fates of the cells in the 1-day embryo. Anterior is to the consistent, as shown in Fig. 9 where data is collected top, the blastoderm margin is to the bottom (except for the last drawing, where there is no margin). The founding from a set of 5 embryos in which the labeled clones were labeled DEL cell had divided a single time by the time the positioned at approximately the same lateral blasto- recording session began. Cells of the subclone generated by derm location at the beginning of gastrulation. 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 Convergent extension movements are mediated by by v's, and arrowheads point to newly involuting cells. mediolateral cell intercalations Initially the stippled cell is nearest the margin. Its progeny involute earliest, and some of them eventually differentiate Convergence is a third morphogenetic cell movement as epithelial cells of the foregut (gut). Other cells in this occurring simultaneously with epiboly and involution. subclone, and cells of the black subclone differentiate as DEL cells move towards the dorsal side of the gastrula somitic muscle (mus). A few cells in the black subclone that (Ballard, 1973; Kimmel and Warga 1987b), 'converging' are present within one somite remain undifferentiated there from their original locations in the blastoderm. (und). Notice that the stippled cells are initially closer to The formation of the embryonic shield, a local dorsal the margin, involute earlier, and then form more anterior thickening of the germ ring (Oppenheimer, 1937; fates, including endoderm. (B) Cell lineage diagram for the Hisaoka and Battle, 1958), is a prominent effect of early same two subclones for the period 3.8-13 h. Hours of convergence movements. In Xenopus, convergence development are shown to the left. Horizontal lines in the diagram indicate divisions and vertical lines indicate cells; cannot be separated from extension, the elongation of the more anterior daughter cells (and farther from the the embryonic axis (Keller and Danilchik, 1988). Cells blastoderm margin) arising at each divisions are shown as move from lateral positions dorsalwards by intercalat- left-side branches in the diagram. Arrowheads indicate ing between neighboring cells that lie more medially when individual cells involuted; in general cells towards the (i.e. towards the axis). Such 'mediolateral intercal- left involute later because they are farther from margin. ations' (Keller and Tibbetts, 1989) produce both nar- The endodermal (end) and mesodermal (mes) cell fates are rowing and elongation of the axis. indicated below the diagram. Notice the symmetrical (generative) form of the lineage: sibling cells tend to divide Convergent extension occurs both in the epiblast at the same time, as is also the case in the examples shown (Kimmel and Warga, 1986; and see below) and in the in Fig. 8. (C) Pathways of movements of selected cells hypoblast. Convergence of hypoblast cells is illustrated present in the same two subclones. aaa refers to the most in Fig. 7C as a drift in the pathways of the cells towards anterior, and ppp to the most posterior cells in the lineages the right side of the figure, the direction towards the (i.e. the left-most and right-most branches in B). During embryonic shield in this example. That intercalations the recording session, the embryo itself was held stationary occur during this movement is revealed by the separ- in agarose, and serves as a fixed reference for the ation and spreading apart of the clonally-related cells movements of these individual cells within it. The pathways along the anterior-posterior axis (Fig. 7A). During this were reconstructed directly from the video tapes. The star dispersion, the labeled cells are intercalating among shows the starting position of the cell, and the arrowhead more medial unlabeled neighbors. shows the time of involution. The orientation is as in A. Individual points along the pathways represent approximate The intercalations are more completely illustrated in one-half hour intervals. Notice that the cells that involute Fig. 10, an example following cells that remained in the earlier (in the stippled subclone in this case) turn anterior epiblast. This case is instructive because intercalations as they involute. Whether or not a turn occurs, cells recede occurred not only between labeled and unlabeled cells, from the margin after involution. Scale bars (in A & C): but also between different labeled cells, and it is clear 100 ^m. 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 cells recede from the margin of the blastoderm, which indicate their lineal relationships. Eventually black and continues to rapidly advance by epiboly towards the hatched cells were positioned between different white vegetal pole. We measured the rates that the involuting cells. The intercalations effecting this intermixing oc- DEL cells moved (18 cells from 3 embryos), but no curred in the gastrula, beginning at about 6.8 h significant differences were found in the speed of (Fig. 10). Shortly after both the black and hatched cells 576 R. M. Warga and C. B. Kimmel divided, one of the two daughter cells from each of the gastrulation, although mixing within each of these divisions inserted between the pair of white sister cells. layers is extensive. In contrast to the radial intercalations that we considered above, the intercalations occurring during convergent extension do not scatter cells indiscriminately. Discussion For example, we have not observed DEL cells in the hypoblast and epiblast to mix with one another during This work has revealed three distinctive cell movements

