Cell lineage of zebrafish blastomeres. I. Cleavage pattern and cytoplasmic bridges between cells

Patterns of cleavage and cytoplasmic connections between blastomeres in the embryo of the zebrafish, Brachydanio rerio have been described. The cell division pattern is often very regular; in many embryos a blastomere's lineage may be ascertained from its position in the cluster through the 64-cell stage. At the 5th cleavage, however, significant variability in pattern is observed, and alternative patterns of the 5th cleavage are described. The early cleavages are partial, incompletely separating blastomeres from the giant yolk cell. The tracer fluorescein-dextran (FD) was injected into blastomeres to learn the extent of the cytoplasmic bridging. It was observed that until the 10th cleavage, blastomeres located along the blastoderm margin maintain cytoplasmic bridges to the yolk cell. Beginning with the 5th cleavage, FD injected into a nonmarginal blastomere either remains confined to the injected cell, or if the injection was early in the cell cycle, the tracer spreads to the cell's sibling, through a bridge persisting from the previous cleavage. On the other hand, injected Lucifer yellow spreads, presumably via gap junctions, widely among blastomeres in a pattern unrelated to lineage.     

An ultrastructural analysis of the dispersed cell phase during development of the annual fish,Cynolebias

Cell ultrastructure was investigated during the dispersion phase of development in the annual fish Cynolebias. Three cellular populations encompass the yolk mass during dispersion, namely, 1) the yolk syncytial layer (YSL) or periblast, which lies directly over the surface of the yolk; 2) the deep blastomeres of the blastoderm, which engage in morphogenetic movements on the surface of the YSL and beneath the enveloping layer prior to forming the future embryo; and 3) the enveloping layer (EVL) of the blastoderm, which is a cohesive epithelium that forms the outermost cell layer of the blastoderm. Deep blastomeres contain numerous mitochondria and scattered glycogen rosettes that appear to function in the utilization of energy reserves. These cells also possess surface extensions such as filopodia and ruffles. Numerous microfilaments running parallel to the plasma membrane occur in cell extensions and in the cortical cytoplasm of neighboring blastomeres. In bleb-like extensions such as ruffles, microfilamentous stress fibers run parallel to the plane of the plasma membrane and prevent cellular organelles from entering the hyaline cap of the ruffle. Deep blastomeres also have basal projections that contain glycogen as well as pits in the basal membrane. Blastomeres move about using the YSL as a substrate. The YSL possesses specializations for nutrient uptake, storage, and transport such as numerous multivesicular bodies and large amounts of glycogen. Glycogen, in the rosette form, occurs in extraordinary amounts, virtually occluding the cytoplasm. Glycogen reserves are postulated to serve as an energy source during diapause. Glycogen is sometimes contained within villous projections that extend from the apical surface of the YSL. This configuration suggests the possibility of glycogen transport to the overlying deep blastomeres. Specializations of the EVL include apical tight junctions and basal lateral zonulae adherentes that interdigitate with those of adjacent EVL cells. The EVL serves as an impermeable membrane that protects the developing egg from the vicissitudes of its environment.     

Mesoderm differentiation in explants of carp embryos

An analysis of carp blastoderm development was carried out in culture after isolation from the yolk cell and its yolk syncytial layer (YSL). The blastoderms were separated from the YSL at four different stages of embryogenesis: the blastula, early epiboly, early gastrula and late gastrula stages. Absence of the YSL in explants was checked by scanning electron microscopy. From observations of living embryos and histological examination of tissues which were formed in explants from all stages studied it was observed that they contained notochordal, muscle and neural tissue as signs of dorsal types of differentiation. Only in explants from the early and late gastrula stages were histotypical tissues organized in an embryonic-like body pattern. The data indicate that mesoderm differentiation in fish embryos is independent from the YSL, contrary to normal pattern formation which needs the presence of the YSL before the onset of gastrulation.     

