Essential role of the yolk syncytial layer for the development of isolated blastoderms from medaka embryos

To investigate a possible role of the yolk syncytial layer (YSL) in the development of the medaka embryo, blastoderms were isolated at different stages of embryogenesis either with or without the layer and were incubated in a culture medium. The blastoderms from cleavage stage embryos (stage 8–9), in which the YSL had not yet formed, developed into an irregular mass of cells. But some of the blastoderms isolated with the YSL from the blastula embryos (stage 10) developed into embryo-like structures with apparent body axes and contained differentiated organs, such as the eye, ear, contractile heart, yolk sac-like sphere and posterior body trunk with notochord. The proportion of such explants increased as the developmental stage proceeded. However, the proportion was much smaller when blastoderms were isolated at the blastula stage without the YSL. These results suggest that the YSL is essential for the development of embryonic structures. At stage 12 (early gastrula), the frequency of formation of such structures was the same among blastoderms with or without the YSL, so that these embryos are apparently committed for pattern formation.     

Overcoming a permeability barrier by microinjecting cryoprotectants into zebrafish embryos (Brachydanio rerio)

The goal of this research was to examine the developmental effects on zebrafish embryos (Brachydanio rerio) when cryoprotectants were directly microinjected into the yolk. Our objectives were to: (i) determine the final concentration of propylene glycol (PG) and dimethyl sulfoxide (Me(2)SO) that the embryos could tolerate without causing teratogenic effects; (ii) determine if the toxicity of Me(2)SO could be reduced by the simultaneous presence of various proportions of amides; and (iii) examine whether this intracellular cryoprotectant incorporation could reduce the cryodamage to the yolk syncytial layer (YSL) after vitrification trials. The rationale for conducting these microinjection experiments was to overcome the permeability barrier of the YSL. Intracellular PG produced better survival than Me(2)SO (P < 0.05). Embryos tolerated both 10- and 30-nl microinjections of PG, yielding final concentrations of 2.3 and 5.0 M within the yolk, resulting in 70 +/- 3 and 35 +/- 4% survival at day 5, respectively. In similar experiments with Me(2)SO, survival was lower than PG at 60 +/- 4 and 14 +/- 4% at 2.4 and 5.2 M. Unlike other cellular systems, the presence of amides, specifically acetamide or formamide, did not reduce the toxicity of Me(2)SO in zebrafish embryos (P > 0.05). During vitrification trials, we estimated a 25% dehydration of the yolk, yielding an effective PG concentration of 5.9 M. However, the incorporation of this vitrifiable concentration of PG was not sufficient to improve the postthaw morphology of the YSL (P > 0.05). Clearly, other factors need to be examined in establishing a successful vitrification protocol for zebrafish embryos.     

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.     

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.     

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.     

The antifreeze protein type I (AFP I) increases seabream (Sparus aurata) embryos tolerance to low temperatures

To date, all attempts at fish embryo cryopreservation have failed. One of the main reasons for this to occur is the high chilling sensitivity reported in fish embryos thus emphasizing the need for further testing of different methods and alternative cryoprotective agents (CPAs) in order to improve our chances to succeed in this purpose. In this work we have used the antifreeze protein type I (AFP I) as a natural CPA. This protein is naturally expressed in sub-arctic fish species, and inhibits the growth of ice crystals as well as recrystallization during thawing. Embryos from Sparus aurata were microinjected with AFP I at different developmental stages, 2 cells and blastula, into the blastomere-yolk interface and into the yolk sac, respectively. Control, punctured and microinjected embryos were subjected to chilling at two different temperatures, 0 degrees C (1h) and -10 degrees C (15min) when embryos reached 5-somite stage. Embryos were subjected to -10 degrees C chilling in a 3M DMSO extender to avoid ice crystal formation in the external solution. Survival after chilling was established as the percentage of embryos that hatch. To study the AFP I distribution in the microinjected embryos, a confocal microscopy study was done. Results demonstrate that AFP I can significantly improve chilling resistance at 0 degrees C, particularly in 2-cell microinjected embryos, displaying nearly 100% hatching rates. This fact is in agreement with the confocal microscopy observations which confirmed the presence of the AFP protein in embryonic cells. These results support the hypothesis that AFP protect cellular structures by stabilizing cellular membranes.     

