Epiboly Stage Research Articles

Type I and type II cytokeratin cDNAs from the zebrafish ( Danio rerio) and expression patterns during early development

Full-length cDNAs of a type I (zfCKI), and a type II (zfCKII) cytokeratin from the adult zebrafish, Danio rerio, were characterized and their expressions studied during early development and in the adult. The 1426 bp long zfCKI cDNA encodes a 46.7 kD protein, whereas the 2398 bp zfCKII cDNA encodes a protein of 58.6 kD. zfCKI and zfCKII each have a central rod domain that is characteristic of intermediate filaments and which share 73 %–91 % and 87 %–93 % similarity, respectively, with those of type I and type II cytokeratins from zebrafish, goldfish, and the rainbow trout. The central rod domains of zfCKI and zfCKII also contain the IF signature motif, IA[T/E]YR[K/R]LL[D/E]. zfCKI has, in addition, a leucine-zipper motif at a.a. residues 184–205 and 191–212. Both zfCKI and zfCKII mRNAs are expressed in the epidermis of the zebrafish. zfCKII mRNA was both maternally inherited and zygotically transcribed and was detected from the one-cell embryo to adult stages. zfCKII was also strongly expressed specifically during the 20-somites, protruding-mouth, and adult stages. In the adult, it was uniformly expressed in the skin, fins and scale epidermis. In contrast, zfCKI mRNA was undetectable in the oocyte but was zygotically transcribed from the epiboly stage onwards. Its expression in the skin was strong only up to the swimming larva stage and was weak and patchy in the adult. Both zfCKI and zfCKII were expressed in the neurons and glial cells of the brain and spinal cord. In the adult eye, zfCKI and zfCKII were expressed in the ganglion cell layer and the retina, but zfCKII was also strongly expressed in the cornea as well as in chondrocytes in the skull.

Zebrafish zic1 expression in brain and somites is affected by BMP and Hedgehog signalling

Here we report the expression of the zebrafish zic1 gene, also known as opl, a homologue to other vertebrate Zic genes and the Drosophila odd-paired gene. zic1 expression starts during epiboly stages in lateral parts of the neural plate and eventually comes to lie in dorsal regions of the developing brain following the morphogenetic movements of neural tube formation. To address the question whether BMP2 signalling affects the extent of zic1 expression, we analysed swirl and chordino mutant embryos. Expanded Zic1 expression in swirl and reduced expression in chordino as well as in bmp2 injected embryos suggest that BMP2 and its antagonists define the extent of zic1 expression in the neural plate. By searching for factors responsible for the dorsal restriction of Zic1 expression, we found zic1 expression is eliminated in sonic hedgehog (shh) injected embryos. The most rostral expression however is not affected by Shh suggesting that Shh plays a different role in dorso-ventral patterning of the future telencephalon. During somitogenesis zic1 is expressed in the dorsal most part of the developing somites. Here zic1 marks cells that are distinct from the main adaxial somite portion, the future myomere. zic1 expression in the somites is expanded in swirl but reduced in shh injected embryos, suggesting these factors have opposing activity in dorsoventral patterning of the somites. Later, a growing mass of zic1 expressing cells occurs in a dorsal mesenchyme that eventually invades the dorsal fin fold, suggesting a somitic contribution to the dorsal fin mesenchyme.

Zebrafish CTH1, a C3H zinc finger protein, is expressed in ovarian oocytes and embryos.

The Zfcth1 gene is, as the previously cloned carp cth1 gene, related to the mammalian TIS 11 family of primary response genes and encodes a protein with two putative CCCH zinc fingers. This report describes the RNA expression of this gene during oogenesis and early embryogenesis up to gastrulation in the zebrafish (Danio rerio). Maternal cth1 message is present in the ovary of 1-month-old fish and of adult fish in oocytes at all stages of maturation. In the youngest oocytes the message is localized in the cytoplasm all around the nucleus, in larger oocytes the message becomes restricted to the future animal pole of the embryo, and in mature oocytes the expression is sharply localized in the cortical layer under the micropyle. After ovulation the cth1 messenger spreads over the cytoplasmic cap and is distributed over the blastomeres during subsequent cleavages. In subsequent stages maternal expression of cth1 gradually disappears. From early epiboly stages onward embryonic cth1 expression is localized to the germ ring and the hypoblast cells in the central part of the embryonic shield. In the shield, cth1 expression largely overlaps with the area of gooscoid expression in the first involuting cells. In stages after 70% of epiboly cth1 expression diminishes and soon can no longer be detected in the embryo. Next to a developmental role in cell fate determination we propose a function for cth1 during oocyte maturation.

