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.     

The Egg Cortex Problem as Seen Through the Squid Eye

In many spiralian embryos it has been possible to demonstratethat embryonic development is partially controlled by cytoplasmicfactors located at or in the surface of the fertilized egg andcleaving embryo. In the embryo of the squid Loligo pealei, apattern of developmental information can be demonstrated toexist at the surface, or the egg cortex, of the fertilized butuncleaved embryo. The informational pattern apparently is releasedor activated during the time of the cytoplasmic streaming whichforms the blastodisc. Eventually this developmental informationalpattern is imposed upon the blastoderm cells that come to coverorgan-specific regions of the yolk syncytium which was derivedfrom the egg cortex. Ultrastructural studies demonstrate manyintercellular connections between the yolk syncytium and theblastoderm and between the cells of the blastoderm itself. Duringoogenesis there are regional differences in the follicular syncytiumwhich suggests that the pattern of developmental informationmay arise in the ovary and be retained in a latent state untiltriggered by fertilization.     

Magnetic resonance imaging study of the structure of the yolk in the developing avian egg

Magnetic resonance imaging (MRI) techniques were used to study the morphology of the latebra and concentric rings seen in the yolk of White Leghorn eggs during development of the avian embryo. Previous studies of the macroscopic structure of avian yolk have revealed the latebra, a vase-shaped structure beneath the blastoderm composed of white yolk. The bulbous portion in the center of the yolk is termed the body of the latebra. The thinner portion extending toward the blastoderm is referred to as the neck of the latebra. As the neck of the latebra approaches the blastoderm, it flares out to become the nucleus of Pander. The remainder of the yolk often features alternating concentric layers of white and yellow yolk. These layers, which appear as rings in sections, are thought to represent the daily accumulation of yolk during oogenesis. In this study eggs were imaged with a single slice spin echo sequence using MRI parameters that maximized the visualization of the latebra and concentric rings in the egg yolk. Some experiments were conducted for 2 to 3 day periods with eggs kept in the bore of the magnet using a small incubator that was constructed using a temperature-controlled water pump. The concentric rings of the yolk and the body of the latebra flatten and become more elliptical during development. The neck of the latebra becomes shorter and disappears around the 7th day of incubation. The body of the latebra starts to become incorporated into the embryo at about the 7th day of incubation and usually disappears by the 13th day. The concentric rings are no longer visible as distinct entities at this time. Histochemical procedures carried out as a result of MRI findings indicate that the latebra is an iron-rich structure.     

A Force Balance Can Explain Local and Global Cell Movements during Early Zebrafish Development

Embryonic morphogenesis takes place via a series of dramatic collective cell movements. The mechanisms that coordinate these intricate structural transformations across an entire organism are not well understood. In this study, we used gentle mechanical deformation of developing zebrafish embryos to probe the role of physical forces in generating long-range intercellular coordination during epiboly, the process in which the blastoderm spreads over the yolk cell. Geometric distortion of the embryo resulted in nonuniform blastoderm migration and realignment of the anterior-posterior (AP) axis, as defined by the locations at which the head and tail form, toward the new long axis of the embryo and away from the initial animal-vegetal axis defined by the starting location of the blastoderm. We found that local alterations in the rate of blastoderm migration correlated with the local geometry of the embryo. Chemical disruption of the contractile ring of actin and myosin immediately vegetal to the blastoderm margin via Ca²⁺ reduction or treatment with blebbistatin restored uniform migration and eliminated AP axis reorientation in mechanically deformed embryos; it also resulted in cellular disorganization at the blastoderm margin. Our results support a model in which tension generated by the contractile actomyosin ring coordinates epiboly on both the organismal and cellular scales. Our observations likewise suggest that the AP axis is distinct from the initial animal-vegetal axis in zebrafish.     