Dye-coupling and the formation and fate of the hypoblast in the teleost fish embryo, Barbus conchonius.

The present report describes Lucifer Yellow (LY) transfer between the syncytial layer of the yolk cell (YSL) and blastodermal cells during epiboly in the teleost fish Barbus conchonius. The fate of a group of labeled cells is described until germ layer formation. At the onset of epiboly, LY seems to be transferred from the YSL to all blastodermal cells. Between 10% and 40% epiboly, dye-coupling appears to be restricted to the marginal region. Within 60 min individually labeled cells are distributed among unlabeled cells within the blastoderm. Between 40% and 60% epiboly, we observed a ring-shaped group of labeled cells, which probably have involuted during early gastrulation. Consequently, this cell group may correlate with the leading edge of the hypoblast layer within the germ ring. At 60% epiboly and later, the blastodermal cells are dye-uncoupled from the YSL. A gradual translocation of the ring-shaped hypoblast towards a dorsally located bar-like structure is observed between 50% and 100% epiboly. At 100% epiboly, fluorescent cells were located in contact with the YSL within the embryo proper, with the brightest fluorescence in the future head region. The translocation is due to dorsalwards convergent cell movements during the gastrulation process. The appearance of the hypoblast as a dye-coupled cell layer may correlate with some restriction in cell fate since the hypoblast differs in fate from the epiblast.     

Zebrafish Dynamin is required for maintenance of enveloping layer integrity and the progression of epiboly

Epiboly, the first morphogenetic cell movement that occurs in the zebrafish embryo, is the process by which the blastoderm thins and spreads to engulf the yolk cell. This process requires the concerted actions of the deep cells, the enveloping layer (EVL) and the extra-embryonic yolk syncytial layer (YSL). The EVL is mechanically coupled to the YSL which acts as an epiboly motor, generating the force necessary to draw the blastoderm towards the vegetal pole though actomyosin flow and contraction of the actomyosin ring. However, it has been proposed that the endocytic removal of yolk cell membrane just ahead of the advancing blastoderm may also play a role. To assess the contribution of yolk cell endocytosis in driving epiboly movements, we used a combination of drug- and dominant-negative-based approaches to inhibit Dynamin, a large GTPase with a well-characterized role in vesicle scission. We show that Dynamin-dependent endocytosis in the yolk cell is dispensable for epiboly of the blastoderm. However, global inhibition of Dynamin function revealed that Dynamin plays a fundamental role within the blastoderm during epiboly, where it maintains epithelial integrity and the transmission of tension across the EVL. The epithelial defects were associated with disrupted tight junctions and a striking reduction of cortically localized phosphorylated ezrin/radixin/moesin (P-ERM), key regulators of epithelial integrity in other systems. Furthermore, we show that Dynamin maintains EVL and promotes epiboly progression by antagonizing Rho A activity.     

Early embryonic development,including germ-disk stage,in the theridiid spider Achaearanea japonica (Bös. et Str.)

Early embryonic development, from the first cleavage to the germ-disk stage, in the theridiid spider Achaearanea japonica was examined by light and electron microscopy. The eggs are syncytial during the first four cleavages, and then invaginations of cell membranes fuse to generate the blastomeres at the sixteen-nucleus stage. The cleavage pattern is a modified type of total cleavage. It appears that radial bundles of microtubules that radiate from the perinuclear cytoplasm may participate in the migration of cleavage nuclei for the formation of the blastoderm. The large yolk granules are sequestered by cell membranes from the blastomeres or blastoderm cells into the interior of the embryo together with various organelles and glycogen granules. Most of the blastoderm cells converge in the upper hemisphere to form the germ disk, whereas a few cells remain in the lower hemisphere. The embryo at the germ-disk stage contains many spherical germ-disk cells. Almost no large yolk granules are found in these cells, but the flat remaining cells each contain several large yolk granules. These remaining cells may preserve a flat shape to cover the surface of the embryo that does not include the germ disk. © 1995 Wiley-Liss, Inc.     

