Regularities of early embryogenesis in teleosts as revealed by studies of the temporal pattern of development

Summary The absolute durations in minutes of the periods from insemination till the onset of gastrulation, the end of epiboly and appearance of the 10th pair of somites, as well as the intervals between these stages, have been determined in 7 species of Teleostei. Their relative duration, in terms of mitotic cycle time units {ie313-1} has also been determined. The biological age (expressed in number of {ie313-2} units) at which the isolated blastoderm becomes capable of differentiating in vitro was also determined in 4 of the species.The relative duration of the periods studied in all the species was shown to be stable at optimal temperatures. The corresponding periods of development have similar duration in closely related teleost species (i.e. belonging to the same genus).The relative duration of the cleavage period varies among the species belonging to different families. These variations are correlated with differences in the amount of yolk in the egg and the degree of psychrophily of the species compared. By contrast, the relative duration of the period between the onset of gastrulation and the stage of 10 pairs of somites was found to be very similar in species which belong to different orders and differ with respect to the amount of yolk in the egg and the temperature ranges of development. The rate of movement of blastoderm cells during epiboly is higher, the larger the eggs in the species under study. The variations in the age at which the isolated blastoderm acquires the ability to differentiate in vitro are not always correlated with the amount of yolk in the egg.The hypothesis is proposed that an interaction between the yolk and active cytoplasm, which determines time of the onset of ribosomal RNA synthesis, is of importance in controlling the temporal relationships in early embryogenesis.     

Temporary patterns in the embryonic development of the beluga]

The data concerning the absolute and relative duration of different periods of the beluga embryogenesis are obtained and the relative durations of the same periods of embryogenesis in beluga, sevryuga (Acipenser stellatus) and sturgeon (Acipenser güldenst?dti) were compared. The relative durations of cleavage, gastrulation and early organogenesis in beluga are rather stable within the zone of optimal temperatures and similar to those in sturgeon and sevryuga. In beluga the relative duration of the period between the insemination and the appearance of the 1st cleavage furrow on the egg surface and the relative duration of the second half of embryogenesis (from the formation of heart rudiment till the hatching of single larvae) are somewhat less.     

Rho mediates cytokinesis and epiboly via ROCK in zebrafish

To study the regulation of embryonic development by Rho, we microinjected Clostridium botulinum C3-exoenzyme (C3) into zebrafish embryos. We found that C3 inhibited cytokinesis during early cleavages. C3 inhibition appeared to be specific on RhoA, since the constitutively active RhoA could partially rescued the C3-induced defects. Distributions of actin and the cleavage furrow associated beta-catenin were disrupted by C3. Belbbistatin, a myosin II inhibitor, also caused blastomeres disintegration. It suggested that Rho mediates cytokinesis via cleavage furrow protein assembly and actomyosin ring constriction. Furthermore, C3 blocked cellular movements during epiboly and gastrulation as evident by the impairment on no tail and goosecoid expression in blastoderm front runner cells and the dorsal lip of blastopore, respectively. Y-27632, an antagonist of Rho-associated kinase (ROK/ROCK), had the similar inhibitory effects on zebrafish development as the C3 treatments. Taken together, these results suggest that Rho mediates cleavage furrow protein assembly during cytokinesis and cellular migration during epiboly and gastrulation via a ROK/ROCK-dependent pathway.     

Role of Yes kinase during early zebrafish development

We have identified the Yes kinase in zebrafish eggs and investigated its role in development of the zebrafish embryo. In situ hybridization as well as immunofluorescence techniques demonstrated that Yes kinase is maternally expressed and is localized to the cortical region of the unfertilized egg. Fertilization resulted in concentration of Yes kinase to the blastodisc where it continued to be localized to the blastoderm cells through cleavage, gastrulation, and later development. Yes kinase activity was found to decrease abruptly at fertilization, then increase progressively during epiboly, and was maintained at high levels throughout gastrulation. The role of Yes kinase in development was tested by treating embryos with chemical protein tyrosine kinase (PTK) inhibitors such as 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d] pyrimidine (PP2) and by injection of antisense morpholinos. Both treatments resulted in the arrest of development at the beginning of the epiboly. Co-immunoprecipitation studies demonstrated that Yes kinase participates in a stable complex with focal adhesion kinase (FAK), which is phosphorylated in vitro. These results demonstrate that Yes kinase plays an important role in epiboly and indicate that Yes kinase participates in signaling by focal adhesion kinase during early development.     