The formation of primordial germ cells from germline cells in spherical embryos derived from the blastodisc of 2-cell embryos in goldfish, Carassius auratus

The property of primordial germ cells (PGCs) in fragmented goldfish embryos was investigated. When 1- and 2- cell embryos were cut at several perpendicular levels at the animal-vegetal axis, cells expressing vas mRNA were observed in the resultant embryos derived from all kinds of animal fragments. Blastodisc fragments from the 1- to 2-cell stage developed to spherical embryos containing yolk body with a yolk syncytial layer (YSL). Germ ring and no tail expression were not observed in the spherical embryo. When the spherical embryo labeled with tracer dye or GFP-nos1 3'UTR mRNA was transplanted onto the animal part of the blastoderm in a host embryo at the blastula stage, PGCs of spherical embryo origin were detected around the gonadal ridges in the resultant embryos which developed normally. These results suggest that small animal fragments should contain factors sufficient for PGC differentiation and that PGCs differentiate without mesoderm induction, since mesoderm is not induced in a spherical embryo.     

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.     

Autoradiographic study of protein and RNA formation during early development of Drosophila eggs

Permeabilized eggs of Drosophila melanogaster were incubated in tritiated uridine, valine, and phenylalanine. The uptake and incorporation into TCA-insoluble material were measured by scintillation counting. There was very little incorporation of uridine before the blastoderm stage. At the blastoderm stage, the egg took up 2.4 pmoles/hr of uridine and incorporated 0.13 pmoles into RNA (assuming no dilution of specific activity of the precursor). The uptake of amino acids varied with the age of the embryo; virgin eggs synthesized about as much protein as fertilized eggs. Autoradiography of eggs incubated in uridine showed a lack of RNA synthesis in nuclei until the start of the blastoderm formation. The small amount of uridine incorporation before this stage was due to mitochondria. Incorporation of amino acids was uniform in the cytoplasm until the blastoderm; there was no incorporation by yolk granules. Regional difference in labeling appeared during gastrulation. The pole cells did not form RNA during the blastoderm stage, formation started during gastrulation. Protein labeling of the pole cells, on the contrary, was very strong in the blastoderm and early gastrula. These results indicate that the expression of zygotic genome before the blastoderm stage is unlikely.     

Cell movements during epiboly and gastrulation in zebrafish

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

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.     

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.     

Embryonic development of the hemipteran insect Rhodnius prolixus

The embryonic development of the hemipteran insect Rhodnius prolixus was studied by use of contemporary light and electron microscopy. Embryos were staged according to days postoviposition. Eggs laid on day one complete blastoderm formation and anatrepsis, the first phase of blastokinesis, by day 5. The embryo develops in a cephalocaudal orientation which is 180° to the anteroposterior axis of the egg. Subsequent development, prior to the second phase of blastokinesis (katatrepsis), leads to segmentation of the germ band, evagination of appendages, and histogenesis of germ layers. Concomitantly with these events, the amnion undergoes dramatic change. By day 7 the embryo begins a 180° revolution while migrating to the ventral surface of the yolk. This restores its polarity with respect to that of the egg and facilitates hatching. The serosa contracts, pulling the amnion and embryo anteriorly. Eventually the serosa is internalized at a point dorsal to the head and the lateral walls of the embryo grow up and surround the yolk. Development continues until day 15 when the embryo hatches as a first instar larva.     

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.     

New Approaches for Studying the Permeability of Fish Embryos: Toward Successful Cryopreservation

This paper describes some new approaches for understanding the permeability of teleost embryos. The dechorionated zebrafish (Brachydanio rerio) was used as a model for basic studies of water and cryoprotectant permeability. These embryos are composed of two compartments, a large yolk (surrounded by the yolk syncytial layer) and differentiating blastoderm cells. Cellular water was distributed unequally in each compartment. Measurements indicated that the total water in the embryo was 74%, while the total water in the yolk was 42%, and total water in the blastoderm was 82%. The internal isosmotic value for the zebrafish embryo is unknown. However, for one-compartment modeling studies of membrane permeability, the mean Lp (±SEM) values were 0.022 ± 0.002 to 0.049 ± 0.008 μm × min⁻¹atm⁻¹at 40 mOsm (assuming this was one possible internal isosmotic value for the entire embryo) and 0.040 ± 0.004 to 0.1 ± 0.017 μm × min⁻¹atm⁻¹at 300 mOsm (assuming this was another possible internal isosmotic value for the entire embryo). When three- and six-somite embryos were placed in 1.5 and 2.0Mcryoprotectants (dimethyl sulfoxide and propylene glycol), osmometric measurements of volume changes indicated no cryoprotectant permeation. However, similar measurements with methanol revealed a small volume decrease (ca. 8%) and recovery (ca. 5%) for six-somite embryos in a 2.0Msolution. Magnetic resonance (MR) images of the spatial distribution of three cryoprotectants (dimethyl sulfoxide, propylene glycol, and methanol) demonstrated that only methanol permeated the entire embryo within 15 min. The other cryoprotectants exhibited little or no permeation into the yolk over 2.5 h. The results from MR spectroscopy and cryoprotectant microinjections into the yolk suggested that the yolk syncytial layer plays the critical limiting role for cryoprotectant permeation throughout the embryo.     