Structural and ultrastructural characteristics of the yolk syncytial layer in Prochilodus lineatus (Valenciennes, 1836) (Teleostei; Prochilodontidae)

The yolk syncytial layer (YSL) has been regarded as one of the main obstacles for a successful cryopreservation of fish embryos. The purpose of this study was to identify and characterize the YSL in Prochilodus lineatus, a fish species found in southeastern Brazil and considered a very important fishery resource. Embryos were obtained through artificial breeding by hormonal induction. After fertilization, the eggs were incubated in vertical incubators with a controlled temperature (28 degrees C). Embryos were collected in several periods of development up to hatching and then fixed with 2% glutaraldehyde and 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.3). Morphological analyses were carried out under either light, transmission or scanning electron microscopy. The formation of the YSL in P. lineatus embryos starts at the end of the cleavage stage (morula), mainly at the margin of the blastoderm, and develops along the embryo finally covering the entire yolk mass (late gastrula) and producing a distinct intermediate zone between the yolk and the endodermal cells. The YSL was characterized by the presence of microvilli on the contact region with the yolk endoderm. A cytoplasmic mass, full of mitochondria, vacuoles, ribosomes, endomembrane nets and euchromatic nuclei, indicated a high metabolic activity. This layer is shown as an interface between the yolk and the embryo cells that, besides sustaining and separating the yolk, acts as a structure that makes it available for the embryo. The structural analyses identified no possible barriers to cryoprotectant penetration.     

Mechanism of Fundulus Epiboly--A Current View

This paper summarizes evidence for the following picture ofFundulus epiboly, with an eye toward laying groundwork for futureinvestigation. The major force in epiboly is the yolk syncytiallayer (YSL). Prior to epiboly, it spreads well beyond the borderof the blastoderm to form the wide external YSL (E-YSL). Thishas contractile properties, which, however, are restrained priorto epiboly by the attached enveloping layer (EVL) of the blastoderm.Epiboly begins when the E-YSL contracts and narrows, throwingits surface into folds and pulling the internal YSL (I-YSL)and the attached EVL vegetally. When the narrowing of the E-YSLhas ceased, it is postulated that its contractility continuesas a circumferential wave of vegetally directed contractionthat moves over the yolk toward the vegetal pole, dragging theI-YSL and the attached EVL (and blastoderm) with it. The mostobvious visible manifestation of this wave is a marked marginalconstriction, where the YSL joins the yolk cytoplasmic layer(YCL). As this contractile wave passes over the yolk, cytoplasmfrom the YCL mingles with that of the advancing E-YSL, and YCLsurface adds to the already highly convoluted surface of theE-YSL. This folded surface is the site of a thin, highly localizedband of rapid endocytosis that encircles the egg and passesover it with the E-YSL in a wave throughout epiboly. This internalization,which is receptor independent and therefore somehow programmed,accompanies the putative contractile wave, and accounts forthe disappearance of the surface of the YCL. Since the YCL surfacestands in the way of the advancing YSL, its internalizationis part of the mechanism of epiboly. As the I-YSL expands inresponse to this marginal pull, its abundant microvilli graduallydisappear, providing surface for its epiboly. The firmly attachedEVL likewise expands toward the vegetal pole in response tothe pull of the autonomously expanding YSL. As epiboly of theEVL progresses, it adjusts to the geometric problems posed bya sheet expanding over a sphere by active cell rearrangementwithin the cell monolayer. Thus, epiboly of the EVL has an activeas well as a passive component. Deep cells are not causallyinvolved in epiboly, but move about in coordinated ways in theconstantly increasing space between the I-YSL and the EVL providedby epiboly and form the germ ring and the embryonic shield andeventually the embryo proper. An attempt is made to pull allof this together, and more, in order to achieve as comprehensivean understanding of epiboly as present evidence will allow.     

