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Transparent things: Cell fates and cell movements during early embryogenesis of zebrafish
Abstract
Development of an animal embryo involves the coordination of cell divisions, a variety of inductive interactions and extensive cellular rearrangements. One of the biggest challenges in developmental biology is to explain the relationships between these processes and the mechanisms that regulate them. Teleost embryos provide an ideal subject for the study of these issues. Their optical lucidity combined with modern techniques for the marking and observation of individual living cells allow high resolution investigations of specific morphogenetic movements and the construction of detailed fate maps. In this review we describe the patterns of cell divisions, cellular movements and other morphogenetic events during zebrafish early development and discuss how these events relate to the formation of restricted lineages.
... The EVL will form a protective cover called the periderm, an extraembryonic structure that will disappear later in development. The deep cells are located between the YSL and the EVL and will give rise to the embryo proper (reviewed in Kimmel et al., 1995;Solnica-Krezel et al., 1995). epiboly the mesoderm layer has migrated anteriorly and is located between the ectoderm and the endoderm. ...
... The com bination of convergence with m ediolateral intercalation results in the extension of the em bryo along an anterior-posterior direction. Gastrulation is essentially complete by 10 hpf when the embryo reaches the tailbud stage (reviewed in Kimmel et al., 1995;Solnica-Krezel et al., 1995). ...
... In the high salt condition (IX Danieau solution), according to this model, EVL cells fail to rapidly close the wound and deep cells are able to re-associate and go on to form normal shield derivatives. The formation of shield derivatives requires not only the presence of adjacent marginal tissue relative to the morphological shield, but also that this tissue heals properly.A zebrafish embryo at gastrula stage consists of around 2 '"^=16,384 cells(Solnica-Krezel et al., 1995) and the amount of cells removed in the morphological shield removal experiments was around 400 cells (estimated by counting nuclei stained with Hoechst, a DNA fluorochrome). Since this corresponds to a loss of only 2%, it is perhaps not surprising that morphological shield-ablated embryos have a normal size by 24 hpf. ...
The Spemann's organizer is a region of the amphibian gastrula that will induce a second body axis when transplanted to ventral or lateral regions of a host embryo. The organizer can dorsalise mesoderm, induce convergent extension movements and specify neuroectoderm. Functional equivalents of the Spemann's organizer have been identified in other vertebrates by transplantation experiments. A region of the fish gastrula called the embryonic shield is thought to function as the dorsal organizer. Using a novel surgical method, I showed that the morphological shield can induce complete secondary axes when transplanted into the ventral germ ring of a host embryo. In induced secondary axes, the donor shield contributed to hatching gland, prechordal plate, notochord, floor plate and hypochord. When explanted shields were divided into deep and superficial fragments and separately transplanted, I found that deep tissue can induce ectopic axes with heads but lacking posterior tissues. I found that when only the morphological shield was removed, embryos recovered and were completely normal by 24 hours-post-fertilisation. Ablation of the morphological shield does not remove all goosecoid- and floating head-expressing cells, suggesting that the morphological shield does not comprise the entire organizer region. Removal of the morphological shield plus adjacent marginal tissue, however, led to loss of all shield derivatives, a cyclopean head, loss of floor plate and primary motomeurons and disrupted somite patterning. Embryos from which only the morphological shield was removed still had some goosecoid- and floating head-expressing cells. I have tested whether these residual shield cells were sufficient to form all shield derivatives or, alternatively, if adjacent non-shield tissues could be recruited to shield fate. After morphological shield removal, I found no increase in cell proliferation. Transplantation studies indicated, however, that non-shield tissue may be recruited to a shield fate. Finally, I have employed the shield removal and transplantation method to study two mutations: sneezy and silberblick/wnt11. Transplantation results indicate that sneezy acts autonomously within the shield derivatives. By contrast, silberblick/wnt11 acts non-autonomously in paraxial tissues to drive the convergent extension movement of axial mesoderm.
... In zebrafish, at least three major types of cell rearrangements shape the embryo during gastrulation: epiboly, involution/ ingression and convergence and extension Kane and Warga, 1994;Solnica-Krezel et al., 1995). Most of the mutants described above and in the following section exhibit an abnormal morphology during gastrulation (Figs 3 and 4). ...
... Epiboly is the first morphogenetic movement involving vegetal expansion of the deep cell layer, the enveloping layer (EVL) and the yolk syncytial layer (YSL; reviewed by Trinkaus, 1984b;Solnica-Krezel et al., 1995). In vol m712 mutants, epiboly of the majority of deep cells is arrested when the blastoderm covers 60% of the yolk cell. ...
One of the major challenges of developmental biology is understanding the inductive and morphogenetic processes that shape the vertebrate embryo. In a large-scale genetic screen for zygotic effect, embryonic lethal mutations in zebrafish we have identified 25 mutations that affect specification of cell fates and/or cellular rearrangements during gastrulation. These mutations define at least 14 complementation groups, four of which correspond to previously identified genes. Phenotypic analysis of the ten novel loci revealed three groups of mutations causing distinct effects on cell fates in the gastrula. One group comprises mutations that lead to deficiencies in dorsal mesodermal fates and affect central nervous system patterning. Mutations from the second group affect formation of ventroposterior embryonic structures. We suggest that mutations in these two groups identify genes necessary for the formation, maintenance or function of the dorsal organizer and the ventral signaling pathway, respectively. Mutations in the third group affect primarily cellular rearrangements during gastrulation and have complex effects on cell fates in the embryo. This group, and to some extent mutations from the first two groups, affect the major morphogenetic processes, epiboly, convergence and extension, and tail morphogenesis. These mutations provide an approach to understanding the genetic control of gastrulation in vertebrates.
... Here, we investigate these questions in zebrafish, analysing how the migration of the anterior axial mesendoderm is directed towards the animal pole. At the onset of gastrulation, the first cells to internalize on the dorsal side of the embryo are precursors of the polster (hereafter referred to as polster cells; Solnica-Krezel et al., 1995). From the embryonic organiser, they migrate in straight towards the animal pole, leading the extension of the axis, and are followed by more posterior axial mesodermal cells, including posterior prechordal plate precursors and notochord precursors (Montero et al., 2005; Figure S1A). ...
... Small groups of migration defective cells are displaced by axis extension ( Figure S3C If what orients polster cells is the presence of migrating cells at their back, we reasoned that they may not require interactions specifically with the posterior axial mesoderm, and that other cells migrating towards them should be able to orient them. To test this, we transplanted polster cells ahead of the animally migrating lateral mesoderm ( Figure S4A, Movie S4, Solnica-Krezel et al., 1995). Before contact, polster cells moved and extended protrusions without a preferred orientation ( Figure S4B-D). ...
Morphogenesis, wound healing, and some cancer metastases depend upon the migration of cell collectives that need to be guided to their destination as well as coordinated with other cell movements. During zebrafish gastrulation, the extension of the embryonic axis is led by the mesendodermal polster that migrates toward the animal pole, followed by the axial mesoderm that undergoes convergence and extension. Here, we investigate how polster cells are guided toward the animal pole. Using a combination of precise laser ablations, advanced transplants, and functional as well as in silico approaches, we establish that each polster cell is oriented by its immediate follower cells. Each cell perceives the migration of followers, through E-cadherin/α-catenin mechanotransduction, and aligns with them. Therefore, directional information propagates from cell to cell over the whole tissue. Such guidance of migrating cells by followers ensures long-range coordination of movements and developmental robustness.
... The zebrafish possesses many characteristics that make it an invaluable model to study human diseases [27]; however, one of its unique advantages is the unparalleled optical clarity of the embryo, which allows for the visualization of individual genes (fluorescently stained or labeled) throughout development, using non-invasive imaging techniques [28][29][30][31] (Figure 1A). This transparency of the embryo also facilitates genetic manipulation, such that gene function can readily be studied by the injection of synthetic mRNA or plasmid DNA into early stage zebrafish embryos generating transgenic zebrafish lines or altering gene function through genome editing techniques, such as the inclusion of zinc finger nucleases (ZFNs), transcription activation-like effector nucleases (TALENs), and the clustered regularly interspaced repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system [32][33][34]. ...
... R PEER REVIEW 4 of 15 cently stained or labeled) throughout development, using non-invasive imaging techniques [28][29][30][31] ( Figure 1A). This transparency of the embryo also facilitates genetic manipulation, such that gene function can readily be studied by the injection of synthetic mRNA or plasmid DNA into early stage zebrafish embryos generating transgenic zebrafish lines or altering gene function through genome editing techniques, such as the inclusion of zinc finger nucleases (ZFNs), transcription activation-like effector nucleases (TALENs), and the clustered regularly interspaced repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system [32][33][34]. ...
Rare diseases are those which affect a small number of people compared to the general population. However, many patients with a rare disease remain undiagnosed, and a large majority of rare diseases still have no form of viable treatment. Approximately 40% of rare diseases include neurologic and neurodevelopmental disorders. In order to understand the characteristics of rare neurological disorders and identify causative genes, various model organisms have been utilized extensively. In this review, the characteristics of model organisms, such as roundworms, fruit flies, and zebrafish, are examined, with an emphasis on zebrafish disease modeling in rare neurological disorders.
... During this period there is an increase in cell cycle length and commencement of zygotic transcription. Later, cell motility begins through radial intercalations and subsequently epiboly (Solnica-Krezel et al., 1995;Warga and Kimmel, 1990). These events are also characteristic of MET in other metazoans. ...
... The blastoderm is pulled down over the embryo through strong attachments between the marginal cells of the EVL and the YSL, which in turn is attached to force-generated microtubules within the yolk cell (Warga and Kimmel, 1990). Other gastrulation movements such as, involution, and convergent extension occur simultaneously with epiboly (Solnica-Krezel et al., 1995). ...
