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.     

Cell adhesion and the actin cytoskeleton of the enveloping layer in the zebrafish embryo during epiboly.

As the zebrafish embryo undergoes gastrulation and epiboly, the cells of the enveloping layer (EVL) expand, covering the entire yolk cell. During the epiboly process, the EVL cells move as a coherent layer, remaining tightly attached to each other and to the underlying yolk syncytial layer (YSL). In view of the central role of the actin cytoskeleton, in both cell motility and cell-cell adhesion, we have labeled these cells in situ with fluorescent phalloidin and anti-actin antibodies. We show that, throughout their migration, the EVL cells retain a conspicuous cortical actin cytoskeletal belt coinciding with cell surface cadherins. At the margins approaching the YSL, the EVL cells extend, from their apicolateral domains, actin-rich filopodial protrusions devoid of detectable cadherin. We have studied the role of the actin cytoskeleton in the maintenance of EVL cohesion during epiboly. Cytochalasin treatment of embryos induces EVL dissociation accompanied by general detachment of the rest of the embryonic cells. In the dissociating EVL cells, the cortical actin belt undergoes fragmentation with the formation of actin aggregates; cadherins, on the other hand, remain evenly distributed at the junctional cell surface. Removal of Ca2+ by ethyleneglycolbis (amino-ethyl-ether)-tetraacetic acid (EGTA) treatment also induces cell dissociation without visible disruption of the cortical actin belt. The protein kinase inhibitor (1-isoquinolinylsulfonyl)-2-methyl-piperazine dihydrochloride (H-7), which blocks acto-myosin contractility and disrupts actin cables in cultured cells, also potentiates cytochalasin-induced dissociation and promotes the projection of numerous actin-rich lamellipodial extensions. The fact that EVL cells produce microspike-like structures towards the YSL and are capable of lamellipodial activity lend further support to the suggestion (R.W. Keller and J.P. Trinkaus. 1987. Dev. Biol. 120: 12-24) that the EVL cells are not passively mobilized on the expanding YSL but actively participate in epiboly.     

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.     

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.     

Programmed Endocytosis During Epiboly of Fundulus heteroclitus

A major question in the analysis of teleost epiboly is the fateof the yolk cytoplasmic layer. It diminishes during epibolyand eventually disappears at the completion of epiboly. Thispaper is concerned with the fate of the surface of the yolkcytoplasmic layer during epiboly. When gastrulae during epibolyare bathed in lucifer yellow (CH) and then observed with fluorescentmicroscopy or bathed in ferritin and then fixed and observedwith TEM, a thin circumferential ring of endocytic vesiclesis observed, confined to the external yolk syncytial layer justperipheral to the advancing margin of the blastoderm. Even thoughthe entire egg is immersed in the marker, endocytosis is confinedto this limited region. More precisely, this endocytosis occursonly within the region of the external yolk syncytial layer,where the surface is most folded. The endocytic vesicles thusformed move downward and settle on the surface of the membraneseparating the yolk from the cytoplasm in the yolk syncytiallayer. They do not join the surface of the internal yolk syncytiallayer; hence they do not contribute to its expansion. Priorto the onset of epiboly there is no such endocytosis at thesurface of the egg. Since this endocytosis occurs only duringepiboly and only at the surface of the external yolk syncytiallayer just peripheral to the advancing margin of the blastoderm,and in the absence of large molecules in the medium, we concludethat it is programmed. We, therefore, present this as a caseof programmed internalization of cell surface serving as themorphogenetic mechanism responsible for the disappearance ofthe surface of the yolk cytoplasmic layer during gastrulationof the teleost Fundulus heteroclitus     

Contractility,differential tension and membrane removal lead zebrafish epiboly biomechanics

Precise tissue remodeling during development is essential for shaping embryos and optimal organ function. Epiboly is an early gastrulation event by which the blastoderm expands around the yolk to engulf it. Three different layers are involved in this process, an epithelial layer (the enveloping layer, EVL), the embryo proper, constituted by the deep cells (DCs), and the yolk cell. Although teleost epiboly has been studied for many years, a clear understanding of its mechanics was still missing. Here we present new information on the cellular, molecular and mechanical elements involved in epiboly that, together with some other recent data and upon comparison with previous biomechanical models, lets conclude that the expansion of the epithelia is passive and driven by active cortical contraction and membrane removal in the adjacent layer, the External Yolk Syncytial Layer (E-YSL). The isotropic actomyosin contraction of the E-YSL cortex generates an anisotropic stress pattern and a directional net movement consequence of the differences in the deformation response of the 2 opposites adjacent domains (EVL and the Yolk Cytoplasmic Layer - YCL). Contractility is accompanied by the local formation of membrane folds and its removal by Rab5ab dependent macropinocytosis. The increase in area of the epithelia during the expansion is achieved by cell-shape changes (flattening) responding to spherical geometrical cues. The counterbalance between the geometry of the embryo and forces dissipation among different elements is therefore essential for epiboly global coordination.     

