Gradient in convergent cell movement during Fundulus gastrulation.

This contribution represents a continuation of our studies of a gradient in convergent cell movement in the germ ring (GR) during Fundulus gastrulation (Trinkaus et al. [1992] J. Exp. Zool., 261:40-61). In our previous study, we discovered that cells in the dorsal GR nearest the embryonic shield (ES) move toward the ES at a net faster rate than those farther away and that this is due to less meandering. Those farther away meander more. These data suggest the hypothesis that there is a gradient of cues that direct cells to the ES. If so, cells in the ventral GR, farthest from the ES, should meander even more and hence show little or no convergence toward the ES. To test this hypothesis, we have traced the trajectories of individual cells in the midventral GR during midepiboly and have found that, although the general motile behavior of ventral GR cells is the same as that of dorsal cells, they do indeed meander much more and, as a result, show little or no directional movement toward the ES. Taken together, these results indicate that cells of the germ ring move up a gradient in directionality as they converge toward their target, the embryonic shield. One possible explanation for this is that the embryonic shield attracts cells to itself.

Ingression during early gastrulation of fundulus.

This study demonstrates that involution does not occur during early gastrulation of Fundulus heteroclitus, prior to and during germ ring formation. This conclusion has been reached by following the motile behavior of large numbers of individual cells. Instead of involution, superficial deep cells of the marginal region of the blastoderm undergo ingression. They do not leave the surface as members of a flowing cohesive sheet, but sink beneath rather haphazardly as individuals. Indeed, during much of ingression, many marginal cells are so loosely arranged that they move about freely on the yolk syncytial layer. A small proportion of the cells initially at the blastoderm margin undergo ingression there, but most recede from the margin and ingress supramarginally one to three cell diameters from the margin. Cells that are initially supramarginal ingress mainly there, sometimes quite far from the margin. Only a small number moves to the margin and ingresses there. Interestingly, although most ingression takes place supramarginally, much occurs close to the margin-up to one to four cells away. Ingression begins immediately after the onset of epiboly and is most active before appearance of the germ ring; it ceases quite soon thereafter. It is also more active dorsally than ventrally, correlating with the earlier formation of the germ ring dorsally. Ingression constitutes the first invasive cellular activity of development. Significantly, it proceeds by blebbing locomotion, a noncontact inhibiting mode of cell movement. The possible broader import of these discoveries is given appropriate attention.

The yolk syncytial layer of Fundulus: its origin and history and its significance for early embryogenesis.

Because of its importance in early embryogenesis, the developmental history of the yolk syncytial layer (YSL) of Fundulus has been investigated in detail. As in other teleosts, the Fundulus YSL forms mainly by collapse of certain marginal blastomeres which then merge with the cytoplasm of the yolk cell peripheral to the blastoderm. Nuclei enter the yolk cell from these open blastomeres variably during cleavages 8-11, but most frequently at cleavages 9 and 10. After entry, the first nuclei divide five times and later nuclei divide with them. Thus, nuclei that have invaded at the next cleavage divide four times, etc. When the first YSL nuclei cease dividing, all other YSL nuclei cease dividing with them. These YSL mitoses occur in metachrony. Two or more metachronous waves progress through the YSL cytoplasm at each mitosis. After each nuclear division, the YSL increases in width and its nuclei are quite evenly spaced. After the 5th and last mitosis, when the YSL is at its widest, it contracts in its animal-vegetal axis. This slow contraction has two major effects: 1) narrowing of the YSL, accompanied by crowding of its nuclei and their disappearance beneath the blastoderm to nucleate the internal YSL; 2) epibolic expansion of the I-YSL and the blastoderm, followed soon after by other cell movements of gastrulation. This YSL transition, therefore, sets the stage for the onset of gastrulation. It is preceded by increased duration and variability of succeeding mitoses and, in particular, duration of their interphases, a decrease and deceleration in the rate of the last metachronous waves, and, finally, by the complete cessation of mitosis and the entry of YSL nuclei into permanent interphase.

The midblastula transition, the YSL transition and the onset of gastrulation in Fundulus.