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.     

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.     

Developmental uncoupling between blastoderm and yolk cell in the embryo of the teleost Fundulus

During cleavage and blastula stages of embryos of the teleost Fundulus heteroclitus all of the cells are both electotonically coupled and dye coupled to one another, as determined by microelectrode impalements and spread of Lucifer Yellow. At about the time that gastrulation begins we observed a specific loss of junctional coupling between the yolk cell and cells of the blastoderm. Passage of Lucifer Yellow between the yolk cell and blastoderm was reduced at stage 12 (late blastula), and not detected at stage 13 and thereafter, although cells of the blastoderm remain dye coupled to one another through gastrula stages. Also, junctional electrical coupling between the yolk cell and blastoderm became substantially reduced at stage 13 and thereafter. The loss of coupling at this specific cell apposition and time and the large size of the yolk cell may prove useful in analyzing the underlying cellular mechanisms.     

Developmental expression of aquaporin-3 in zebrafish embryos (Danio rerio)

Fish embryos have never been successfully cryopreserved because of the low permeability of cryoprotectants into the yolk. Recently, we used aquaporin-3 fused with a green fluorescent protein (AQP3GFP) to modify the zebrafish embryo, and demonstrated that the pores functioned physiologically. This increased the water and cryoprotectant permeability of the membranes. We have continued our work on AQP3-modified embryos and here we report their developmental expression of AQP3, the success of various culture media on their survival and development, and their reproductive success. The AQP3GFP expression begins within 30 m after the mRNA AQP3GFP injection into the yolk of the 1- to 4-cell embryo. This expression is distributed in the membranes throughout the blastoderm and the yolk syncytial layer within 24 h. It diminishes after 96 h. We found no difference in the survival or normal development of embryos from AQP3GFP or wild-type adults. Additionally, zebrafish embryos did not require special culture medium to survive after AQP3GFP modification. In fact, they survived best in embryo medium (ca. 40 mOsm). Embryos reared entirely in embryo medium had a higher percent survival and a higher percent normal development than those exposed to a high osmolality sucrose culture medium (ca. 330 mOsm). The mechanism whereby these embryos can maintain their internal osmolality in a hypoosmotic solution with water channels in their membranes is unknown.     

The amnioserosa is an apomorphic character of cyclorrhaphan flies

In developing insect eggs the cells of the blastoderm adopt either an embryonic or an extraembryonic fate. The extraembryonic tissue consists of epithelia, termed amnion and serosa, which wrap the germ band embryo. The serosa develops directly from part of the blastoderm and surrounds the embryo as well as the yolk. The amnion develops from the margins of the germ band and in most insect species generates a transient ventral cavity for the developing embryo. The amniotic cavity and the serosa have been reduced in the course of dipteran evolution. The insect order of Diptera includes the paraphyletic Nematocera, including gnats and mosquitoes, and the more derived monophyletic Brachycera, the true flies. Nematocera develop within an amniotic cavity and the surrounding serosa, whereas cyclorrhaphan Brachycera do not. This observation implies that the amnion and serosa have been reduced before the radiation of the monophyletic cyclorrhaphan flies. Here I show that an amniotic cavity is formed during embryogenesis of the horsefly Haematopota pluvialis (Tabanidae) and the dancefly Empis livida (Empididae). The results suggest that extraembryonic tissue was reduced in the stem lineage of cyclorrhaphan flies, with consequences for the molecular basis of pattern formation along the anterior-posterior axis of the embryo. Received: 21 October 1999 / Accepted: 17 January 2000