The notochord is critical for the normal development of vertebrate embryos. Zebrafish mutants, bashful (bal), grumpy (gup) and sleepy (sly) were identified in mutagenesis screens and have defects in notochord differentiation. By positional cloning I identified lama1, encoding Laminin α1, as a candidate for the bal gene. Analysis of mRNA expression, antisense knockdown and sequencing of the mutant lama1, confirmed that mutations in this gene are responsible for the bal phenotype. A similar approach was taken to identify the gup gene. Meiotic mapping identified a region containing lamb1, encoding Laminin β1. Cloning and sequencing of lamb1 from mutants revealed a non-sense mutation. These results identify a role for Laminin αl and Laminin β1 in notochord differentiation. Concurrent work in our laboratory revealed that the third mutant, sly, encodes the Laminin γl chain (Parsons et al., 2002). Thus, the laminin 1 isoform, a heterotrimer comprising the α1β1γ1 chains, is necessary for notochord differentiation. bal mutants differ from gup and sly, as failure of notochord differentiation is not as extensive, occurring only in anterior regions. We hypothesised this was due to a redundant role of another a chain. Characterisation of lama4 and lama5, encoding Laminin α4 and α5 chains, respectively, showed that both control aspects of notochord differentiation. Furthermore, lama5 has a functionally redundant role with lama1 during CNS development. Characterisation of lama2 identifies a role for Laminin α2 chain in muscle function, consistent with loss-of-function phenotypes in human and mouse. Hence, I demonstrate different roles for specific laminin chains during zebrafish development, confirming and extending results from studies in other vertebrate systems. Finally, I studied integrin α6, integrin β4, and integrin-linked kinase (ILK), to determine their role during development. The results suggest these genes control cell movements during gastrulation, rather than notochord differentiation.
... The fish embryo, being optically clear, offers direct access to these processes [12]. Among the diverse movements taking place during gastrulation, cells of the organiser (termed 'shield' in zebrafish) internalise and migrate collectively toward the animal pole of the embryo, forming the prechordal plate [9,13,14]. ...
... Sterile-filtered water (Sigma, W3500) for diluting RNAs. 12. Injection needle: needles should be long enough to reach the embryo within its chorion, without creating too large a hole in the chorion. ...
... The prechordal plate is a group of cells composed of the first internalised cells on the dorsal side of the embryo. During gastrulation, the prechordal plate migrates from the embryonic organiser to the animal pole, to later give rise mainly to the hatching gland, the anterior-most structure in the zebrafish embryo ( Kimmel et al., 1995;Solnica-Krezel et al., 1995). Being a cohesive group, the prechordal plate, also referred to as mesendoderm, is a very good model to study the mechanisms of oriented collective migration. ...
... Whether cells internalise by involution of a sheet of cells around the margin or by ingression of individual cells has been long debated. Initial descriptions, reporting that marginal cells undergo a coordinated movement to form the hypoblast, pleaded in favour of an involution process (Solnica- Krezel et al., 1995;Warga and Kimmel, 1990). However, it had also been suggested that ingression movements contribute to the internalisation of mesendodermal cells (Shih and Fraser, 1995;Trinkaus, 1996). ...
During development, cells are progressively separated into distinct territories, delimited by embryonic boundaries. The first segregation event occurs during gastrulation, when the embryo is organised in three germ-layers, the ectoderm, the mesoderm and the endoderm. The molecular and cellular mechanisms ensuring this segregation have not yet been elucidated. During my PhD thesis, I have focused on the endoderm internalisation in the zebrafish embryo. Based on in vitro results, it has been suggested that germ-layer progenitors would be segregated by a passive cell sorting. Combining cell transplantation, live confocal microscopy and functional analyses, I have shown that endodermal cell internalisation actually results from an active migration process dependent on Rac1 and its effector Arp2/3, a direct regulator of actin. Strikingly, endodermal cells are not attracted to their internal destination but rather appear to migrate out of their neighbouring cells. This process is dependent on the Wnt/PCP pathway and N-cadherin. Furthermore, N-cadherin is sufficient to trigger the internalisation of ectodermal cells, without affecting their fate. Overall, these results lead to a new model of germ-layer formation, in which endodermal cells actively migrate out of the epiblast to reach their internal position.
... The emphasis on the study of sectioned material detracted from the observation of living embryos, especially cell behavior during early development, a field that was just beginning to be explored by His (vide infra). This trend led to subsequent difficulties in the interpretation of morphogenetic movements and embryo formation (Trinkaus, 1984a;Solnica-Krezel et al, 1995). Even as this trend was established, its advantages and limitations were becoming obvious. ...
... The results contradict His's theory. In retrospect, His's concrescence theory was an excessively rigid statement of the process of confluence of germ-ring cells and their incorporation into the em-bryo (Solnica-Krezel et al., 1995;Ballard et al., 1993). Before leaving the subject of concrescence, I wish to point out that His restated the theory in 1894 (His, 1894), but, more importantly, in that paper he introduced physical models as a means of illustrating the mechanical basis of morphogenesis. ...
SYNOPSIS. The nineteenth century was critical for the empirical and conceptual growth of developmental biology. Fishes played a central role in this process. The study of fish development, mainly that of teleosts but also chondrichthyans, can be traced back to classical times. In the nineteenth century, it merged with modern descriptive embryology, continued with the rise of comparative embryology associated with evolutionary studies, and moved into the experimental and physiological analysis of development. Any consideration of fish development must take into account that fishes phylogenetically are the most diverse group of the vertebrates and also the most speciose. These features are reflected in the diversity of their development. The descriptive embryology of fishes is reviewed from Aristotle to the beginning of the nineteenth century. The study of chondrichthyans, especially viviparous species, was characteristic of this period. During the nineteenth century, there was a progressive development of knowledge of the descriptive embryology of teleosts and chondrichthyans. Teleosts came to the fore because artificial fertilization ensured a ready supply of material and their transparent eggs were well suited for microscopy. The subsequent development of embryological microtechnique made possible the examination of sectioned material and moved research to a more cellular level. By the end of the century, an in-depth description of development was in place. Interest in the comparative embryology of fishes was stimulated by Haeckel's melding of embryology and evolution and led to a description of development of agnaths, chimaeras, lungfish, and primitive actinopterygian fishes. Experimental and analytical methods of inquiry began to be used at mid-century. The experiments of Ransom on the contractility of egg cytoplasm, Lereboullet's experimental teratology, chemical studies of embryonic nutrition in viviparous fishes, in vitro observation of blastomeres, His's concrescence theory of embryo formation and Kastschenko's and Morgan's testing of it are considered.
... In zebrafish, at least three major types of cell rearrangements shape the embryo during gastrulation: epiboly, involution/ ingression and convergence and extension Kane and Warga, 1994;Solnica-Krezel et al., 1995). Most of the mutants described above and in the following section exhibit an abnormal morphology during gastrulation (Figs 3 and 4). ...
... Epiboly is the first morphogenetic movement involving vegetal expansion of the deep cell layer, the enveloping layer (EVL) and the yolk syncytial layer (YSL; reviewed by Trinkaus, 1984b;Solnica-Krezel et al., 1995). In vol m712 mutants, epiboly of the majority of deep cells is arrested when the blastoderm covers 60% of the yolk cell. ...
One of the major challenges of developmental biology is understanding the inductive and morphogenetic processes that shape the vertebrate embryo. In a large-scale genetic screen for zygotic effect, embryonic lethal mutations in zebrafish we have identified 25 mutations that affect specification of cell fates and/or cellular rearrangements during gastrulation. These mutations define at least 14 complementation groups, four of which correspond to previously identified genes. Phenotypic analysis of the ten novel loci revealed three groups of mutations causing distinct effects on cell fates in the gastrula. One group comprises mutations that lead to deficiencies in dorsal mesodermal fates and affect central nervous system patterning. Mutations from the second group affect formation of ventroposterior embryonic structures. We suggest that mutations in these two groups identify genes necessary for the formation, maintenance or function of the dorsal organizer and the ventral signaling pathway, respectively. Mutations in the third group affect primarily cellular rearrangements during gastrulation and have complex effects on cell fates in the embryo. This group, and to some extent mutations from the first two groups, affect the major morphogenetic processes, epiboly, convergence and extension, and tail morphogenesis. These mutations provide an approach to understanding the genetic control of gastrulation in vertebrates.
... The water is also vital for the rearing of freshwater aquarium fish, since the health of fish and their breeding success depend critically on the properties and quality (Westerfield 2000). Of the freshwater aquarium fish, zebrafish (Danio rerio) has been the most commonly used in a variety of scientific research areas, from developmental biology (Metscher and Ahlberg 1999;Mork and Crump 2015;Rawls et al. 2001;Solnica-Krezel et al. 1995;Staudt and Stainier 2012) and molecular genetics (Fuentes et al. 2018;Kawakami 2005;Lieschke and Currie 2007;Shin and Fishman 2002;Varshney et al. 2015) to toxicology (Cui et al. 2023;Dai et al. 2014;He et al. 2014;Hill et al. 2005;Horzmann and Freeman 2018). It is estimated that more than 1,000 laboratories of the world use more than 5 million zebrafish annually (Aleström et al. 2019;Lidster et al. 2017). ...
The health of freshwater aquarium fish and their breeding success depend critically on the quality of tap water. In general, tap water in Japan is potable, although the properties of tap water vary among regions in Japan. The city of Niigata is located in the largest rice production region of Japan. We have faced challenges concerning the reproduction of freshwater aquarium fish in Niigata. To determine whether water properties and quality affect the reproduction of aquarium fish in Niigata, we investigated the chemical properties of water and raised zebrafish in water from three different sources, namely tap water of Niigata in May, artificial freshwater (i.e., prepared via reverse osmosis), and natural spring water of Gosen, to document any effects on their sexual maturation and reproduction. We found that the tap water of Niigata was not stable throughout a year (median electrical conductivity = 147.1 μS/cm; SD = 25.6), with springtime lower than the first quartile. We also found that low concentrations of four pesticides in the tap water have been detected in May (max. concentration in 2020, bromobutide 2,000 ng/L, butachlor 600 ng/L, pyraclonil 200 ng/L, ipfencarbazone 20 ng/L). Moreover, rearing zebrafish in tap water negatively influenced both fish growth and reproduction: The sex ratio of adults was male biased (proportion of F0 male 70.8%); the average total length (30.5 mm) and weight (182 mg) of F0 males was decreased; the GSI of F0 females (9.7%) was decreased; the fecundity (the mating success 58.7%; the number of F1 eggs 63.1) of adults was reduced. Rearing in artificial freshwater could improve these outcomes (the sex ratio 55.7%; the total length of F0 males 31.8 mm; the weight of F0 males 211 mg; the GSI of F0 females 11.7%; the mating success 72.6%; the number of F1 eggs 99.0), whereas rearing in natural spring water from Gosen could improve the sex ratio (56.3%) and the weight of F0 males (200 mg), but not the others. Therefore, artificial freshwater made via reverse osmosis should be used for breeding freshwater aquarium fish in rice production region like Niigata. Finally, our results demonstrate that the reproduction of freshwater aquarium fish can serve as a bioindicator of low levels of organic pollutants in tap water and thus provide a basis for evaluating the safety of tap water for human consumption.