Characterization of two frizzled8 homologues expressed in the embryonic shield and prechordal plate of zebrafish embryos

We have isolated and characterized two complete cDNA clones, Zfz8a and Zfz8b, which encode zebrafish Frizzled (Fz) homologues. The predicted protein sequences, spanning 579 and 576 amino acid residues for ZFz8a and ZFz8b, respectively, were highly homologous (78%) to each other and contained an extracellular cysteine-rich domain and seven transmembrane domains that are well conserved in Fz receptor protein members. In comparison with other Fz family members, ZFz8a and ZFz8b showed the highest homology with mouse Fz8 (MFz8), sharing 84 and 76% amino acid identity, respectively. The presence of Zfz8a and Zfz8b transcripts was detected by in situ hybridization in zebrafish embryos from the 512 cell stage, and their appearance in the future dorsal region could be observed before embryos reached the 30% epiboly stage. At shield stage, Zfz8a transcripts were expressed in both epiblast and shield whereas expression of Zfz8b was only detected in the embryonic shield. During gastrula stages, both Zfz8a and Zfz8b transcripts were found in anterior dorsal regions of the involuting mesendoderm (future prechordal plate). By the 2- to 3-somite stage, expression of both Zfz8a and Zfz8b was restricted to the prechordal plate and prospective anterior neurectoderm, although expression of the Zfz8a gene was no longer present in the most anterior portion of the prechordal plate, the polster. In one-eyed pinhead mutant embryos, which lack prechordal plate, both Zfz8a and Zfz8b transcripts were reduced, confirming the prechordal plate specificity of Zfz8a and Zfz8b gene expression. These results provide an additional evidence supporting the role of Wnt signaling in organizer-mediated axial patterning.

Regional cell movement and tissue patterning in the zebrafish embryo revealed by fate mapping with caged fluorescein

Determination of fate maps and cell lineage tracing have previously been carried out in the zebrafish embryo by following the progeny of individual cells injected with fluorescent dyes. We review the information obtained from these experiments and then present an approach to fate mapping and cell movement tracing, utilizing the activation of caged fluorescein-dextran. This method has several advantages over single-cell injections in that it is rapid, allows cells at all depths in the embryo to be marked, can be used to follow cells starting at any time during development, and allows an appreciation of the movements of cells located in a coherent group at the time of uncaging. We demonstrate that the approach is effective in providing additional and complementary information on prospective mesoderm and brain tissues studied previously. We also present, for the first time, a fate map of placodal tissues including the otic vesicle, lateral line, cranial ganglia, lens, and olfactory epithelium. The prospective placodal cells are oriented at the 50% epiboly stage on the ventral side of the embryo with anterior structures close to the animal pole, and posterior structures nearer to the germ ring.

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.

Manipulation of Gap Junctional Communication Between the Yolk Syncytial Layer and Blastoderm Cells and Its Effect On Embryonic Development of Cyprinus Carpio

In an attempt to block gap junctional communication during early development antibodies against connexins were injected into the YSL of carp embryos at the 30% epiboly stage. No effect on development in terms of survival or specific aberrations was observed. It appeared that no antibodies could be demonstrated in the YSL bordering the blastoderm. Connexin translation was aimed to be interrupted by antisense connexin RNA injection into the yolk just beneath the blastoderm cells of the 2- to 16-cell stage carp embryos. Preliminary results indicate a relatively small body volume in a number of treated embryos, which might indicate a disturbance of cell convergence during early gastrulation. The overexpression of connexin RNA by the injection of sense connexin RNA formed another approach to study gap junctional communication. This treatment resulted in aberrant development, which most seriously affected embryos after injection at the 2- to 8-cell stage.