Regulation of hhex expression in the yolk syncytial layer, the potential Nieuwkoop center homolog in zebrafish

The Nieuwkoop center is the earliest signaling center during dorsal-ventral pattern formation in amphibian embryos and has been implied to function in induction of the Spemann-Mangold organizer. In zebrafish, Nieuwkoop-center-like activity resides in the dorsal yolk syncytial layer (YSL) at the interface of the vegetal yolk cell and the blastoderm. hex homologs are expressed in the anterior endomesoderm in frogs (Xhex), the anterior visceral endoderm in mice, and the dorsal YSL in zebrafish (hhex). Here, we investigate the control of hhex expression in the YSL. We demonstrate that bozozok (boz) is absolutely required for early hhex expression, while overexpression of boz causes ectopic hhex expression. Activation of Wnt/beta-catenin signaling by LiCl induces hhex expression in wild-type YSL but not in boz mutant embryos, revealing that boz activity is required downstream of Wnt/beta-catenin signaling for hhex expression. Further, we show that the boz-mediated induction of hhex is independent of the Boz-mediated repression of bmp2b. Our data reveal that repressive effects of both Vega1 and Vega2 may be responsible for the exclusion of hhex expression from the ventral and lateral parts of the YSL. In summary, zebrafish hhex appears to be activated by Wnt/beta-catenin in the dorsal YSL, where Boz acts in a permissive way to limit repression of hhex by Vega1 and Vega2.     

Embryonic stages from cleavage to gastrula in the loach Misgurnus anguillicaudatus

Early developmental staging from the zygote stage to the gastrula is a basic step for studying embryonic development and biotechnology. We described the early embryonic development of the loach, Misgurnus anguillicaudatus, based on morphological features and gene expression. Synchronous cleavage was repeated for 9 cycles about every 27 min at 20 degrees C after the first cleavage. After the 10th synchronous cleavage, asynchronous cleavage was observed 5.5 h post-fertilization (hpf), indicating the mid-blastula transition. The yolk syncytial layer (YSL) was formed at this time. Expressions of goosecoid and no tail were detected by whole-mount in situ hybridization from 6 hpf. This time corresponded to the late-blastula period. Thereafter, epiboly started and a blastoderm covered over the yolk cell at 8 hpf. At 10 hpf, the germ ring and the embryonic shield were formed, indicating the stage of early gastrula. Afterward, the epiboly advanced at the rate of 10% of the yolk cell each hour. The blastoderm covered the yolk cell completely at 15 hpf. The embryonic development of the loach resembled that of the zebrafish in terms of morphological change and gene expression. Therefore, it is possible that knowledge of the developmental stages of the zebrafish might be applicable to the loach.     

Morphogenetic consequences of partial removal of blastomere cytoplasm during early embryonic development of the loach, Misgurnus fossilis L.

The development of loach embryos is successfully regulated (normalized) after partial removal of the cytoplasm from one blastomere at the two- or four-cell stage or complete removal of one or two blastomeres at the stage of 8?C16 cells. Using time-lapse video imaging and morphometric analysis, it has been shown that this regulation is a two-stage process. At the first stage, the ratio between the volumes of the blastodisk and yolk sac is rapidly (within one or two cell cycles) restored almost to the initial level; at the second stage, morphogenesis of the embryo is modified according to its new structural features acquired after the operation. After several rounds of cytokinesis, the cytoplasm remaining in the operated blastomere fuses with the marginal yolk syncytium (periblast), which at the blastula stage forms a distinct extension at the operation site. This extension marks the site of embryonic shield formation. The results of morphometric analysis show that restoration of the initial blastoderm volume in operated embryos leads to a reduction of active tension at the blastoderm-yolk boundary and an increase in the ratio of blastoderm surface to its volume at the moment of epiboly initiation. As a result, the convergence of blastoderm cells to the operation site and the embryonic shield formation begin at a lesser degree of epiboly, compared to the control.     