... This screen demonstrates that mutations affecting a variety of developmental processes can be efficiently recovered from zebrafish. opment of the zebrafish embryo has been studied in detail, from pre-gastrula and gastrula stages (reviewed by Driever, 1995;Kuwada, 1995;Solnica-Krezel et al., 1995;Woo et al., 1995) through organogenesis (Schmitt and Dowling, 1994;Stainier and Fishman, 1994), lending a solid base of knowledge for the detection and interpretation of mutant phenotypes. Prior to our screen, nine zebrafish mutations affecting embryogenesis have been published and their effect on development studied in detail, providing important insights into mechanisms of vertebrate development (Grunwald et al., 1988;Kimmel et al., 1989;Felsenfeld et al., 1990;Westerfield et al., 1990;Hatta et al., 1991;Halpern et al., 1993;Abdelilah et al., 1994;Stainier et al., 1995;Talbot et al., 1995). ...
Systematic genome-wide mutagenesis screens for embryonic phenotypes have been instrumental in the understanding of invertebrate and plant development. Here, we report the results from the first application of such a large-scale genetic screening to vertebrate development.
Male zebrafish were mutagenized with N-ethyl N-nitrosourea to induce mutations in spermatogonial cells at an average specific locus rate of one in 651 mutagenized genomes. Mutations were transmitted to the F1 generation, and 2205 F2 families were raised. F3 embryos from sibling crosses within the F2 families were screened for develop-mental abnormalities. A total of 2337 mutagenized genomes were analyzed, and 2383 mutations resulting in abnormal embryonic and early larval phenotypes were identified. The phenotypes of 695 mutants indicated involvement of the identified loci in specific aspects of embryogenesis. These mutations were maintained for further characterization and were classified into categories according to their phenotypes. The analyses and genetic complementation of mutations from several categories are reported in separate manuscripts. Mutations affecting pig-mentation, motility, muscle and body shape have not been extensively analyzed and are listed here. A total of 331 mutations were tested for allelism within their respective categories. This defined 220 genetic loci with on average 1.5 alleles per locus. For about two-thirds of all loci only one allele was isolated. Therefore it is not possible to give a reliable estimate on the degree of saturation reached in our screen; however, the number of genes that can mutate to visible embryonic and early larval phenotypes in zebrafish is expected to be several-fold larger than the one for which we have observed mutant alleles during the screen. This screen demonstrates that mutations affecting a variety of developmental processes can be efficiently recovered from zebrafish.
... The extraordinary optical clarity of embryos that shows fluorescently labelled individual genes during the developmental progress with non-invasive imaging technique is truly a unique advantage of these models. [3][4][5][6][7][8] Due to the small size of larvae and transparency of the embryo the genetic manipulations and screening of nueroactive compounds are effortlessly achieved. Scientific experimentations are usually reiterated manifold in order to substantiate that the outcomes are precise, so having an animal that can yield a hefty number of issues over and over is obliging. ...
Genetic roots of human ailments were studied by using various laboratory methods. Animal models are required to define genetic mutations involved in manifestation of symptoms in patients. Obesity is a clinical condition characterized by subclinical inflammation due to the predominance of adipokines, often causing tissue damage and if not treated with adequate care can even lead to organ damage. The microarray investigation has demonstrated a specific gene pattern that was similar to human non-alcoholic fatty liver disease (NAFLD) in diet-induced obesity zebrafish model. It is now well documented that there is an eighty percent resemblance with human genome, with complete sequencing of zebrafish genome as indication ropes the theory of positive association amid increased lipid content in food items, obesogenic pathways, abnormal regulation and weight gain and the development of cardiovascular diseases and hence findings in zebrafish could be extrapolated to human beings. Zebrafish model is now being preferred over other commonly used animal models because of its cost effectiveness and good genetic compatibility when compared with human species as evident from success of various translational research with zebrafish on disorders of nervous system in humans. Review (J Int Dent Med Res 2020; 13(4): 1665-1671)
... Thus, MondoA function in the YSL indeed is important for correct epiboly movements. Impaired YSL structure or function may lead to impaired vesicle trafficking at the YSL-EVL border (Lepage and Bruce, 2010;Solnica-Krezel et al., 1995; but see also or to defective patterning of the overlying blastoderm (Carvalho and Heisenberg, 2010), both of which can perturb epiboly. However, we did not find any evidence for major perturbations of marginal endocytosis or blastoderm patterning in the morphants ( Transcriptome analysis points to the cholesterol synthesis enzyme nsdhl as a main target gene of MondoA in the early embryo To obtain cues how MondoA function might impact on epiboly we carried out an unbiased examination of differential gene expression upon mondoa knockdown by applying RNA-seq. ...
The glucose-sensing Mondo pathway regulates expression of metabolic genes in mammals. Here, we characterized its function in the zebrafish and revealed an unexpected role of this pathway in vertebrate embryonic development. We showed that knockdown of mondoa impaired the early morphogenetic movement of epiboly in zebrafish embryos and caused microtubule defects. Expression of genes in the terpenoid backbone and sterol biosynthesis pathways upstream of pregnenolone synthesis was coordinately downregulated in these embryos, including the most downregulated gene nsdhl. Loss of Nsdhl function likewise impaired epiboly, similar to MondoA loss of function. Both epiboly and microtubule defects were partially restored by pregnenolone treatment. Maternal-zygotic mutants of mondoa showed perturbed epiboly with low penetrance and compensatory changes in the expression of terpenoid/sterol/steroid metabolism genes. Collectively, our results show a novel role for MondoA in the regulation of early vertebrate development, connecting glucose, cholesterol and steroid hormone metabolism with early embryonic cell movements.
... Thus, MondoA function in the YSL indeed is important for correct epiboly movements. Impaired YSL structure or function may lead to impaired vesicle trafficking at the YSL-EVL border (Lepage and Bruce, 2010;Solnica-Krezel et al., 1995; but see also or to defective patterning of the overlying blastoderm (Carvalho and Heisenberg, 2010), both of which can perturb epiboly. However, we did not find any evidence for major perturbations of marginal endocytosis or blastoderm patterning in the morphants ( Transcriptome analysis points to the cholesterol synthesis enzyme nsdhl as a main target gene of MondoA in the early embryo To obtain cues how MondoA function might impact on epiboly we carried out an unbiased examination of differential gene expression upon mondoa knockdown by applying RNA-seq. ...
The glucose-sensing Mondo pathway regulates expression of metabolic genes in mammals. Here, we characterized its function in the zebrafish and revealed an unexpected role of this pathway in vertebrate embryonic development. We showed that knockdown of mondoa impaired the early morphogenetic movement of epiboly in zebrafish embryos and caused microtubule defects. Expression of genes in the terpenoid backbone and sterol biosynthesis pathways upstream of pregnenolone synthesis was coordinately downregulated in these embryos, including the most downregulated gene nsdhl. Loss of Nsdhl function likewise impaired epiboly, similar to MondoA loss of function. Both epiboly and microtubule defects were partially restored by pregnenolone treatment. Maternal-zygotic mutants of mondoa showed perturbed epiboly with low penetrance and compensatory changes in the expression of terpenoid/sterol/steroid metabolism genes. Collectively, our results show a novel role for MondoA in the regulation of early vertebrate development, connecting glucose, cholesterol and steroid hormone metabolism with early embryonic cell movements.
... Thus, MondoA function in the YSL indeed is important for correct epiboly movements. Impaired YSL structure or function may lead to impaired vesicle trafficking at the YSL-EVL border (Lepage and Bruce, 2010;Solnica-Krezel et al., 1995; but see also or to defective patterning of the overlying blastoderm (Carvalho and Heisenberg, 2010), both of which can perturb epiboly. However, we did not find any evidence for major perturbations of marginal endocytosis or blastoderm patterning in the morphants ( Transcriptome analysis points to the cholesterol synthesis enzyme nsdhl as a main target gene of MondoA in the early embryo To obtain cues how MondoA function might impact on epiboly we carried out an unbiased examination of differential gene expression upon mondoa knockdown by applying RNA-seq. ...
The glucose-sensing Mondo pathway regulates expression of metabolic genes in mammals. Here, we characterized its function in the zebrafish and revealed an unexpected role of this pathway in vertebrate embryonic development. We showed that knockdown of mondoa impaired the early morphogenetic movement of epiboly in zebrafish embryos and caused microtubule defects. Expression of genes in the terpenoid backbone and sterol biosynthesis pathways upstream of pregnenolone synthesis was coordinately downregulated in these embryos, including the most downregulated gene nsdhl. Loss of Nsdhl function likewise impaired epiboly, similar to MondoA loss of function. Both epiboly and microtubule defects were partially restored by pregnenolone treatment. Maternal-zygotic mutants of mondoa showed perturbed epiboly with low penetrance and compensatory changes in the expression of terpenoid/sterol/steroid metabolism genes. Collectively, our results show a novel role for MondoA in the regulation of early vertebrate development, connecting glucose, cholesterol and steroid hormone metabolism with early embryonic cell movements.
... In addition, when the morphological shield is ablated and replaced by a ventral tissue graft, some of the grafted cells become part of notochord derived tissues like notochord, floor plate or hypochord. Fate maps (Solnica-Krezel et al., 1995;Woo et al., 1995) and transplantation experiments (Saude, L. ...
Mutant alleles of sneezy were identified during the first Tubingen and Boston large-scale systematic screens for recessive-zygotic mutations affecting embryogenesis in zebrafish. It affects differentiation of the notochord, pigmentation, fin formation and leads to widespread degeneration of the embryo in the mid hatching period at about 60 hpf. Using a positional cloning approach, I have identified coatomer subunit α (copa) as the gene mutated in sneezy. The coatomer complex, together with the small GTPase ARF1, constitutes the protein coat of COPI vesicles, an essential component of the early secretory pathway. In zebrafish, copa is expressed maternally and during the first 24 hpf shows ubiquitous zygotic expression. This maternal wild-type component is responsible for absence of defects prior to ±24 hpf By tissue transplantation, I show that α-COP function is required within the shield derivatives for normal notochord differentiation. In addition, we find that α-COP activity is required within the neural tube for normal melanophore development. At 24 hpf sneezy mutant embryos display an abnormal maintenance of early chordamesoderm marker gene expression. This correlates with a failure of the chordamesoderm to differentiate into notochord. EM studies of notochord cells in sneezy mutants and wild-type siblings show that the early secretory pathway is blocked in sneezy. This results in disruption of formation or maintenance of the perinotochordal basal lamina. This general block in transport, which may affect the elaboration of integral membrane receptors, leads to a failure in notochord differentiation and subsequent apoptosis. In addition, abnormally high levels of apoptosis occur in the floorplate and posterior dorsal neural tube. Apoptosis in the posterior dorsal neural tube correlates with the lack of pigmentation, in the posterior trunk of mutant embryos. At more anterior levels, where melanophores may survive, the failure to become pigmented probably arises from a failure of the Golgi apparatus, which normally generates the melanosomes.