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.     

Cell lineage of zebrafish blastomeres. II. Formation of the yolk syncytial layer

Dye coupling and cell lineages of blastomeres that participate in the formation of the yolk syncytial layer (YSL) in the zebrafish Brachydanio rerio have been examined. The YSL is a multinucleate layer of nonyolky cytoplasm underlying the cellular blastoderm at one pole of the giant yolk cell. It forms at the time of the 10th (sometimes 9th) cleavage by a collapse of a set of blastomeres, termed marginal blastomeres, into the yolk cell. Marginal blastomeres possess cytoplasmic bridges to the yolk cell before the YSL forms, and injections of fluorescein-dextran into the cells revealed that bridges between the yolk cell and blastoderm do not persist after this time. Injections of Lucifer yellow revealed that shortly after the YSL forms the yolk cell and blastoderm are dye coupled, presumably by gap junctions, and that this coupling disappears gradually during early gastrulation. Lineage analyses revealed that not all of the progeny of early marginal blastomeres participate in YSL formation. Although some descendants of marginal blastomeres remained on the margin during successive cleavages, neither "compartment" nor "strict lineage" models are sufficient to explain the origin of the YSL. It is proposed that the position of a cell on the blastoderm margin, and not the cell's lineage, determines YSL cell fate.     

SEM observation of the External Yolk Syncytial Layer in Blastopore Closure of the Medaka, Oryzias latipes

The embryonic surface of the teleost, Oryzias latipes , was observed by scanning electron microscopy (SEM) to examine the last phase of epiboly or blastopore closure. The surface of the external yolk syncytial layer (E–YSL), a surface cytoplasmic layer encompassing the yolk sphere situated beyond the blastoderm, was highly undulated with surface folds of random orientation throughout most of epiboly (st. 14–20). Scattered microvilli were observed on the surface of the margin of the yolk plug in st. 18–20. The microvilli, 1 to 6 μm in length, were projected in a bunch at the end of blastopore closure (st. 20–21). The appearance of these microvilli in the last phase of epiboly is discussed with respect to the mechanism of epiboly.     

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.     

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.     

A Novel Role for MAPKAPK2 in Morphogenesis during Zebrafish Development

One of the earliest morphogenetic processes in the development of many animals is epiboly. In the zebrafish, epiboly ensues when the animally localized blastoderm cells spread, thin over, and enclose the vegetally localized yolk. Only a few factors are known to function in this fundamental process. We identified a maternal-effect mutant, betty boop (bbp), which displays a novel defect in epiboly, wherein the blastoderm margin constricts dramatically, precisely when half of the yolk cell is covered by the blastoderm, causing the yolk cell to burst. Whole-blastoderm transplants and mRNA microinjection rescue demonstrate that Bbp functions in the yolk cell to regulate epiboly. We positionally cloned the maternal-effect bbp mutant gene and identified it as the zebrafish homolog of the serine-threonine kinase Mitogen Activated Protein Kinase Activated Protein Kinase 2, or MAPKAPK2, which was not previously known to function in embryonic development. We show that the regulation of MAPKAPK2 is conserved and p38 MAP kinase functions upstream of MAPKAPK2 in regulating epiboly in the zebrafish embryo. Dramatic alterations in calcium dynamics, together with the massive marginal constrictive force observed in bbp mutants, indicate precocious constriction of an F-actin network within the yolk cell, which first forms at 50% epiboly and regulates epiboly progression. We show that MAPKAPK2 activity and its regulator p38 MAPK function in the yolk cell to regulate the process of epiboly, identifying a new pathway regulating this cell movement process. We postulate that a p38 MAPKAPK2 kinase cascade modulates the activity of F-actin at the yolk cell margin circumference allowing the gradual closure of the blastopore as epiboly progresses.     