Variability of quantitative morphogenetic parameters during early morphogenesis of the loach, <Emphasis Type="Italic">Misgurnus fossilis</Emphasis> L.

Analysis of normal variation in quantitative morphological characters during the early embryonic development of the loach, based on observations on individual developmental trajectories of living embryos, shows that the dorsoventral differentiation of the blastoderm proceeds in two stages. Initially, at the onset of epiboly, the sagittal (short) and transverse (long) blastoderm meridians are marked off, and only then, upon germ ring (GR) formation, differentiation between the opposite poles of the sagittal meridian takes place. The embryonic shield (ES) usually appears in the segment of the blastoderm where the radius of its external curvature reaches a maximum and, therefore, the active surface tension at the blastoderm boundary with the YSL periblast) and yolk is the highest. In this case, the convergence of inner cells toward the future dorsal segment (leading to ES formation) is a mechanical consequence of surface tension anisotropy. The normal course of epiboly is associated with periodic changes in the curvature of the blastoderm external surface, with new structures (the dorsal segment, GR, and ES) are marked off only when the surface curvature becomes maximally uniform. Although the ES in most embryos appears within the initial dorsal segment, individual developmental trajectories have been traced where the GR starts to form at the dorsal pole of the blastoderm but the ES develops on its opposite site, at the point of GR closure. In both cases, GR formation is initiated at the point of convergence of centrifugal cell migration flows that arise in the marginal zone of the blastoderm upon GR initiation or closure.     

Origin and organization of the zebrafish fate map

We have analyzed lineages of cells labeled by intracellular injection of tracer dye during early zebrafish development to learn when cells become allocated to particular fates during development, and how the fate map is organized. The earliest lineage restriction was described previously, and segregates the yolk cell from the blastoderm in the midblastula. After one or two more cell divisions, the lineages of epithelial enveloping layer (EVL) cells become restricted to generate exclusively periderm. Following an additional division in the late blastula, deep layer (DEL) cells generate clones that are restricted to single deep embryonic tissues. The appearance of both the EVL and DEL restrictions could be causally linked to blastoderm morphogenesis during epiboly. A fate map emerges as the DEL cell lineages become restricted in the late blastula. It is similar in organization to that of an amphibian embryo. DEL cells located near the animal pole of the early gastrula give rise to ectodermal fates (including the definitive epidermis). Cells located near the blastoderm margin give rise to mesodermal and endodermal fates. Dorsal cells in the gastrula form dorsal and anterior structures in the embryo, and ventral cells in the gastrula form dorsal, ventral and posterior structures. The exact locations of progenitors of single cell types and of local regions of the embryo cannot be mapped at the stages we examined, because of variable cell rearrangements during gastrulation.     

Morphogenetic consequences of partial removal of blastomere cytoplasm during early embryonic development of the loach, Misgurnus fossilis L

The development of loach embryos is successfully regulated (normalized) after partial removal of the cytoplasm from one blastomere at the two- or four-cell stage or complete removal of one or two blastomeres at the stage of 8-16 cells. Using time-lapse video imaging and morphometric analysis, it has been shown that this regulation is a two-stage process. At the first stage, the ratio between the volumes of the blastodisk and yolk sac is rapidly (within one or two cell cycles) restored almost to the initial level; at the second stage, morphogenesis of the embryo is modified according to its new structural features acquired after the operation. After several rounds of cytokinesis, the cytoplasm remaining in the operated blastomere fuses with the marginal yolk syncytium (periblast),which at the blastula stage forms a distinct extension at the operation site. This extension marks the site of embryonic shield formation. The results of morphometric analysis show that restoration of the initial blastoderm volume in operated embryos leads to a reduction of active tension at the blastoderm--yolk boundary and an increase in the ratio of blastoderm surface to its volume at the moment of epiboly initiation. As a result, the convergence of blastoderm cells to the operation site and the embryonic shield formation begin at a lesser degree of epiboly, compared to the control.