... Thus, it facilitates the simultaneous testing of huge number of animals at different environmental conditions. The ocular transparency of the zebrafish embryos permits to visualize the individual genes during its developmental process by coupling with non-invasive imaging techniques (Cooper, D'Amico, & Henry, 1999;Kimmel 1989;Solnica-Krezel, Stemple, & Driever, 1995). ...
Zebrafish (Danio rerio ), is a well‐established vertebrate animal model widely used in developmental biology and toxicological research. In the present study, foldscope is used as an innovative tool to study the developmental stages and toxicological analysis of the Zebrafish embryos. Briefly, the developmental stages such as zygote, cleavage, blastula, gastrula, segmentation and pharyngula formation are observed and documented using simple foldscope. Toxicological parameters upon exposure to different concentration of ethanol extract of Curcuma longa and its lead compound, ar‐turmerone along with Rhodamine B (bio‐coupler) on Zebrafish embryos are analyzed upto 72 h using foldscopes in live condition. The lethal endpoints such as coagulation, lack of somite formation, non‐detachment of tail, and lack of heartbeat are clearly monitored and documented using foldscope. Bio‐evaluation of test compounds with the aid of foldscope confirms that the toxicity is directly proportional to the concentration. Our results conclude that, ethanol extract of C.longa , ar‐turmerone and Rhodamine B exposed embryos remains healthy up to 96 µg, 48 µg and 24 µg concentrations, respectively. Embryos exposed to higher concentrations become coagulated, however normal physiological active movement of tail lashing and heartbeat are evident in lower concentration exposed embryos. Except coagulation, no other abnormalities are observed and interestingly, the hatching ability is not delayed, when compared to the control embryos. It is confirmed that the test compounds are not highly toxic to Zebrafish embryos. Hence it can be used for further analysis, especially for studying the neural‐regeneration and its neuronal development in Zebrafish embryos.
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... Zebrafish are much easier to maintain in a laboratory and can also be grown in large numbers (Kimmel, 1989). The mode of fertilization is external, and their embryos are optically clear allowing examination of individual genes (fluorescently labeled or dyed; Bernasconi, 2004;Cendes, 2005;Kimmel & Warga 1988;Solnica-Krezel, Stemple, & Driever, 1995;Tran & Gerlai, 2015). The small size of zebrafish larvae allows easy manipulation of gene activities and screening of neuroactive compounds (Kuzniecky & Knowlton, 2002). ...
... Third, the stress granules could be easily monitored in real-time, and the dynamics could be analyzed. Given that zebrafish (Danio Rerio) has become a valuable tool in biological research to visualize physiological changes using live imaging due to its transparent body at embryonic and early larval stages (Cooper et al., 1999a;Cooper et al., 1999b;Kimmel, 1989;Kimmel and Warga, 1988;Solnica-Krezel et al., 1995;Spitsbergen and Kent, 2003), we decided to genetically knock in a GFP tag to the zebrafish G3BP1, which shares 65% protein sequence homology with its human counterpart. With this approach, the expression of GFPtagged G3BP1 will be under the control of endogenous G3BP1 gene promoter, and the stress granule formation will not be affected by G3BP1 overexpression. ...
Although the regulation of stress granules has become an intensely studied topic, current investigations of stress granule assembly, disassembly and dynamics are mainly performed in cultured cells. Here, we report the establishment of a stress granule reporter to facilitate the real-time study of stress granules in vivo. Using CRISPR/Cas9, we fused a green fluorescence protein (GFP) to endogenous G3BP1 in zebrafish. The GFP–G3BP1 reporter faithfully and robustly responded to heat stress in zebrafish embryos and larvae. The induction of stress granules varied by brain regions under the same stress condition, with the midbrain cells showing the highest efficiency and dynamics. Furthermore, pre-conditioning using lower heat stress significantly limited stress granule formation during subsequent higher heat stress. More interestingly, stress granule formation was much more robust in zebrafish embryos than in larvae and coincided with significantly elevated levels of phosphorylated eIF2α and enhanced heat resilience. Therefore, these findings have generated new insights into stress response in zebrafish during early development and demonstrated that the GFP–G3BP1 knock-in zebrafish could be a valuable tool for the investigation of stress granule biology.
This article has an associated First Person interview with the first author of the paper.
... The high fecundity, short generation time and rapid development of the externally fertilized, translucent embryos make it an excellent vertebrate genetic model system (Driever et al., 1994), allowing for cost-effective investigations. Zebrafish embryo development has been studied in detail (Kimmel et al., 1995), from pregastrula and gastrula stages (Solnica-Krezel et al., 1995) and organogenesis through (Schimitt and Dowling, 1994;Stainier and Fishman, 1994), lending a solid base of knowledge for the detection and interpretation of mutant phenotypes. ...
Evaluation of the toxic effects of a widely used synthetic pyrethroid, deltamethrin (DM), was carried out in this study. This pesticide is preferred for pest control because of its low environmental persistence and toxicity. We investigated the expression pattern of four genes, namely, you (you), yot (you-too), momo (mom) and ubo (u-boot) during early development of zebrafish, that is, from 12 hpf to 48 hpf stages. These stages are selected as most of the important developmental aspects take place during this period. All four genes are known to play a vital role in development of notochord and somites. To understand the effect of DM on development, embryos of 4 hpf stage were exposed to two concentrations (100 and 200 µg/L) of DM, and observations were made at 12, 24 and 48 hpf stages. Our earlier studies have shown phenotypic abnormalities such as notochord bending, tail deformation, yolk sac and pericardial edema, lightening of body and eye pigmentation and interfered in somite patterning, during these stages of development. Understanding the relationship of phenotypic abnormalities with these four genes has been our primary objective. These four genes were analyzed by Reverse transcription (RT)-polymerase chain reaction and intensity of the bands has shown induction in their expression after exposure to the toxicant. In spite of the expression of genes, it was noticed that DM caused abnormalities. It can be said from the results that translational pathway could have been affected.
... Zebrafish have relatively high fertility rates, rapid development with short intergenerational time, and a well-annotated genome with ~70% concordance with mammalian species (Postlethwait et al., 2000;Howe et al., 2013). For these reasons, zebrafish have been used extensively in several fields, including environmental health science (Lieschke and Currie, 2007;Perkins et al., 2013;Aguirre-Martínez et al., 2017;Horie et al., 2017), ecotoxicology (Martins et al., 2007;Almond and Trombetta, 2016;de Oliveira et al., 2016), developmental biology (Solnica-Krezel et al., 1995;Lele and Krone, 1996;Elkouby, 2017), and genetics (Kinth et al., 2013;Varshney et al., 2015;White, 2015;Ceol and Houvras, 2016). Kinth et al. (2013) provides a recent review of literature for use of zebrafish as a model organism, including prominent areas of research, researchers, and research facilities. ...
There is a need for fast, efficient, and cost-effective hazard identification and characterization of chemical hazards. This need is generating increased interest in the use of zebrafish embryos as both a screening tool and an alternative to mammalian test methods. A Collaborative Workshop on Aquatic Models and 21st Century Toxicology identified the lack of appropriate and consistent testing protocols as a challenge to the broader application of the zebrafish embryo model. The National Toxicology Program established the Systematic Evaluation of the Application of Zebrafish in Toxicology (SEAZIT) initiative to address the lack of consistent testing guidelines and identify sources of variability for zebrafish-based assays. This report summarizes initial SEAZIT information-gathering efforts. Investigators in academic, government, and industry laboratories that routinely use zebrafish embryos for chemical toxicity testing were asked about their husbandry practices and standard protocols. Information was collected about protocol components including zebrafish strains, feed, system water, disease surveillance, embryo exposure conditions, and endpoints. Literature was reviewed to assess issues raised by the investigators. Interviews revealed substantial variability across design parameters, data collected, and analysis procedures. The presence of the chorion and renewal of exposure medium (static versus static-renewal) were identified as design parameters that could potentially influence study outcomes and should be investigated further with studies to determine chemical uptake from treatment solution into embryos. The information gathered in this effort provides a basis for future SEAZIT activities to promote more consistent practices among researchers using zebrafish embryos for toxicity evaluation.
... • One of the most unique advantages of the zebrafish is the unrivalled optical clarity of the embryos, allowing visualisation of individual genes (fluorescently labelled or dyed) throughout the developmental process using non-invasive imaging techniques [3][4][5][6][7][8] . This transparency of the embryo also helps in genetic manipulations. ...
Zebrafish (Danio rerio) is emerging as an increasingly successful model for translational research on human neurological disorders. In this review, we appraise the high degree of neurological and behavioural resemblance of zebrafish with humans. It is highly validated as a powerful vertebrate model for investigating human neurodegenerative diseases. The neuroanatomic and neurochemical pathways of zebrafish brain exhibit a profound resemblance with the human brain. Physiological, emotional and social behavioural pattern similarities between them have also been well established. Interestingly, zebrafish models have been used successfully to simulate the pathology of Alzheimer’s disease (AD) as well as Tauopathy. Their relatively simple nervous system and the optical transparency of the embryos permit real-time neurological imaging. Here, we further elaborate on the use of recent real-time imaging techniques to obtain vital insights into the neurodegeneration that occurs in AD. Zebrafish is adeptly suitable for Ca2+ imaging, which provides a better understanding of neuronal activity and axonal dystrophy in a non-invasive manner. Three-dimensional imaging in zebrafish is a rapidly evolving technique, which allows the visualisation of the whole organism for an elaborate in vivo functional and neurophysiological analysis in disease condition. Suitability to high-throughput screening and similarity with humans makes zebrafish an excellent model for screening neurospecific compounds. Thus, the zebrafish model can be pivotal in bridging the gap from the bench to the bedside. This fish is becoming an increasingly successful model to understand AD with further scope for investigation in neurodevelopment and neurodegeneration, which promises exciting research opportunities in the future.
... This involves a wide array of migration events, including random walks, directed migrations and collective movements. The fish embryo, being optically clear, offers direct access to these processes [12]. Among the diverse movements taking place during gastrulation, cells of the organizer (termed "shield" in zebrafish) internalize and migrate collectively toward the animal pole of the embryo, forming the prechordal plate [9,13,14]. ...