Spatial-temporal dynamics of morphogenetic blastoderm potencies in early embryogenesis of the loach

The degree of differentiation of axial structures (notochord, neuroectoderm, and somites) in 24-hour explants (a total of 380) of the loach embryonic blastoderm was determined on histological sections according to a developed scale of estimates. Before the beginning of epiboly, axial structures were formed only from fragments of the dorsal sector of the blastoderm marginal zone. Its other sectors acquired the capacity of forming axial structure only with the beginning of epiboly, as the germ ring was formed in the marginal zone, unlike the cells outside the germ ring. The degree of differentiation of axial structures in the dorsal sector of marginal zone increased reliably with the appearance of embryonic shield, i.e. area of the convergence of cell flows. Here, statistically significant regional differences in morphogenetic potencies of the marginal zone first appeared, which corresponded to the differences in prospective significance of its materials; notochord and neuroectoderm better differentiate from the dorsal sector material, while somites better differentiate from the ventral sector material. Thus, distribution of morphogenetic potencies reflects precisely the spatial-temporal dynamics of collective movement of the blastoderm cells during the normal course of morphogenesis.     

Spatial-temporal dynamics of morphogenetic blastoderm potencies in early embryogenesis of the loach

The degree of differentiation of axial structures (notochord, neuroectoderm, and somites) in 24-hour explants (a total of 380) of the loach embryonic blastoderm was determined on histological sections according to a developed scale of estimates. Before the beginning of epiboly, axial structures were formed only from fragments of the dorsal sector of the blastoderm marginal zone. Its other sectors acquired the capacity of forming axial structure only with the beginning of epiboly, as the germ ring was formed in the marginal zone, unlike the cells outside the germ ring. The degree of differentiation of axial structures in the dorsal sector of marginal zone increased reliably with the appearance of embryonic shield, i.e. area of the convergence of cell flows. Here, statistically significant regional differences in morphogenetic potencies of the marginal zone first appeared, which corresponded to the differences in prospective significance of its materials; notochord and neuroectoderm better differentiate from the dorsal sector material, while somites better differentiate from the ventral sector material. Thus, distribution of morphogenetic potencies reflects precisely the spatial-temporal dynamics of collective movement of the blastoderm cells during the normal course of morphogenesis.     

A radioautographic study of protein synthesis in loach blastoderm]

The dynamics of protein synthesis in the loach embryos has been studied by means of autoradiography at the stages of cleavage, blastula and gastrula. During the synchronous cleavage divisions, nuclear proteins are mainly synthesized. From the early blastula stage until the early gastrula stage, the intensity of nuclear protein synthesis increases 2.5 times whereas the intensity of cytoplasmic and total protein synthesis is low and relatively constant. After the onset of gastrulation the intensity of nuclear and cytoplasmic protein synthesis increases 3-4 times and at the late gastrula stage it decreases twice as compared with that at the midgastrula stage. During blastulation, no regional differences in the intensity of nuclear and cytoplasmic protein synthesis were found. With the onset of gastrulation, a vegeto-animal gradient of labeled aminoacid incorporation into nuclear and cytoplasmic proteins appears. During gastrulation, reliable differences were found between the intensity of labeled aminoacid incorporation into proteins of the cells of intact and dissociated blastoderms. During this period, the intensity of protein synthesis in embryonic shield is higher than that in the extraembryonic part of blastoderm.     