The first signs of cell motility appear in Fundulus toward the end of cleavage, after cleavages 11 and 12. When blastomers cease cleaving, their surfaces undulate and form blebs. At first, these blebbing cell remain in place. Gradually thereafter they begin movement, with blebs and filolamellipodia serving as organs of locomotion. Non-motile cleaving blastomeres have thus differentiated into motile blastula cells. This transformation corresponds to the midblastula transition of amphibian embryos. Gastrulation in Fundulus begins with vegetalward contraction of the external yolk syncytial layer. This causes narrowing of the E-YSL and initiates the epibolic expansion of the blastoderm. Convergent movements of deep cells within the blastoderm begin toward the end of this contraction. The YSL forms as a result of invasion of the yolk cell cytoplasm by nuclei from open marginal blastomers during cleavage. These YSL nuclei then undergo five metachronous divisions. After this, they divide no more. YSL contraction begins approximately 1.5 hours after cessation of these divisions (21-22 degrees C). This cessation of nuclear divisions is preceded by a gradual decrease in rate. (1) The duration of each succeeding mitosis increases steadily and often some nuclei do not divide at mitosis V. (2) The duration of interphases between succeeding mitoses also increases, but to a much greater degree, and the longest interphase by far is the last one, I-IV, between mitoses IV and V. (3) The mitotic waves responsible for mitosis V move much more slowly than those for the first four mitoses and invariably decelerate.(ABSTRACT TRUNCATED AT 250 WORDS)

On the convergent cell movements of gastrulation in Fundulus.

Mainly because of its transparency, the Fundulus gastrula constitutes ideal material for direct study of morphogenetic cell movements in vivo. Marking studies show that deep cells of the germ ring converge toward and enter the embryonic shield, where they undergo extension. Those close to the shield move faster. Analysis of videotapes reveals that all deep cells of the dorsal germ ring move toward the shield. But none moves in a direct line. All meander considerably. Germ ring cells nearer the shield move toward it at a higher net rate than those farther away because they meander less. This suggests that exogenous factors promote their directionality. Cells in the prospective yolk sac adjacent to the germ ring also show net convergence, but they meander more. Directional forces are apparently stronger in the germ ring. Converging deep cells move both by filolamellipodia and, less frequently, by blebs. However, there is very little individual cell movement; all cells are almost always in adhesive contact with other cells in moving cell clusters. Clusters vary constantly in size, continually aggregating with other cells and other clusters and splitting. Filolamellipodial cells show contact inhibition of cell movement. Nevertheless, they move and do so directionally, presumably in part because, as members of cell clusters, much of their movement is passive. They also show intercalation or invasive activity, but, consistent with their contact-inhibiting properties, only when neighboring cells separate and provide free space. Cells moving by blebbing locomotion are non-contact inhibiting and intercalate readily. Cell division continues during convergence. Although this temporarily arrests their movement, the daughter cells soon join in the mass convergent movement.

Directional cell movement during early development of the teleost Blennius pholis: II. Transformation of the cells of epithelial clusters into dendritic melanocytes, their dissociation from each other, and their migration to and invasion of the pectoral fin buds.

After clusters of pigmented epithelial cells have rested immobile in the yolk sac of Blennius pholis for 2-4 days (Trinkaus, '88), their constituent cells transform into mesenchymal, dendritic melanocytes. Then these melanocytes dissociate from one another and migrate directionally toward the developing pectoral fin bud (PFB) on either side. Each of these changes takes place in a proximodistal sequence, starting with the epithelial cluster closest to each PFB. Even individual clusters conform to this sequence, the proximal side dissociating first. Eventually, all melanocytes reach and invade the PFB. This is a 100% efficient morphogenetic cell movement. At the completion of this developmental sequence, each PFB is filled with melanocytes arranged in an arc with their filopodia extending outward and the yolk sac is bereft of pigment cells. The form and surface activity of these cells in relation to their motility and to their rate of movement are considered in detail. Attempts to understand the forces involved in giving directionality to these cell migrations are described. Finally, the significance of these observations for morphogenetic cell movements generally and for the relation between epithelial and mesenchymal cells is discussed briefly.

Fundulus deep cells: directional migration in response to epithelial wounding.