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.

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.

Neurulation in the anterior trunk region of the zebrafish Brachydanio rerio.

We have studied the process of neurulation within the anterior trunk region of the zebrafish by means of serial sectioning of staged embryos and labelling cells by applications of the dye Dil and intracellular injections of fluoresceine dextran amine. The first morphological manifestation of the prospective neural plate is a dorsomedial ectodermal thickening which becomes visible immediately after gastrulation. Within 1-2 h, by the time somatogenesis begins, two bilaterally symmetrical thickenings have appeared more laterally, which eventually fuse with the medial thickening to form the neural keel. The central canal forms next by separation of the cells on either side of the midline of the neural keel, beginning ventrally at the 17-somite stage and progressing towards dorsal levels. By means of fluorescent dye labelling in the late gastrula, we found that both the medial and lateral thickenings contribute to the nerve cord. The medial thickening was found to contain, exclusively, neural progenitor cells from the 90-100% epiboly stage on, whereas the adjacent regions contained a mixture of neural and epidermal progenitor cells, as well as prospective neural crest cells. Between the 90-100% epiboly and 2-somite stages, this heterogeneity of developmental capabilities is resolved into territories, with epidermogenic and neurogenic cells clearly separated from each other. To achieve this segregation into neural and epidermal anlagen, cells from the lateral thickenings have to move over a distance of roughly 400 μm within 1-2 h. Epidermal overgrowth of the nerve cord occurs during the morphogenetic movements that accompany nerve cord formation.

A study on cell lineage, especially the germ cell line, in embryos of the teleost fish,Barbus conchonius

Lucifer Yellow-Dextran labelling of lower layer cells (LLC), sometimes together with upper layer cells (ULC), of the 64-cellBarbus conchonius embryo resulted in labelled primordial germ cells (PGCs) at 12 h after fertilization (a.f.) in about 25% of cases. The presence of labelled PGCs was independent of the location of the injected blastomere with respect to the later orientation of the embryonic axis. After injection of an ULC alone, however, labelled PGCs were never found. Also, the distribution of labelled somatic cells differed between the ULC- and LLC-injected embryos. When we found fluorescent PGCs, only a few of them were labelled, suggesting that either a single predecessor exists earlier than the 64-cell stage or that the formation of germ cells is a polyclonal process. Tracing the fluorescent cells at successive stages of development shows an extensive mixing with unlabelled cells during the epiboly stage, which might well be the cause of partly unpredictable cell lineages. The chance of being committed to a specific fate is different for the ULC and LLC descendants. This might be due to relatively limited cell mixing between these two cell populations.

Cell Morphologies and Contacts in Annual Fish Embryos

During epiboly and early dispersed stages, the deep blastomeres of annual fish embryos emigrate from the blastodisc to wander as easily visible single cells in the narrow space between the yolk synctial layer (YSL) and the enveloping cell layer (ECL). Subsequently these deep blastomeres reaggregate and eventually form the definitive embryo. We are studying these morphogen-etic cell movements and the controls that may exist upon cell movement in vivo. Time-lapse cinemicrophotography showed three types of cell movement by the deep blastomeres during epiboly through reaggregation stages [1], The present electron microscopic investigation was undertaken to reveal further details of the intercellular contacts during these developmental stages.