Relationship between annual variation in reservoir conditions and year-class strength of peled (Coregonus peled) and whitefish (C. lavaretus)

We estimated year-class strength (YCS) of whitefish and peled by VPA in two boreal reservoirs Lokka and Porttipahta with mean regulation of water level amplitudes of 2.34 and 2.49 m, respectively. YCS of peled Coregonus peled(Gmelin) was related to winter drawdown most probably via egg desiccation. In the relatively shallow Lokka dissolved oxygen deficiency in late winter also had a negative impact on survival of eggs and year-class strength. In contrast to peled, whitefish Coregonus lavaretus(L.) indicated no significant relationship with winter drawdown or dissolved oxygen level. The obvious reason for the pattern is that peled spawn on the reservoir bottom, but whitefish spawn in the ascending rivers, which are mainly unaffected by reservoir regulation.     

Embryonic development of the sea bass Dicentrarchus labrax

The embryonic development of the sea bass Dicentrarchus labrax during the endotrophic period is discussed. An 8 cells stage, not reported for other studied species, results from two rapid successive cleavages. Blastula occurs at the eighth division when the embryo is made of 128 cells. During gastrulation, the infolded blastoderm creates the endomesoblastic layer. The Kupffer??s vesicle is reported to drive the left/right patterning of brain, heart and digestive tract. Heart formation starts at 8 pairs of somites, differentiation of myotomes and sclerotomes starts at the stage 18 pairs of somites; main parts of the digestive tract are entirely formed at 25 pairs of somites. At 28 pairs of somites, a rectal region is detected, however, the digestive tube is closed at both ends, the jaw appears the fourth day after hatching, but the mouth is not opened before the fifth day. Although cardiac beating and blood circulation are observed, gills are not reported in newly hatched individuals; eye melanization appears concomitant with exotrophic behavior.     

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.     

Piscivory and prey selection of four predator species in a whitefish dominated subarctic lake

According to logistic regressions derived for pike Esox lucius and burbot Lota lota , the probability of ingesting fishes in Lake Muddusjärvi, northern Finland, was 50% at 19·3 and 22·1 cm L _T, whereas Arctic charr Salvelinus alpinus and brown trout Salmo trutta shifted to piscivory at the lengths of 25·7 and 26·4 cm L _T. The specialist piscivores, pike and burbot, consumed more prey species and took a wider range of prey sizes than Arctic charr and brown trout. The prey length for all predators increased in relationship to predator length. Whitefish Coregonus lavaretus was the dominant prey species in the lake and in the diet of all the piscivorous species. The whitefish population was divided into three forms, of which the slow-growing, and the most numerous densely rakered whitefish form (DR), was selected by all predator species. This form also had the smallest average size and widest habitat range, utilizing both pelagic and epibenthic habitats. Two sparsely rakered whitefish forms (LSR and SSR) occupied only epibenthic habitats and had lower relative densities than DR. These forms, LSR and SSR, had a minor importance in the diet of predator species.     

Dorso-ventral polarity of the zebrafish embryo is distinguishable prior to the onset of gastrulation

We describe a set of observations on developing zebrafish embryos and discuss the main conclusions they allow:(1) the embryonic dorso-ventral polarity axis is morphologically distinguishable prior to the onset of gastrulation; and (2) the involution of deep layer cells starts on the prospective dorsal side of the embryo. An asymmetry can be distinguished in the organization of the blastomeres in the zebrafish blastula at the 30% epiboly stage, in that one sector of the blastoderm is thicker than the other. Dye-labelling experiments with DiI and DiO and histological analysis allow us to conclude that the embryonic shield will form on the thinner side of the blastoderm. Therefore, this side corresponds to the prospective dorsal side of the embryo. Simultaneous injections of dyes on the thinner side of the blastoderm and on the opposite side show that involution of deep layer cells during gastrulation starts at the site at which the embryonic shield will form and extends from here to the prospective ventral regions of the germ ring.     

Taxonomic Status of Four Rare Alien Fish Species of the Kapchagay Reservoir (Balkhash Basin,Central Asia)

Journal of Ichthyology - Based on the morphological features, four rare alien species in the Kapchagai reservoir were identified: Coregonus peled, Parasalmo mykiss, Megalobrama mantschuricus, and...     