During the normal embryogenesis of the killifish Fundulus heteroclitus deep cells migrate in an apparently random fashion throughout the subepithelial space of the yolk sac. These cells migrate by blebbing locomotion, and individual cells show tendencies for persistence in the directionality of their movement. Immediately after the wounding of the yolk sac epithelium (the enveloping layer), these deep cells reorient and migrate directionally toward the site of wound closure. This directional migration results in the aggregation of a large number of cells at the wound site. The response is both rapid and widespread; cells as far away as 800 micron respond as quickly as those nearby, and by 100 min after wounding up to 90% of the blebbing deep cells within this radius have clustered about the wound site. Then, cells begin to disperse, and by 150 min after wounding, it is almost impossible to tell where the wound had been made. Because of the transparency of the Fundulus yolk sac, this phenomenon can be utilized as a model system for observing details of in vivo directional cell movements. Time-lapse video micrography has revealed that the modes, rates, and overall cell morphologies during locomotion are identical for cells migrating in both unwounded and wounded embryos. What is different in the wounded embryos is that a single directionality is imposed upon a large population of cells, resulting in aggregation. Several aspects of the aggregation phenomenon suggest that a possible attractant originating at the wound site may travel through the subepithelial space by diffusion.

Directional cell movement during early development of the teleost Blennius pholis: I. Formation of epithelial cell clusters and their pattern and mechanism of movement.

Embryos of the teleost Blennius pholis provide exceptional material for observation of the formation and movement of cell clusters in vivo because the clusters are packed with melanosomes and migrate beneath the transparent enveloping layer. These clusters arise from two pigmented cell masses (PCM) which appear precociously on either side of the embryonic axis at 3/5 epiboly, at the future level of somites 1 and 2. As development proceeds, each PCM enlarges and spreads on its lateral margins to form an epithelial sheet. As spreading continues, the sheet fragments, forming small cell clusters that move in a distad direction in the yolk sac. The highly motile lateral marginal cells of the spreading PCM, as well as those of the marginal cells of each moving cluster, invariably protrude highly flattened lamellipodia, which terminate in long, fine, often branched filopodia. As cell clusters leave the PCM, they form long, taut retraction fibers. The rate of spreading of both the lateral edge of the PCM and the initial phase of cluster movement, is higher (1.0 micron/min or greater) than the later rate of cluster movement, apparently because at this phase, motile activity is confined to the distal borders of each. This directional migration ceases in 24 h at 16 degrees - 18 degrees C, when the farthest clusters have reached the ventral region of the yolk sac. By then, all clusters are spaced more or less evenly, apparently due to cessation of all cluster movement at about the same time. Once movement ceases, the clusters remain immobile for 2-4 days, depending on the temperature.

Rearrangement of enveloping layer cells without disruption of the epithelial permeability barrier as a factor in Fundulus epiboly.

Silver nitrate staining of blastoderms of Fundulus heteroclitus gastrulae shows that the number of marginal cells of the enveloping layer (EVL) is reduced from 160 to 25 during epiboly. To determine whether this decrease in the number of marginal cells was due to ingression, cell death, or rearrangement of cells, marginal and submarginal regions of the late gastrula were observed directly by time-lapse cinemicrography. Marginal cells rearrange to occupy submarginal positions by first narrowing their boundary with the external yolk syncytial layer (E-YSL), thus becoming tapered in shape. Then, the narrowed marginal boundary retracts from the E-YSL and moves submarginally in the plane of the epithelium. Concurrently, the marginal cells on both sides come into apposition; no gap or break appears in the circum-apical continuity of the epithelial sheet. Marginal cells leave the margin of the EVL during epiboly at a rate of about six per hour. The rate of movement of the EVL cells with respect to one another is about 0.5 to 1.0 micron/min at 21 degrees C. Submarginal cells rearrange in a similar fashion. Although no protrusive activity was seen at the lateral aspects of rearranging cells, the tapering or narrowing associated with rearrangement was accompanied by formation of microfolds on their apical surfaces, and separating or recently separated submarginal cells form "flowers" of microfolds on their apices adjacent to the site of separation. Morphometric analysis shows that about half the narrowing of the margin of the EVL during epiboly is accounted for by cell rearrangement and the other half by the associated tapering and narrowing. These results suggest that epiboly of the EVL may have an active component as well as a passive one.

Further thoughts on directional cell movement during morphogenesis.

This essay discusses the directional movements of metazoan tissue cells generally, with special emphasis on neurons, in an attempt to show that the directional movements of all share fundamental similarities.

Significance of cell-to cell contacts for the directional movement of neural crest cells within a hydrated collagen lattice.