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11 - end end 12 - 13 - nnnJ, 6 mus- 23 gut 3hrt mes end 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 577

Fig. 9. The time during gastrulation when a cell involutes

23 . correlates directly with the later anterior-posterior position of its progeny. The five different symbols represent cells in clones from five separate embryos, from the set shown in C 20 . 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 o 15 . embryonic shield (at the dorsal side; see Kimmel el al. 2 1 1990). The horizontal axis indicates when involution •L « occurred, relative to the onset of gastrulation at 50%- 10 . epiboly. The vertical axis indicates position along the 1 anterior-posterior axis of the embryo at 24 h. Vertical lines < above the individual symbols indicate the extent of 0 5 . anterior-posterior spread within the subclone derived from • aa [ 1 the involuting cell, in those cases where the spread was extensive. 1 . i "i 3 . • • 1 . along with an expansion and change in shape of the yolk 1 . cell that occur simultaneously, these cell movements «n . o appear to mediate the thinning of the blastoderm that 1 1 —t— H 1 1 1 1 occurs rapidly during this period of development. 0 12 3 Intercalations thoroughly scatter DEL cells and are Hours after 50%-eplboly responsible for marked dispersion of clonally related cells that we have described elsewhere (Kimmel and that accompany, and appear to produce, the early Law, 1985b). It is interesting, however, that DEL cells changes in shape of the zebrafish embryo. These do not intercalate outward into the EVL. The EVL, by movements are epiboly, involution and convergent this stage, has acquired the form of a highly flattened extension. They are diagrammed respectively in squamous epithelium. In Fundulus at a comparable Figs 11-13. stage of development, junctional complexes are present between cells of the EVL (Betachaku and Trinkaus, Epiboly 1978), and it may be that such junctions mediate high Radial intercalations (Keller, 1980) among DEL blasto- adhesivity among the EVL cell, such that the underly- meres occur first, in the late blastula (Fig. 11), and ing DEL cells are unable to penetrate this layer.

25.0h

12.7h

9.2 h

8.7 h

6.8h 6.5h 5.0 h

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

\ yolk Fig. 13. Diagram of mediolateral intercalations. (A) shield cell stage, early gastrula. (B) 80%-epiboly, late gastrula. Dorsal views. DEL cells converge towards the dorsal midline, Fig. 11. Cut-away diagram of radial intercalations moving from lateral to medial positions. This movement beginning in the. midblastula (A), and through the onset of (convergent extension) lengthens the embryonic axis gastrulation (B). Deep DEL cells move outward (radially), (dashed line). intercalating among more superficial DEL cells but not among EVL cells (See Fig. 3). This movement contributes to epiboly, thinning and spreading the blastoderm* 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 Trin- kaus, 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 epiblast 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 hypoblast carefully examined cell movements within the embryonic shield (at the dorsal side of the embryo).