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.     

Early differentiation in zebra fish blastula: Role of yolk syncytial layer

Summary The blastomeres of the zebra fish embryo can be classified into two types-cells stained densely (D) or lightly (L) with a mixture of toluidine and methylene (T-M) blue. The dense staining of D cells is largely due to the high density of mitochondria, rough endoplasmic reticulum and polyribosomes. The presence of partially dense stained cells during early blastula stage shows that L cells are transformed into D cells. That the yolk syncytial layer (YSL) plays some role in this transformation is suggested by the proximity of these cells to the YSL and by their distinct spatial orientation with densely stained cytoplasmic regions always facing towards the interior of the embryo.     

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.     

Origin of primordial germ cells, as characterized by the presence of nuage, in embryos of the teleost fish Barbus conchonius.

The presence, location and morphology of cells containing nuage, an ultrastructural characteristic of primordial germ cells (PGCs), is described from the moment of first morphological recognition of PGC (around 100% epiboly) in embryos of the teleost fish Barbus conchonius. Thus characterized cells were studied in relation to their cellular contacts with somatic germ layer cells, possibly involved in the determination of PGCs. The results show that from the very moment that cells, likely to be PGCs, can be light microscopically identified with morphological and positional criteria (from 10 h post fertilization (p.f.) onwards), they contain nuage near the nuclear envelope, which is a strong indication of their PGC-identity. During the studied period (9-12 h and 24 h p.f.) nuage-containing cells seem to translocate from the mesoderm towards the yolk syncytial layer (YSL). These PGCs usually appear not to be directly connected with the YSL but to remain separated from the YSL by one or more endodermal extensions, at least up to 12 h p.f. Also at 24 h p.f. somatic cells separate the PGCs from the YSL.     

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.     

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.     

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.     

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.     

Quantification of the maternal-embryonal nutritional relationship of elasmobranchs: case study of wobbegong sharks (genus Orectolobus)

The present study used wobbegong sharks (genus Orectolobus) to assess the threshold value proposed by previous research to categorize strict lecithotrophic from incipient histotrophic species. Totals of 236 and 135 ornate wobbegong Orectolobus ornatus and spotted wobbegong Orectolobus maculatus, respectively, were collected from the New South Wales commercial fishery between June 2003 and May 2006. Eight pregnant gulf wobbegong Orectolobus halei were also recorded outside the sampling period for the first time. The three species were reproductively synchronous with a gestation of c. 10-11 months. Embryos started to be macroscopically visible during January and external yolk sacs were fully absorbed by June to July when embryos were c. 200 mm total length (L(T) ). Internal yolk sacs were first observed during April to May when embryos were c. 160 mm L(T) , reached a peak during June and persisted in embryos immediately prior to parturition. The total wet mass from uterine egg to full-term embryos increased by 44-89% and 45-62%, whereas the total organic mass decreased by 32-33% and 26%, for O. ornatus and O. maculatus, respectively, suggesting that these species are strict lecithotrophic yolk-sac viviparous sharks with no maternal nutrient input. A review of the literature identified various issues and suggested that the previously proposed organic mass loss threshold value separating strict lecithotrophic species from incipient histotrophic species might not be appropriate. Instead, it is recommended that a combination of methods (e.g. estimation of organic mass gain or loss between ovarian egg and developed embryo, histology and electron microscopy of the uterus, radio-tracer assay and uterine fluid analysis throughout gestation) is used to discern between strict lecithotrophic and incipient histotrophic species.