Feeding of peled <Emphasis Type="Italic">Coregonus peled</Emphasis> (Coregonidae) in lakes of Bol’shezemel’skaya tundra

Feeding of peled Coregonus peled in lakes of Bol’ shezemel’ skaya tundra has been studied. Basic attention was paid to the change in the importance of dominant food components in the feeding of peled in different ecosystems throughout the vegetation period in years with different weather conditions.     

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.     

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.     

Characterization and expression of a presomitic mesoderm-specific<Emphasis Type="Italic"> mespo</Emphasis> gene in zebrafish

A complete zebrafish mespo cDNA encoding a protein of 131 amino acids with a bHLH domain in the C-terminal has been isolated. The bHLH domain of zebrafish Mespo is highly similar to those in the mouse, chick and Xenopus, sharing 82.4%, 80.4% and 74.5% amino acid identity, respectively. At 50% epiboly, the zebrafish mespo is first detected in the marginal zone of the blastoderm but excluding the prospective shield. Subsequently, mespo expression is intensified in the involuting mesoderm at 60% epiboly, and then restricted to the presomitic mesoderm (PSM) at 95% epiboly. At the 1-somite stage, mespo expression becomes reduced in the most rostral PSM. During segmentation, mespo expression is gradually downregulated at the most rostral segmental plate where cells are being coalesced to form somites. In spadetail mutant embryos, most of mespo-expressing cells were missing.     

Geometry and mechanics of teleost gastrulation and the formation of primary embryonic axes

Examination of normal shaping dynamics and immediate and long-term responses to blastoderm cutting in zebrafish and loach embryos prior to the onset of gastrulation and during the course of epiboly revealed that anteroposterior (AP) and dorsoventral (DV) polarity formation is connected with shaping of the blastoderm circumferential region, which stretches along and shrinks across its movement axes and originates the non-isotropic fields of tensile stresses. Based on data from cutting experiments and quantitative morphology, we reconstructed the movement-shaping patterns of epiboly and embryonic shield formation. We revealed that AP and DV axes originate as a mass cell movement subject to the movement-shaping equivalence principle, which means the spatial series of differently shaped areas corresponding to the time succession of the same area shaping. Maintenance of the main body axes in orthogonal orientation depends on the mechanical equilibrium principle allowing for converting shape asymmetry into that of tensile stresses and vice versa. The causal relationship between the main movement-shaping axes and that of embryonic polarity was proved in cutting experiments in which the DV axis direction was subject to rearrangement so as to adjust to the new direction of mass cell movement axes induced by healing the wound in the blastoderm circumferential region.     

Spatial-temporal characteristics of intercellular junctions in early zebrafish and loach embryos before and during gastrulation

 Injections of lucifer yellow and fluorescein dyes into loach (Misgurnus fossilis) and zebrafish (Danio rerio) embryos were used to analyse the intercellular communication via gap junctions (GJs) and their role in morphogenetic processes during the period from early blastula to late gastrula. It is shown that the efficiency of dye transfer between the superficial blastomeres increases by the late blastula stage. Blastomeres of the basal layer, on the other hand, become gradually uncoupled from the yolk cell (YC). This process is spatially uneven and finishes by the late gastrula stage. Prior to it, at the early epiboly stage, a local increase in dye transfer is observed in the circular zone of the blastoderm margin. During gastrulation, GJ communication between blastomeres and the YC in this zone and also in the newly-formed germ ring region (the prospective mesoderm domain) persists for a longer period of time (up to the stage of 60–70% epiboly) than in the remaining part of the basal layer (the prospective ectoderm domain). Taking into account the data on changes in the adhesive properties of blastomeres during normal development and observations on embryos with retarded epiboly, we hypothesize that changes in GJ communication between superficial blastomeres, on one hand, and between basal blastomeres and the YC, on the other, are the consequences of the same, more general morphogenetic process of compaction occurring within the blastoderm, which supports epiboly and is probably responsible for the distinction between mesodermal and ectodermal fates of cells differently located within the forming epithelioid sheet. Received: 18 October 1996 / Accepted: 4 April 1997