Fig. 12. Cut-away diagram of involution. (A) Onset of Wood and Timmermans (1988) recently also ob- gastrulation. (B) Germ-ring stage, 20min later. Side views. served involution in the rosy barb, another teleost in the A DEL cell first at the blastoderm margin (black) is at the same family as zebrafish. However, Ballard could not front of the wave of involuting cells (See Fig. 5). This find involution in his careful and extensive studies of a movement generates the hypoblast. EVL cells do not variety of other (and larger) teleost embryos involute. (1966a,b,c; 1973; 1981; 1982). It may be that gastru- Epiboly and gastrulation in zebrafish 579 lation is dramatically different in large and small teleost and Warga, 1986; Kimmel et al. 1990). We showed embryos, but we think it more likely that all teleosts earlier (Kimmel and Warga, 19876) that the EVL cells gastrulate as the zebrafish and rosy barb do, and that do not undergo convergence, at least in the sense used the cell marking procedures available to Ballard were here to mean a specific dorsalwards movement. inadequate to reveal all the cell movements that occur The EVL may be a relatively passive participant in in the DEL. blastoderm epiboly; as revealed by studies in Fundulus, Involution is special in teleost fish, as compared to it seems to be pulled and stretched across the yolk cell other types of vertebrates, in that the EVL is not by the yolk syncytial layer of the yolk cell itself involved (see below). Moreover, involution doesn't (Betchaku and Trinkaus, 1978; Trinkaus, 1984a). How- seem to be initiated first at the dorsal side of the embryo ever, perhaps active rearrangements among EVL cells (as it does for example in amphibians). As judged from have recently been shown to occur both in Fundulus the time-course of appearance of the germ ring, invol- (Keller and Trinkaus, 1987) and the medaka ution in the zebrafish begins more-or-less simul- (Kageyama, 1982), where they serve to continuously taneously around the circumference of the blastoderm. decrease the diameter of the EVL as epiboly is completed and the marginal ring of EVL cells closes at the Convergent extension vegetal pole of the yolk cell. It is likely that this During gastrulation DEL cells also undergo mediolat- rearrangement also occurs in the zebrafish, for EVL eral intercalations (Fig. 13), producing a general dorsal- cells in single clones do sometimes become dispersed wards drift of the cells that has been described pre- from one another, rather than being present in a single viously in other teleosts (e.g. Ballard, 1973; 1982). The coherent patch (e.g. Kimmel and Warga, 1987b). The cells accumulate dorsally to form the embryonic shield, dispersion is, however, markedly less than that occur- and the subsequent narrowing (convergence) and ring in the DEL. lengthening (extension) of the shield produces a well- defined embryonic axis within about two hours after the Control and patterning of cell movements shield first forms. Our studies are descriptive, and do not reveal the We have shown that cells in both the hypoblast and mechanisms that underlie these morphogenetic move- epiblast undergo convergent extension. The intercal- ments. However, the rearrangements appear to be ations appear to be regulated such that extensive mixing active ones, for DEL cells constantly change in shape occurs among the cells within both of these layers, but and they move relative both to neighboring cells and to not between the layers. Moreover, the fact that most a fixed point on the yolk cell. The yolk cell and EVL gastrula lineages are tissue-restricted (Kimmel and cells both participate in epiboly and, as we have shown Warga, 1986) shows that mixing among cells must occur here, so do DEL cells. The gastrulation movements of within, but not between, the primordia of different involution and convergence may also depend upon tissues. However, the boundaries of the primordia are interactions among cells of all three classes. Recently invisible in the gastrula, such that we could not hope to Symes and Smith (1987) suggested that activation of observe distinctive cellular behaviors in their vicinities. gastrulation movements in amphibians is an early Later in embryogenesis the boundaries become recog- consequence of mesodermal induction. This might nizable, and no mixing occurs across at least one of involve the yolk cell; Long (1983) obtained evidence them - the boundary separating the axial (prospective from transplantation experiments in