Being optically clear, the zebrafish embryo is a nice model system to analyze cell migration in vivo. This chapter describes a combination of injection and cell transplant procedures that allows creation of mosaic embryos, containing a few cells labeled differently from their neighbors. Rapid 5D confocal imaging of these embryos permits to simultaneously track and quantify the movement of large cell groups, as well as analyze the cellular or subcellular dynamics of transplanted cells during their migration. In addition, expression of a candidate gene can be modified in transplanted cells. Comparing behavior of these cells to control or neighboring cells allows determination of the role of the candidate gene in cell migration. We describe the procedure, focusing on one specific cell population during gastrulation, but it can easily be adapted to other cell populations and other migration events during early embryogenesis.
... In addition to altering cell fates, the BMP gradient is proposed to link the DV and AP axes by regulating dorsal convergence and extension movements 37 . These movements involve the migration of ventrolateral cells towards the dorsal side of the embryo together with their intercalation, which drives elongation of the body axis 38,39 . The highest level of BMP signalling (encompassing 20°-30°of the ventral margin at the shield stage 37 ) creates a 'no convergence no extension zone' (NCEZ) resulting in the ventralmost cells moving vegetally, but not dorsally, and contributing to the tailbud and other posterior fates. ...
Despite the fundamental importance of patterning along the dorsal-ventral (DV) and anterior-posterior (AP) axes during embryogenesis, uncertainty exists in the orientation of these axes for the mesoderm. Here we examine the origin and formation of the zebrafish kidney, a ventrolateral mesoderm derivative, and show that AP patterning of the non-axial mesoderm occurs across the classic gastrula stage DV axis while DV patterning aligns along the animal-vegetal pole. We find that BMP signalling acts early to establish broad anterior and posterior territories in the non-axial mesoderm while retinoic acid (RA) functions later, but also across the classic DV axis. Our data support a model in which RA on the dorsal side of the embryo induces anterior kidney fates while posterior kidney progenitors are protected ventrally by the RA-catabolizing enzyme Cyp26a1. This work clarifies our understanding of vertebrate axis orientation and establishes a new paradigm for how the kidney and other mesodermal derivatives arise during embryogenesis.
... Prospective prechordal plate (also known as anterior mesendoderm) is a group of cells forming at the onset of gastrulation on the dorsal side of the embryo. During gastrulation this group collectively migrates towards the animal pole of the embryo [6][7][8] , to form the prechordal plate, a mesendodermal thickening, anterior to the notochord, and underlying the neural plate. The anterior part of the prechordal plate will give rise to the hatching gland, while its posterior part likely contributes to head mesoderm 9 . ...
Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.
... To study the Afp18 virulence effector of Y. ruckeri, we employed the zebrafish embryo as a model, in which cell biological processes during vertebrate development can be visualized at high spatial and temporal resolution. The early cleavage, blastula and gastrula stages of zebrafish development are an excellent model to study individual and collective cellular behaviours 25 . Analyses of infection of zebrafish with Y. ruckeri and other pathogens have provided insights into disease mechanism 6,26 as well as function of the innate immune system 27,28 . ...
Yersinia species cause zoonotic infections, including enterocolitis and plague. Here we studied Yersinia ruckeri antifeeding prophage 18 (Afp18), the toxin component of the phage tail-derived protein translocation system Afp, which causes enteric redmouth disease in salmonid fish species. Here we show that microinjection of the glycosyltransferase domain Afp18(G) into zebrafish embryos blocks cytokinesis, actin-dependent motility and cell blebbing, eventually abrogating gastrulation. In zebrafish ZF4 cells, Afp18(G) depolymerizes actin stress fibres by mono-O-GlcNAcylation of RhoA at tyrosine-34; thereby Afp18(G) inhibits RhoA activation by guanine nucleotide exchange factors, and blocks RhoA, but not Rac and Cdc42 downstream signalling. The crystal structure of tyrosine-GlcNAcylated RhoA reveals an open conformation of the effector loop distinct from recently described structures of GDP- or GTP-bound RhoA. Unravelling of the molecular mechanism of the toxin component Afp18 as glycosyltransferase opens new perspectives in studies of phage tail-derived protein translocation systems, which are preserved from archaea to human pathogenic prokaryotes.
... Prechordal plate is composed of a group of cells that, during gastrulation, leads the forming embryonic axis. The prechordal plate migrates from the organiser (the node or shield in fish) to the animal pole, and later gives rise to the hatching gland [25][26][27][28]. Our analysis reveals that Sin1 controls both cell speed and persistence, and is essential for emission of actin-rich cell protrusions. ...
TORC2 is a serine-threonine kinase complex conserved through evolution that recently emerged as a new regulator of actin dynamics and cell migration. However, knockout in mice of its core components Sin1 and Rictor is embryonic lethal, which has limited in vivo analyses. Here, we analysed TORC2 function during early zebrafish development, using a morpholino-mediated loss of function of sin1. Sin1 appears required during gastrulation for migration of the prechordal plate, the anterior most mesoderm. In absence of Sin1, cells migrate both slower and less persistently, which can be correlated to a reduction in actin-rich protrusions and a randomisation of the remaining protrusions. These results demonstrate that, as established in vitro, the TORC2 component Sin1 controls actin dynamics and cell migration in vivo. We furthermore establish that Sin1 is required for protrusion formation downstream of PI3K, and is acting upstream of the GTPase Rac1, since expression of an activated form of Rac1 is sufficient to rescue sin1 loss of function.
... Whether marginal deep cells enter the hypoblast through involution or ingression has been debated (Warga and Kimmel, 1990;Shih and Fraser, 1995;Solnica-Krezel et al., 1995). Do marginal cells enter the hypoblast mostly at the margin in an involution-type behavior or are they entering it up to tens of cell diameters away in an ingression mechanism? ...
The yolk syncytial layer (YSL) of the teleostean yolk cell is known to play important roles in the induction of cellular mesendoderm, as well as the patterning of dorsal tissues. To determine how this extraembryonic endodermal compartment is subdivided and morphologically transformed during early development, we have examined collective movements of vitally stained YSL nuclei in axiating zebrafish embryos by using four-dimensional confocal microscopy. During blastulation, gastrulation, and early segmentation, zebrafish YSL nuclei display several highly patterned movements, which are organized into spatially distinct morphogenetic domains along the anterior-posterior and dorsal-ventral axes. During the late blastula period, with the onset of epiboly, nuclei throughout the YSL initiate longitudinal movements that are directed along the animal-vegetal axis. As epiboly progresses, nuclei progressively recede from the advancing margin of the epibolic YSL. However, a small group of nuclei is retained at the YSL margin to form a constricting blastoporal ring. During mid-gastrulation, YSL nuclei undergo convergent-extension behavior toward the dorsal midline, with a subset of nuclei forming an axial domain that underlies the notochord. These highly patterned movements of YSL nuclei share remarkable similarities to the morphogenetic movements of deep cells in the overlying zebrafish blastoderm. The macroscopic shape changes of the zebrafish yolk cell, as well as the morphogenetic movements of its YSL nuclei, are homologous to several morphogenetic behaviors that are regionally expressed within the vegetal endodermal cell mass of gastrulating Xenopus embryos. In contrast to the cellular endoderm of Xenopus, the dynamics of zebrafish YSL show that a syncytial endodermal germ layer can express a temporal sequence of morphogenetic domains without undergoing progressive steps of cell fate restriction. © 2001 Wiley-Liss, Inc.
... D uring vertebrate gastrulation, the embryonic body plan is established by coordinated movements of large populations of cells to generate the ectodermal, mesodermal, and endodermal layers (22,49,66). A key driving force of gastrulation is convergence and extension (CE) movements. ...
During vertebrate gastrulation, both concurrent inductive events and cell movements are required for axis formation. Convergence
and extension (CE) movements contribute to narrowing and lengthening the forming embryonic axis. MicroRNAs (miRNAs) play a
critical role in regulating fundamental cellular functions and developmental processes, but their functions in CE movements
are not well known. Zebrafish mir206 is maternally expressed and present throughout blastulation and gastrulation periods. Either gain or loss of function of
mir206 leads to severe defects of convergent extension movements both cell autonomously and non-cell autonomously. Mosaic lineage
tracing studies reveal that the formation of membrane protrusions and actin filaments is disturbed in mir206-overexpressing embryos or mir206 morphants. Mechanistically, mir206 targets prickle1a (pk1a) mRNA and as a result regulates c-Jun N-terminal protein kinase 2 (JNK2) phosphorylation. pk1a overexpression or knockdown can rescue convergent extension defects induced by mir206 overexpression or knockdown, respectively. Therefore, mir206 is an essential, novel regulator for normal convergent and extension movements by regulating mitogen-activated protein kinase
(MAPK) JNK signaling.
... For instance, we have purposely omitted most recent investigations related to the study of embryogenesis. Instead, we refer interested readers to excellent reviews by Driever et al. (1994), Kimmel et al. (1995 , Krezel et al. (1995) , to the references therein , and to the extensive compilation of literature in The Zebrafish Book (Westerfield 1995) and on The Fish Net at http://zfish.uoregon.edu/. ...
The zebrafish has long been the favorite organism in many scientific disciplines. Although its attributes as a model were expounded for many years and thus were no secret, the zebrafish sat in the wings while other more popular vertebrates such as chick, amphibians, and mouse were examined at length. We cannot say there was a resurgence in popularity, but more an explosion of research utilizing the zebrafish beginning in the late 1970s when investigators at the University of Oregon began using it as their model in neuroscience. Prior to this reawakening, the zebrafish was one of the significant organisms in the study of teratology and toxicology, development, and, to some extent, behavior. Recently, however, the field of zebrafish genetics has gained immense popularity and success, in part owing to the fact that zebrafish are diploid and are amenable to genetic manipulations. Here we present an overview of the multidisciplinary research that has laid some of the foundation of our present understanding of the biochemical, cell biological, and molecular genetic events accompanying zebrafish development.
... This study showed transient high levels of apoptosis in various cells and tissues during specific phases of development (16,18). Because the zebrafish is very well suited for live fluorescence imaging due to the optical transparency during early development (19), a transgenic fluorescent marker allowing in vivo imaging of apoptotic cells would greatly benefit research into the in vivo dynamics of apoptotic cell death. ...