Contact inhibition of cell movement in living embryos of an annual fish, Nothobranchius korthausae: Its role in the switch from persistent to random cell movement

AbstractThe deep blastomeres of annual fish embryos emigrate from the blastodisc to wander as single cells, during epiboly and early dispersed stages, between the periblast and the enveloping layer cells. Time‐lapse films of the deep blastomere movements were analyzed to determine whether the cells move persistently or randomly and whether or not they show contact inhibition of cell movement. Certain predictions which follow from the random‐walk model (uniform distribution of intersegmental angles, exponential distribution of squared segmental lengths, and a straight‐line plot – passing through the origin – of mean square displacement against time) were tested and, in general, found to apply to these deep blastomeres during dispersed phase 1 but not during epiboly stages. These results indicate that the cells tend to persist in their direction of movement during epiboly, but not during dispersed phase 1, when their movement is random. Upon completion of epiboly, there is a decrease in cell motility, as measured by the augmented diffusion constant, D*, and an increase in the average collision rate. Numerous cases of cell contact between deep blastomeres were observed (297 during epiboly and 167 during dispersed phase 1); in every case one or both cells changed their direction of movement. The role this contact inhibition might play in the persistent movement of epiboly and the random movement of dispersed phase 1 is discussed.

Cell patterns and cell movements during early development of an annual fish, Nothobranchius neumanni.

During epiboly stages the cells (called deep blastomeres) which will form the definitive embryo disperse over the surface of the yolk sphere, only later aggregating and developing an embryonic axis. Five different statistical tests were used to study the pattern formed by the deep blastomeres during epiboly and early dispersed stages. The two most reliable tests, based on the distance from each deep blastomere within a selected area to its nearest neighboring cell, indicate that the distribution pattern changes from regular during epiboly stages to random during dispersed stages 1 and 2. Careful observation and time-lapse microphotography revealed some aspects of how the cells set up the regular pattern. The deep blastomeres exhibit a variety of cell extensions, with which they often contact one another. When two deep blastomeres make contact during epiboly stages, they soon break the contact and move apart; they overlap one another only rarely. Deep blastomeres are frequently located at, and are even elongated along, borders of the overlying flat cells (enveloping layer cells). These two mechanisms, one similar to contact inhibition of cell movement, the other to contact guidance, may contribute to the rather regular spacing of the deep blastomeres as well as to their arrangement in rows during epiboly stages.

Cytokinesis during early development of a teleost embryo: Brachydanio rerio

During early development of the teleost Brachydanio rerio , both furrow cleavage and vesicle cleavage were found in the same blastula. Furrow cleavage, characterized by an “in-pinching” of the plasma membrane with a fine deposit on the cytoplasmic side of the encroaching furrow, and vesicle cleavage, characterized by a row of vesicles across the plane of daughter cell separation, are found until one-third epiboly stage embryos, when only furrow cleavage was found.

SYPHILIS AND MENTAL TREATMENT ACT

<h3>Abstract</h3> Few models exist that allow for rapid and effective screening of anti-metastasis drugs. Here, we present a phenotype-based chemical screen utilizing gastrulation of zebrafish embryos for identification of anti-metastasis drugs. Based on the evidence that metastasis proceeds through utilizing the molecular mechanisms of gastrulation, we hypothesize that chemicals which interrupt zebrafish gastrulation might suppress metastasis of cancer cells. Thus, we developed a drug screening protocol which uses epiboly, the first morphogenetic movement in gastrulation, as a marker. The screen only needs zebrafish embryos and enables hundreds of chemicals to be tested in five hours through observing epiboly progression of a test chemical-treated embryos. In the screen, embryos at the two-cell stage are firstly corrected and then developed to the sphere stage. The embryos are treated with a test chemical and incubated in the presence of the chemical until vehicle-treated embryos develop to 90% epiboly stage. Finally, positive ‘hit’ chemicals that interrupt epiboly progression are selected through comparing epiboly progression of the chemical-treated embryos with that of vehicle-treated embryos under a stereoscopic microscope. Previous study subjected 1,280 FDA-approved drugs to the screen and identified Adrenosterone and Pizotifen as epiboly-interrupting drugs. These drugs were validated to suppress metastasis of breast cancer cells in mice models of metastasis. Furthermore, 11β–Hydroxysteroid Dehydrogenase 1 (HSD11β1) and serotonin receptor 2C (HTR2C), which are primary target of Adrenosterone and Pizotifen respectively, promotes metastasis through induction of epithelial-mesenchymal transition (EMT). That indicates the screen could be diverted to a chemical genetic screening platform for identification of metastasis-promoting genes.