the trout that the notochord) and paraxial (prospective somite) meso- yolk cell can induce dorsoventral polarity of the blastoderm - as recently shown for Xenopus (Wilson et al. derm. 1989) and rosy barb (Thorogood and Wood, 1987). Progress in understanding how such specific movements are produced may come through mutational The enveloping layer analysis in zebrafish. We have recently described a All of the movements we have described appear to mutation, spt-1, that appears to selectively disrupt closely resemble their counterparts that have been convergence of laterally positioned mesodermal cells thoroughly described in Xenopus (Keller, 1986). Radial during gastrulation (Kimmel et al. 1989). Convergence intercalations, involution movements and mediolateral of ectoderm is not disturbed, suggesting that different intercalations occur at the equivalent stages, relative to genes control dorsalwards movements of cells that gastrulation onset, in zebrafish and Xenopus and they occupy different germ layers. Furthermore, mosaic produce equivalent changes in shape and organization analysis suggests that the wild-type gene is required in of the embryo. There is a single important difference, the mesoderm specifically (Ho et al. 1989). The gene however; the outside layer of cells in the teleost could code for, or regulate the expression of, a receptor blastoderm does not participate in any of them. DEL or adhesion molecule required for the convergence cells do not enter the EVL during their radial intercal- movements of a subset of mesodermal cells. ations, as we have shown in this study. We also An important finding from our study is that cells that confirmed that EVL cells do not undergo involution, as involute to enter the hypoblast then give rise to endo- was first convincingly shown by Ballard for the trout derm or mesoderm and, conversely, that the epiblast is (1966a). This finding was expected in zebrafish since the the equivalent of ectoderm in other vertebrates. This exclusive fate of the EVL is the periderm - an outer- observation is in accord with the interpretations of early most epithelial cell layer covering the embryo (Kimmel 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 KELLER, R. E. (1980). The cellular basis of epiboly: an SEM study cell in the hypoblast will form endoderm or mesoderm, of deep cell rearrangement during gastrulation in Xenopus laevis. J. Embryol. exp. Morph. 60, 201-234. and where its clonal descendants will come to lie along KELLER, R. E. (1986). The cellular basis of amphibian gastrulation. the anterior-posterior axis of the embryo is directly In Developmental Biology: A Comprehensive Synthesis. Vol. 2. correlated with when it entered the hypoblast. Further- The Cellular Basis of Morphogenesis (ed. L. Browder), pp. more, we detected no differences in direction or rate of 241-327. movements of DEL cells as they approached the KELLER, R. E. (1987). Cell rearrangement in morphogenesis. Zool. Sci. 4, 763-779. margin, before involution, that correlated with their KELLER, R. E. AND DANILCHIK, M. (1988). Regional expression, future fates. Together, these observations lead to the pattern and timing of convergence and extension during suggestion that whether and when a cell involutes is a gastrulation of Xenopus laevis. Development 103, 193-209. direct function of how far from the margin it was KELLER, R. E. AND TIBBETS, P. (1989). Mediolateral cell positioned prior to the onset of gastrulation. We ad- intercalation in the dorsal, axial mesoderm of Xenopus laevis. dress this issue in the accompanying paper (Kimmel et Devi Biol. 131, 539-549. KELLER, R. E. AND TRINKAUS, J. P. (1987). Rearrangement of al. 1990), examining in more detail how cell fate is enveloping layer cells without disruption of the epithelial related to cell position in the early gastrula. permeability barrier as a factor in Fundulus epiboly. Devi Biol. 120, 12-24. We thank D. A. Kane, A. Felsenfeld and D. Frost for their KIMMEL, C. B., KANE, D. A., WALKER, C, WARGA, R. M. AND critical comments on early versions of this paper, and for ROTHMAN, M. B. (1989). A mutation that changes cell movement stimulating discussion throughout the course of the study. C. and cell fate in the zebrafish embryo. Nature 337, 358-362. Cogswell, P. Myers, H. Howard, and R. Kimmel provided KIMMEL, C. B. AND LAW, R. D. (1985a). Cell lineage of zebrafish technical assistance. The research was supported by NSF blastomeres I. 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