Many debilitating diseases, including neurodegenerative diseases, involve apoptosis. Several methods have been developed for visualizing apoptotic cells in vitro or in fixed tissues, but few tools are available for visualizing apoptotic cells in live animals. Here we describe a genetically encoded fluorescent reporter protein that labels apoptotic cells in live zebrafish embryos. During apoptosis, the phospholipid phosphatidylserine (PS) is exposed on the outer leaflet of the plasma membrane. The calcium-dependent protein Annexin V (A5) binds PS with high affinity, and biochemically purified, fluorescently labeled A5 probes have been widely used to detect apoptosis in vitro. Here we show that secreted A5 fused to yellow fluorescent protein specifically labels apoptotic cells in living zebrafish. We use this fluorescent probe to characterize patterns of apoptosis in living zebrafish larvae and to visualize neuronal cell death at single-cell resolution in vivo.
... The zebrafish is an increasingly popular model organism used to understand early vertebrate developmental processes and model diseases [10][11][12]. The amenability to forward genetics and the ability to produce large numbers of translucent embryos that rapidly develop externally has enabled the genetic dissection of most vertebrate organ systems [13,14]. While significant advances have been made in genetic methods [15,16], the paucity of high quality antibodies is considered a significant limitation for zebrafish research [17]. ...
Monoclonal antibodies with high affinity and selectivity that work on wholemount fixed tissues are valuable reagents to the cell and developmental biologist, and yet isolating them remains a long and unpredictable process. Here we report a rapid and scalable method to select and express recombinant mouse monoclonal antibodies that are essentially equivalent to those secreted by parental IgG-isotype hybridomas.
Increased throughput was achieved by immunizing mice with pools of antigens and cloning - from small numbers of hybridoma cells - the functionally rearranged light and heavy chains into a single expression plasmid. By immunizing with the ectodomains of zebrafish cell surface receptor proteins expressed in mammalian cells and screening for formalin-resistant epitopes, we selected antibodies that gave expected staining patterns on wholemount fixed zebrafish embryos.
This method can be used to quickly select several high quality monoclonal antibodies from a single immunized mouse and facilitates their distribution using plasmids.
... These marginal cells undergo but limited mixing and therefore keep their position near the margin of the blastoderm (Wilson et a/.. 1993;Heide et al., 1994) and then occupy the germ ring. Cells in the germ ring involute or ingress during gastrulation (reviewed by Solnica-Krezel et al., 1995) and become the hypoblast layer. Though inducing signals are provided to the blastomeres before the onset of epiboly and gastrulation (Bozhkova et al" 1994;Mizuno et a/., 1996; they are not determined until midgastrulation (Ho, 1992;Ho ----and Kimmel, 1993). ...
The insulin-like growth factor (IGF) family is essential for normal embryonic growth and development and it is highly conserved through vertebrate evolution. However, the roles that the individual members of the IGF family play in embryonic development have not been fully elucidated. This study focuses on the role of IGF-2 in zebrafish embryonic development. Two igf-2 genes, igf-2a and igf-2b, are present in the zebrafish genome. Antisense morpholinos were designed to knock down both igf-2 genes. The neural and cardiovascular defects in IGF-2 morphant embryos were then examined further using wholemount in situ hybridisation, TUNEL analysis and O-dianisidine staining. Knockdown of igf-2a or igf-2b resulted in ventralised embryos with reduced growth, reduced eyes, disrupted brain structures and a disrupted cardiovascular system, with igf-2b playing a more significant role in development. During gastrulation, igf-2a and igf-2b are required for development of anterior neural structures and for regulation of genes critical to dorsal-ventral patterning. As development proceeds, igf-2a and igf-2b play anti-apoptotic roles. Gene expression analysis demonstrates that igf-2a and igf-2b play overlapping roles in angiogenesis and cardiac outflow tract development. Igf-2b is specifically required for cardiac valve development and cardiac looping. Injection of a dominant negative IGF-1 receptor led to similar defects in angiogenesis and cardiac valve development, indicating IGF-2 signals through this receptor to regulate cardiovascular development. This is the first study describing two functional igf-2 genes in zebrafish. This work demonstrates that igf-2a and igf-2b are critical to neural and cardiovascular development in zebrafish embryos. The finding that igf-2a and igf-2b do not act exclusively in a redundant manner may explain why both genes have been stably maintained in the genome.
... To prevent this rolling from occurring, fluorescently lathe distal edge of the blastoderm. By 60%-epiboly, deep beled embryos were imbedded in ERM gelled with 2% ultra-low- NEM cells contribute to a domain of cells known as the melting agarose (Type IX) (Sigma, St. Louis, MO) using the proce''forerunner'' cells (Solnica-Krezel et al., 1995 By loading fluorescent dextrans into the cortical cytoplasm of the yolk cell in mid-blastula-stage embryos, we have found ...
In zebrafish embryos, the nascent embryonic shield first appears as a thickening in the germ ring of the mid-epiboly blastoderm. This site defines the dorsal side of the developing embryo. In this paper, we report that the site of embryonic axis formation is marked earlier at the late-blastula stage by the appearance of a cluster of cells with unique endocytic activities. This cluster of cells is composed of enveloping layer epithelial cells and one to two layers of underlying deep cells. Unlike other marginal blastomeres, cells in this cluster do not participate in involution as the blastoderm undergoes epiboly. These noninvoluting endocytic marginal (NEM) cells can be selectively labeled by applying membrane impermeant fluorescent probes to pre-epiboly and mid-epiboly embryos. During embryonic shield formation, deep cells in the NEM cell cluster rearrange and are displaced forward to the leading edge of the blastoderm. As deep NEM cells move into this location, they become a group of cells known as "forerunner cells." Between 60%- and 80%-epiboly, the forerunner cells coalesce into a coherent cell cluster that forms a wedge-shaped cap at the leading edge of the blastoderm. During embryonic axis formation, deep cells migrate and converge toward the embryonic midline, which is defined by the center of the forerunner cell cluster. At approximately 90% epiboly, the forerunner cell cluster becomes overlapped by the constricting germ ring. At tailbud stage, forerunner cells form the dorsal roof of Kupffer's vesicle, which is located ventral to the nascent chordoneural hinge. On the basis of previous grafting studies and known dorsal gene expression patterns, we discuss possible roles that the NEM/forerunner cell cluster may play in teleost axis formation.
Zebrafish (Danio rerio) is currently in vogue as a prevalently used experimental model for studies concerning neurobehavioural disorders and associated fields. Since the 1960s, this model has succeeded in breaking most barriers faced in the hunt for an experimental model. From its appearance to its high parity with human beings genetically, this model renders itself as an advantageous experimental lab animal. Neurobehavioural disorders have always posed an arduous task in terms of their detection as well as in determining their exact etiology. They are still, in most cases, diseases of interest for inventing or discovering novel pharmacological interventions. Thus, the need for a harbinger experimental model for studying neurobehaviours is escalating. Ensuring the same model is used for studying several neuro-studies conserves the results from inter-species variations. For this, we need a model that satisfies all the pre-requisite conditions to be made the final choice of model for neurobehavioural studies. This review recapitulates the progress of zebrafish as an experimental model with its most up-to-the-minute advances in the area. Various tests, assays, and responses employed using zebrafish in screening neuroactive drugs have been tabulated effectively. The tools, techniques, protocols, and apparatuses that bolster zebrafish studies are discussed. The probable research that can be done using zebrafish has also been briefly outlined. The various breeding and maintenance methods employed, along with the information on various strains available and most commonly used, are also elaborated upon, supplementing Zebrafish's use in neuroscience.
To investigate the role of mechanical constraints in morphogenesis and development, we develop a pipeline of techniques based on incompressible elastic sensors. These techniques combine the advantages of incompressible liquid droplets, which have been used as precise in situ shear stress sensors, and of elastic compressible beads, which are easier to tune and to use. Droplets of a polydimethylsiloxane (PDMS) mix, made fluorescent through specific covalent binding to a rhodamin dye, are produced by a microfluidics device. The elastomer rigidity after polymerization is adjusted to the tissue rigidity. Its mechanical properties are carefully calibrated in situ, for a sensor embedded in a cell aggregate submitted to uniaxial compression. Thelocal shear stress tensor is retrieved from the sensor shape, accurately reconstructed through an active contour method. In vitro, within cell aggregates, and in vivo, in the prechordal plate of the Zebrafish embryo during gastrulation,our pipeline of techniques demonstrates its efficiency to directly measure the three dimensional shear stress repartition within a tissue.
There is a need for fast, efficient, and cost-effective hazard identification and characterization of chemical hazards. This need is generating increased interest in the use of zebrafish embryos as both a screening tool and an alternative to mammalian test methods. A Collaborative Workshop on Aquatic Models and 21st Century Toxicology identified the lack of appropriate and consistent testing protocols as a challenge to the broader application of the zebrafish embryo model. The National Toxicology Program established the Systematic Evaluation of the Application of Zebrafish in Toxicology (SEAZIT) initiative to address the lack of consistent testing guidelines and identify sources of variability for zebrafish-based assays. This report summarizes initial SEAZIT information-gathering efforts. Investigators in academic, government, and industry laboratories that routinely use zebrafish embryos for chemical toxicity testing were asked about their husbandry practices and standard protocols. Information was collected about protocol components including zebrafish strains, feed, system water, disease surveillance, embryo exposure conditions, and endpoints. Literature was reviewed to assess issues raised by the investigators. Interviews revealed substantial variability across design parameters, data collected, and analysis procedures. The presence of the chorion and renewal of exposure media (static versus static-renewal) were identified as design parameters that could potentially influence study outcomes and should be investigated further with studies to determine chemical uptake from treatment solution into embryos. The information gathered in this effort provides a basis for future SEAZIT activities to promote more consistent practices among researchers using zebrafish embryos for toxicity evaluation.
Zebrafish develop in a manner typical of teleosts. In freshly laid eggs, yolk and cytoplasm are intermixed, and the egg is surrounded by a transparent chorion, which swells and lifts away from the egg on contact with water. The animal–vegetal axis is already preset during oogenesis, and sperm can enter the egg only at the future animal pole through the micropyle, a specialization in the otherwise sperm-impermeable chorion. After fertilization, cytoplasm streams to the animal pole as it segregates from the yolk. About 30 minutes after fertilization, the cytoplasm forms the blastodisc at the animal pole, and surrounds the vegetal yolk mass as a thin yolk cytoplasmic layer. As in amphibians, zebrafish embryonic cells are readily marked by cell lineage tracers. At the onset of gastrulation, analysis of cellular fates using this technique reveals that the endoderm will derive from the vegetalmost marginal blastomeres. Mesoderm forms from the vegetal third of the blastoderm, whereas ectoderm originates from the animal half of the blastoderm. Neuroectoderm in particular derives from the dorsal section of the animal half. Notochord derives from the dorsal side, where the shield forms, whereas somitic mesoderm, heart and blood, develop from lateral and ventral positions, respectively. The organization of the zebrafish fate map, therefore, is similar to the one of Xenopus.