Polyploidization of nuclei in the yolk syncytial layer of the embryo of the medaka, Oryzias latipes, after the halt of mitosis

It has been reported that nuclei repeat parasynchronous mitosis four or five times in the yolk syncytial layer (YSL) of the embryo of the medaka, Oryzias latipes , during the blastula stage and that no mitosis occurs in the YSL after the gastrula stage. The present investigation demonstrated the size of nuclei and the number of nucleoli and their staining properties with DNA binding dye. The results indicate that the YSL nuclei actively transcribe RNA and that their DNA content is greater than that of somatic nuclei. The onset and subsequent time course of polyploidization were examined in embryos stained with 4',6-diamidino-2-phenylindole (DAPI) by epifluorescence microspectrophotometry from the cessation of mitosis through hatching. Embryos included YSL nuclei whose DNA content spanned from diploid (2C), tetraploid (4C) to octaploid (8C) at the end of the late blastula stage. The last two populations are produced probably by their early cessation of mitosis and the subsequent duplication of DNA without mitosis or by endoreduplication. The frequency distribution of the DNA content examined during epiboly of the blastoderm suggests that each population is duplicated again until the beginning of the gastrula stage and then once more until the end of epiboly. Eventually these nuclei include polyploid DNA between 8C and 64C or more during later embryonic development.     

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.     

ApoA-II directs morphogenetic movements of zebrafish embryo by preventing chromosome fusion during nuclear division in yolk syncytial layer

The high density lipoprotein (HDL) represents a class of lipid- and protein-containing particles and consists of two major apolipoproteins apoA-I and apoA-II. ApoA-II has been shown to be involved in the pathogenesis of insulin resistance, adiposity, diabetes, and metabolic syndrome. In embryo, apoa2 mRNAs are abundant in the liver, brain, lung, placenta, and in fish yolk syncytial layer (YSL), suggesting that apoa2 may perform a function during embryonic development. Here we find out that apoa2 modulates zebrafish embryonic development by regulating the organization of YSL. Disruption of apoa2 function in zebrafish caused chromosome fusing, which strongly blocked YSL nuclear division, inducing disorders in YSL organization and finally disturbing the embryonic epiboly. Purified native human apoA-II was able specifically to rescue the defects and induced nuclear division in zebrafish embryos and in human HeLa cells. The C terminus of apoA-II was required for the proper chromosome separation during nuclear division of YSL in zebrafish embryos and in human HeLa cells. Our data indicate that organization of YSL is required for blastoderm patterning and morphogenesis and suggest that apolipoprotein apoA-II is a novel factor of nuclear division in YSL involved in the regulation of early zebrafish embryonic morphogenesis and in mammalian cells for proliferation.     

Mutations in half baked/E-cadherin block cell behaviors that are necessary for teleost epiboly

Epiboly, the spreading of the blastoderm over the large yolk cell, is the first morphogenetic movement of the teleost embryo. Examining this movement as a paradigm of vertebrate morphogenesis, we have focused on the epiboly arrest mutant half baked (hab), which segregates as a recessive lethal, including alleles expressing zygotic-maternal dominant (ZMD) effects. Here we show that hab is a mutation in the zebrafish homolog of the adhesion protein E-cadherin. Whereas exclusively recessive alleles of hab produce truncated proteins, dominant alleles all contain transversions in highly conserved amino acids of the extracellular domains, suggesting these alleles produce dominant-negative effects. Antisense oligonucleotides that create specific splicing defects in the hab mRNA phenocopy the recessive phenotypes and, surprisingly, some of the ZMD phenotypes as well. In situ analyses show that during late epiboly hab is expressed in a radial gradient in the non axial epiblast, from high concentrations in the exterior layer of the epiblast to low concentrations in the interior layer of the epiblast. During epiboly, using an asymmetric variant of radial intercalation, epiblast cells from the interior layer sequentially move into the exterior layer and become restricted to that layer; there they participate in subtle cell shape changes that further expand the blastoderm. In hab mutants, when cells intercalate into the exterior layer, they tend to neither change cell shape nor become restricted, and many of these cells 'de-intercalate' and move back into the interior layer. Cell transplantation showed all these defects to be cell-autonomous. Hence, as for the expansion of the mammalian trophoblast at a similar developmental stage, hab/E-cadherin is necessary for the cell rearrangements that spread the teleost blastoderm over the yolk.