Neural tubes whose neural crest had just begun migration were isolated from stage-14 chick embryos, cleaned with 0.1% trypsin, and cultured in transparent hydrated collagen lattices (HCL) in an effect to stimulate in part the three-dimensional environment through which neural crest cells migrate in situ, in the embryo. The concentration of collagen in the lattices varied from 50 microgram/ml to 390 microgram/ml. The mode of movement and contact behaviour of neural crest cells migrating from the neural tube under these conditions were recorded directly with time-lapse cinemicrography. Both their shape and their rate of translocation were dependent on the concentration of collagen in the HCL. In low concentrations (50 microgram/ml to 105 microgram/ml), neural crest cells have elongate spindle shapes and translocate at an average rate of 1 micrometer/min, whereas in high concentrations (190 microgram/ml to 390 microgram/ml), their shape is rounded, and they translocate at an average rate of only 0-5 micrometer/min. Neural crest cells migrate from neural tubes in these preparations principally in loose clusters, with a few single cells in the lead. The cells in these groups display leading-to-trailing edge adhesions and form tongues or streams of cells directed away from the neural tube. The paths of migration of both individual cells and groups of cells are aligned with the collagen fibrils of the HCL, which radiate from the neural tube. The classical visible characteristic of contact inhibition of movement, change in direction of cell movement after contact with other cells, was not observed; neither the rate of translocation nor the time spent migrating away from the tube is dependent on the number of contacts between cells. It is concluded that the directional movement of neural crest cells in HCL cultures does not depend on contact inhibition of movement.

Formation of protrusions of the cell surface during tissue cell movement.

The various forms of protrusions of the surface of tissue cells are described in relation to their role in cell locomotion. It is proposed that these cells have enough cell surface (plasma membrane and linked microfilamentous cortical cytoplasm) at any given moment during interphase of the cell cycle to satisfy their need for the local increases in cell surface area that accompany protrusive activity. Thus, a local increase in protrusive activity in one region of the cell surface would be accompanied by a corresponding decrease elsewhere; that is, formation of new protrusions would require retraction of other protrusions already present and the area retracted would be equivalent to the area protruded. Evidence marshaled in support of this hypothesis includes disappearance of microvilli and other microprotrusions during cell spreading, increase in protrusive activity of uncontacted regions of the cell surface during contact inhibition of cell movement, antagonism between blebbing and spreading, accelerated protrusive activity at the leading edge upon abrupt retraction of the trailing edge, surface flow during bleb formation, and antagonism between various protrusive activities associated with cell movement and cytokinesis. Finally, the relevance of these findings to two important developmental problems is explored: the commencement of gastrulation and directional cell movements during morphogenesis.

Intercellular junctions, intramembranous particles and cytoskeletal elements of deep cells of the Fundulus gastrula.

The fine structure of motile deep cells of the gastrula stage of Fundulus heteroclitus was studied with transmission electron microscopy, using both thin sectioning and freeze-cleave techniques. Gastrula deep cells form extensive non-junctiona appositions with each other, in which the apposed plasma membranes are parallel and separated by a distance of 26-28 nm. They also form gap junctions. Tight junctions, desmosomes, and extensive interdigitations of apposed plasma membranes were not observed. The plasma membranes of deep cells contain numerous unclustered intramembranous particles. Cytoplasmic microtubules were found, but they appear to be small in number, sparsely distributed, and mainly randomly oriented. Microfilaments are also present and are localized largely in the cortical cytoplasm and in thin cell extensions. The significance of these findings for the contact and locomotory behavior of deep cells is discussed.

Some clues as to the formation of protrusions by Fundulus deep cells.

One of the ways in which Fundulus deep cells move in vivo is by putting out long, fingerlike protrusions. This involves a change in the shape of the cell as a whole, with cytoplasmic flow, and is not just a local phenomenon. Moreover, particles on the cell surface move toward a protrusion as it is forming, suggesting surface flow. The role of surface flow is discussed both on a grown level and in respect to molecular fluidity. Long, stable protrusions can be pulled from cells by the application of negative pressure at a constant rate and these behave in a similar way to those formed during cell locomotion. Such long protrusions must be structured. The importance of contractile properties of the cytoplasm in the formation of protrusions was studied by treating cells with media that modify cellular contractility.