The signal transducer and activator of transcription (STAT) molecules mediate biological actions, such as cell proliferation, differentiation, and survival, in various biological processes in response to cytokines (1–3). In skin-wound healing, Stat3 is required for the migration but not the proliferation of keratinocytes (4), showing that Stat3 plays a crucial role in cell migration. Stat3 knockout (stat3-/-) mice die before 8.5 days postcoitum (5), indicating that Stat3 plays a role in the early development of the mouse embryo. During zebrafish gastrulation, Stat3 is activated in the dorsal organizer, and its activity is essential for convergence and extension movements, but is not required for early cell-fate specification. These requirements are cell-autonomous for the anterior migration of dorsal organizer cells, and non-cell-autonomous for the convergence of neighboring cells (6). Morphogenetic functions for STAT signaling pathways have also been described for Drosophila and Dictyostelium. The Drosophila JAK/STAT pathway functions in border cell migration during oogenesis (7), and the establishment of planar polarity during eye development (8). Dictyostelium STAT is required for normal chemotaxis during early development, and for the correct movement of prestalk cells during terminal differentiation (9). These observations raised the possibility that the role of STAT signaling in morphogenetic movements is conserved throughout evolution. In this review, we discuss what is known about STAT signaling requirements in morphogenetic movement processes, in particular, the role of STATs in the cell-autonomous epithelial-mesenchymal transition and the non-cellautonomous establishment of planar cell polarity.
Nodal, a member of the transforming growth factor β (TGF-β) superfamily, has been shown to play a role in mesendoderm induction and gastrulation movements. The activity of Nodal signaling can be modulated by microRNAs (miRNAs) as previously reported, but little is known about which miRNAs are regulated by Nodal during gastrulation. In the present study, we found that the expression of mir206, one of the most abundant miRNAs during zebrafish early embryo development, is regulated by Nodal signaling. Abrogation of Nodal signal activity results in defective convergence and extension (CE) movements, and these cell migration defects can be rescued by supplying an excess of mir206, suggesting that mir206 acts downstream of Nodal signaling to regulate CE movements. Furthermore, in mir206 morphants, the expression of cell adhesion molecule E-cadherin is significantly increased, while the key transcriptional repressor of E-cadherin, snail1a, is depressed. Our study uncovers a novel mechanism by which Nodal-regulated mir206 modulates gastrulation movements in connection with the Snail/E-cadherin pathway.
This chapter reviews the dynamic changes in the organization and function of the cytoskeletal components during early zebrafish embryogenesis. It focuses on actin microfilaments in egg and zygote and microtubules during cleavage stages and gastrulation. Gastrulation involves a set of stereotyped cellular rearrangements including epiboly, involution/ingression, and convergent extension. These concurrent morphogenetic processes create the three-germ-layer organization of the embryo, place tissues, and organ rudiments in the proper position for further inductive interactions and development, and are the major forces underlying the change in the embryonic shape. Although the entire spectrum of morphogenetic cellular behaviors involved in vertebrate embryogenesis is still not known, directed cell migration, cell shape changes, cell intercalation, and oriented cell divisions are important components. In other systems, both intracellular morphogenetic processes and cellular morphogenetic behaviors employ cytoskeletal systems of microfilaments, microtubules, and intermediate filament. The chapter also reviews the first experiments analyzing cytoskeletal dynamics within cells of living zebrafish embryos. It surveys available methods for staining actin filaments and microtubules in fixed material, and provides an overview of drug and physical treatments interfering with different cytoskeletal functions during embryogenesis.
During development, vertebrate embryos undergo dramatic changes in shape. The lengthening and narrowing of a field of cells, termed convergent extension, contributes to a variety of morphogenetic processes. Focusing on frogs and fish, we review the different cellular mechanisms and the well-conserved signaling pathways that underlie this process.
Zebrafish (Danio rerio) has been extensively studied and well described for environmental toxicity studies. Molecular biology and genetics have recently been used to elucidate the underlying mechanisms of toxicity in zebrafish and to predict effects in mammals. The versatile zebrafish is now incorporated in many areas of toxicological programs for assessing human risk and for preclinical drug discovery and screening.
Collective cell migration is key to morphogenesis, wound healing, or cancer cell migration. However, its cellular bases are just starting to be unraveled. During vertebrate gastrulation, axial mesendo-derm migrates in a group, the prechordal plate, from the embry-onic organizer to the animal pole. How this collective migration is achieved remains unclear. Previous work has suggested that cells migrate as individuals, with collective movement resulting from the addition of similar individual cell behavior. Through extensive analyses of cell trajectories, morphologies, and polarization in zebrafish embryos, we reveal that all prechordal plate cells show the same behavior and rely on the same signaling pathway to migrate, as expected if they do so individually. However, by using cell transplants, we demonstrate that prechordal plate migration is a true collective process, as isolated cells do not migrate toward the animal pole. They are still polarized and motile but lose directionality. Directionality is restored upon contact with the endogenous prechordal plate. This contact dependent orientation relies on E-cadherin, Wnt-PCP signaling, and Rac1. Importantly, groups of cells also need contact with the endogenous plate to orient correctly, showing an instructive role of the plate in establishing directionality. Overall, our results lead to an original model of collective migration in which directional information is contained within the moving group rather than provided by extrinsic cues, and constantly maintained in cells by contacts with their neighbors. This self-organizing model could account for collective invasion of new territories, as observed in cancer strands, without requirement for any attractant in the colonized tissue. embryogenesis | cell movement | 3D tracking
IntroductionHistory:Classic Experiments in the Study of GastrulationGastrulation and Neurulation in Different Vertebrate SpeciesMechanistic Aspects, Molecules and Molecular NetworksConclusion and Outlook
Zebrafish are simple, rapidly-developing animals that are amenable to detailed developmental and genetic analyses. Despite their relatively simple morphology, they share many developmental features with other vertebrates. By combining developmental studies in zebrafish with those in other animals, we will begin to understand the complex events that occur as an embryo develops from a one-celled zygote to a complex, multicellular organism.
The carp cth1 gene, related to the mammalian TIS11 family of primary response genes, encodes a novel fish protein with two putative CCCH zinc fingers. This report describes the RNA expression of this gene during cleavage, blastula and gastrula stages of carp embryos. Cth1 mRNA is present in all cleavage stage blastomeres as a maternal message. After the late blastula stage, the maternal expression decreases, revealing a spot of higher expression at the margin of the blastoderm of the dome stage embryo. Further decrease of the maternal message reveals a ring of cth1 expressing cells at the blastoderm margin from the stage of 40% epiboly onwards. By alpha-amanitin treatment we established that this local cth1 expression is of zygotic origin. At the onset of gastrulation the cells of the cth1 ring involute, starting with those in the shield region, and at approximately 60% epiboly the ring is fully involuted and occupies the hypoblast layer. All cth1 transcripts have disappeared at completion of epiboly. We discuss a possible role for the putative cth1 protein during cleavage and gastrulation.
Recent advances in our understanding of axis formation and patterning in zebrafish relate the developmental mode of this aspiring genetic model organism to higher vertebrates. The effect of UV irradiation and lithium treatment, as well as detailed early lineage analyses, have shed some light on dorsoventral axis formation. However, the molecular mechanism of axis formation, as well as the identity of a fish Nieuwkoop center, are still open issues. A Vg1 homolog is expressed in zebrafish, and activin as well as the mouse nodal gene product have been demonstrated to induce mesoderm and ectopic axes, respectively, in zebrafish. The zebrafish organizer is defined by the expression domains of goosecoid, axial, and lim1. The cyclops gene is involved in maintaining goosecoid expression in axial mesoderm of the head. Large mutagenesis screens provide the basis for a genetic analysis of axis formation.
The Drosophila homeobox gene tinman and its vertebrate homologs Nkx-2.5 and Nkx-2.3 are critical determinants of cardiac development. We report here the identification of a new tinman-related gene, nkx2.7, as well as orthologs of Nkx-2.5 and Nkx-2.3 in the zebrafish. Analysis of their expression in the developing zebrafish embryo reveals that nkx2.7 transcripts are the first to appear in cardiac mesodermal and pharyngeal endodermal precursors of the anterior hypoblast, anticipating both temporally and spatially the later expression of nkx2.5 and nkx2.3 in these lineages. The preeminence of nkx2.7 in these embryonic lineages is consistent with a key role in cell fate determination, perhaps in part through the induction of nkx2.5 and nkx2.3. The findings provide the first molecular clues as to the spatial organization of endodermal and cardiac mesodermal precursors in the zebrafish hypoblast immediately following gastrulation. They suggest a coordinate role for these three tinman-related genes in the development of the heart and pharyngeal arches, and reinforce the paradigm of gene duplication and subspecialization between Drosophila and vertebrate species. The results provide a framework in which to analyze potential changes in tinman-related gene expression during abnormal zebrafish development.
Early morphogenesis of the teleost embryo is characterized by three orchestrated cell movements. Epiboly leads to spreading of the blastoderm over an uncleaved yolk cell while involution around the blastoderm margin and convergence movements towards the dorsal side generate the mesendodermal inner cell sheet and the axis rudiment, respectively. Irradiation of zebrafish zygotes with ultraviolet light selectively impairs epiboly resulting in embryos with open blastopores but well-formed anterior axes. Gastrulation movements are only marginally affected by ultraviolet irradiation. Involution of marginal cells in epiboly-retarded embryos takes place prior to 50% epiboly and thus appears independent of epiboly. Expression of dorsal and anterior marker genes is unaffected by ultraviolet irradiation.
The ultraviolet light effect is not restricted to the zygote stage as irradiation of later embryonic stages also impairs epiboly. The ultraviolet-sensitive targets may thus be maternally encoded components of the machinery driving epiboly. These targets appear to be microtubules: firstly, irradiated embryos show disorganized and less microtubules in the cytoplasmic layer of the yolk sphere; secondly, the ultraviolet light effect can be mimicked by the microtubule-depolymerising agent nocodazole. We suggest that epiboly is driven, at least partially, by motors that use microtubules radiating from the yolk syncytial layer into the yolk cytoplasmic layer. Together with an observed constrictive behaviour of the blastoderm margin, we propose a two-force model of epiboly: epiboly is initiated and driven by a pulling force dependent on microtubules in the yolk cytoplasmic layer; contraction at the margin operates in addition to aid closure of the blastopore.
In this article we review the extensive, often conflicting literature on teleost gastrulation and attempt to summarize the current view and place it in a phylogenetic context. Teleosts have evolved a unique pattern of gastrulation that correlates with the presence of meroblastic cleavage. Meroblastic cleavage, on the basis of its phylogenetic distribution, appears to have evolved five times in craniates and is unique in teleosts in not being associated with an increase in egg size. Two developmental transformations, occurring at different points along the actinopterygian clade, may account for the origin of the teleost mode of gastrulation: loss of the bottle cells that appear at the beginning of gastrulation as has occurred between the Ginglymodi (Lepisosteus) and Halecostomi (Amia and Teleostei), and change in the structure of the yolk, resulting in the formation of a continuous mass from the ancestral state of a yolk segregated into platelets within individual cells as has occurred between Amia and Teleostei. These early developmental changes are explored from functionalist and structuralist perspectives for possible explanations of the evolutionary patterns seen. Functionalist explanations include the osmotic advantages offered by the telolecithal egg and the impermeable enveloping layer. Structuralist explanations encompass historical factors such as phylogenetic and developmental constraints.
The processes thought to function in Xenopus gastrulation include bottle cell formation, migration of cells on the roof of the blastocoel, and autonomous convergent extension of the circumblastoporal region. A review of recent and classical results shows that only the last accounts for the bulk of the tissue displacement of gastrulation, including spreading of the marginal zone toward the blastopore, involution of the marginal zone, and closure of the blastopore. Microsurgical manipulation and explantation studies, analysed by time-lapse video and cine microscopy, shows that the dorsal circumblastoporal region contains two regions which show either autonomous or semiautonomous convergent extension. The dorsal involuting marginal zone (IMZ) undergoes convergence (narrowing) and extension (lengthening) after its involution, beginning at the midgastrula stage and continuing through neurulation, such that it simultaneously extends posteriorly across the yolk plug and narrows the blastoporal circumference. Concurrently, the corresponding region of the overlying non-involuting marginal zone (NIMZ) begins a complementary convergent extension, but at a greater rate, which spreads vegetally to occupy surface area vacated by the IMZ. Tissue recombination experiments show that the deep cells of the dorsal IMZ bring about convergent extension. Labelling of small populations of these cells with a cell lineage tracer shows that convergent extension involves intercalation of deep cells to form a longer, narrower array. Direct time-lapse video and cine micrography of deep cells in cultured explants show that convergent extension involves radial and circumferential intercalation. Removal of the entire blastocoel roof of the early gastrula, including all or part of the NIMZ, shows that convergent extension of the IMZ alone can bring about its involution and blastopore closure. The role of convergent extension in gastrulation of other amphibians and other metazoans and its significance to related problems in early development are discussed.
The structure of the junctional complex between surface cells was investigated in blastula, mid gastrula, late gastrula, and early embryo of the teleost fish Fundulus heteroclitus. In blastulae, the intercellular complex is simple and consists of an apical region where the adjacent membranes are closely apposed (40-60 A) and in places touch, an intermediate zone with a wider intercellular space (> 100 A), and incipient desmosomes. In gastrulae, there are frequent points of fusion of membranes along the apical zone of the complex. Dilatations and an increased number of desmosomes in different stages of development are found along the intermediate zone. In mid gastrula, a close or gap junction with an intercellular space of 20 A occurs below the level of the desmosomes. In late gastrula, the gap junction is reduced in extent and desmosomes are better developed. In the early embryo, the basic organization of the complex is the same, although the deeply situated close junctions are no longer apparent and desmosomes and their associated system of filaments are well developed. At this time, the junctional complex is comparable to that of many epithelia and consists of an apical zonula occludens, a short zonula adherens, and deeply situated maculae adherentes.
The availability of molecular markers now permits the analysis of the common elements of vertebrate gastrulation. While gastrulation appears to be very diverse in the vertebrates, by analyzing a head-organizer marker, goosecoid, and a marker common to all forming mesoderm, Brachyury, we attempt to identify homologous structures and equivalent stages in Xenopus, zebrafish, chick and mouse gastrulation. Using a tail-organizer marker, Xnot-2, we also discuss how the late stages of gastrulation lead to the formation of the postanal tail, a structure characteristic of the chordates.
We have used carbocyanine dyes (DiI and DiO) to generate fate maps for the epiblast layer of the chick embryo between stage X and the early primitive streak stage (stages 2 –3).
The overall distribution of presumptive cell types in these maps is similar to that described for other laboratory species (zebrafish, frog, mouse). Our maps also reveal certain patterns of movement for these presumptive areas. Most areas converge towards the midline and then move anteriorly along it. Interestingly, however, some presumptive tissue types do not take part in these predominant movements, but behave in a different way, even if enclosed within an area that does undergo medial convergence and anterior movement. The apparently independent behaviour of certain cell populations suggests that at least some presumptive cell types within the epiblast are already specified at preprimitive streak stages.
We present a fate map for the 16-cell-stage blastomeres of the zebrafish embryo Brachydanio rerio. We injected high molecular weight fluorescent dextran into cleavage-stage cells to observe the contributions of the descendants of the first 16 cells to the adult. The patterns derived from these early cells are similar, but not identical among different embryos. Furthermore, two-color injections showed that sister blastomeres at the 16-cell stage regularly contribute to different sets of adult structures. A few of the scored tissues could not be mapped in a manner consistent with the predicted axes. Other tissues were mapped to several of the 16 blastomeres. Many of the tissues map with high probability to a few 16-cell-stage blastomeres. Thus, based on 112 injections in 56 different embryos, we have constructed a fate map by assigning probabilities for the contribution of each blastomere to each of 31 tissues in the 26-hour embryo.
In zebrafish (Danio rerio), meroblastic cleavages generate an embryo in which blastomeres cover the animal pole of a large yolk cell. At the 500-1000 cell stage, the marginal blastomeres fuse with the yolk cell forming the yolk syncytial layer. During epiboly the blastoderm and the yolk syncytial layer spread toward the vegetal pole. We have studied developmental changes in organization and function during epiboly of two distinct microtubule arrays located in the cortical cytoplasm of the yolk cell. In the anuclear yolk cytoplasmic layer, an array of microtubules extends along the animal-vegetal axis to the vegetal pole. In the early blastula the yolk cytoplasmic layer microtubules appear to originate from the marginal blastomeres. Once formed, the yolk syncytial layer exhibits its own network of intercrossing mitotic or interphase microtubules. The microtubules of the yolk cytoplasmic layer emanate from the microtubule network of the syncytial layer.
At the onset of epiboly, the external yolk syncytial layer narrows, the syncytial nuclei become tightly packed and the network of intercrossing microtubules surrounding them becomes denser. Soon after, there is a vegetal expansion of the blastoderm and of the yolk syncytial layer with its network of intercrossing microtubules. Concomitantly, the yolk cytoplasmic layer diminishes and its set of animal-vegetal microtubules becomes shorter.
We investigated the involvement of microtubules in epiboly using the microtubule depolymerizing agent nocodazole and a stabilizing agent taxol. In embryos treated with nocodazole, microtubules were absent and epibolic movements of the yolk syncytial nuclei were blocked. In contrast, the vegetal expansion of the enveloping layer and deep cells was only partially inhibited. The process of endo-cytosis, proposed to play a major role in epiboly of the yolk syncytial layer (Betchaku, T. and Trinkaus, J. P. (1986) Am. Zool. 26, 193-199), was still observed in nocodazole-treated embryos. Treatment of embryos with taxol led to a delay in all epibolic movements.
We propose that the yolk cell microtubules contribute either directly or indirectly to all epibolic movements. However, the epibolic movements of the yolk syncytial layer nuclei and of the blastoderm are not coupled, and only movements of the yolk syncytial nuclei are absolutely dependent on microtubules. We hypothesize that the microtubule network of the syncytial layer and the animal-vegetal set of the yolk cytoplasmic layer contribute differently to various aspects of epiboly. Models that address the mechanisms by which the two microtubule arrays might function during epiboly are discussed.
The disposition of prospective areas and the course of morphogenetic movements during gastrulation and neurulation were investigated by vital staining. The prospective lining of the archenteron, the prospective neural area, and the prospective epidermal area are represented on the surface of the early gastrula. The prospective lining of the archenteron occupies the area within 65–70° of the vegetal pole and is divided into prospective archenteron roof and prospective archenteron floor by the blastopore pigment line which functions as the locus of invagination. A crescent-shaped neural area lies immediately above the prospective archenteron roof, rising from it at 125° lateral to the dorsal midline to a point 130° above the vegetal pole in the dorsal midline. In the early gastrula, most, if not all, mesoderm is deep to the surface layer and is mapped by the insertion of dyed agar spikes. Results thus far indicate that the prospective notochord lies in the dorsal deep marginal zone, followed laterally by the medial region of the somites, the lateral region of the somites, and the lateral plate.
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.
Using the prominent cell nucleus as an intrinsic marker, individual deep cell blastomeres have been monitored in vivo using Nomarski differential inter-ference contrast microscopy during spreading of the teleost blastoderm. Involution of these cells has been recorded during early to mid stages of epiboly about an apparent point of shear located centrally within the germ ring. This involuting movement involves super-ficial deep cells, adjacent to the enveloping layer, as well as those located more centrally within the germ ring and is associated with a continuous vegetal displacement of the outer strata of deep cell blasto-meres towards the edge of the blastodisc. During the early stages of epiboly this process is qualitatively similar at any location around the entire circumfer-ential margin of the blastodisc. Postinvoluting deep cells are found close to the yolk syncytial layer, are surrounded by considerable intercellular space and illustrate less directional displacement. In contrast to the deep cell layer, the enveloping layer was never observed to invaginate. These results contradict the current view that no involution or global rearrange-ment of deep cells occurs during teleost gastrulation and present the first direct evidence of involution within the deep cell population during early epiboly.
A major question in the analysis of teleost epiboly is the fate of the yolk cytoplasmic layer. It diminishes during epiboly and eventually disappears at the completion of epiboly. This paper is concerned with the fate of the surface of the yolk cytoplasmic layer during epiboly. When gastrulae during epiboly are bathed in lucifer yellow (CH) and then observed with fluorescent microscopy or bathed in ferritin and then fixed and observed with TEM, a thin circumferential ring of endocytic vesicles is observed, confined to the external yolk syncytial layer just peripheral to the advancing margin of the blastoderm. Even though the entire egg is immersed in the marker, endocytosis is confined to this limited region. More precisely, this endocytosis occurs only within the region of the external yolk syncytial layer, where the surface is most folded. The endocytic vesicles thus formed move downward and settle on the surface of the membrane separating the yolk from the cytoplasm in the yolk syncytial layer. They do not join the surface of the internal yolk syncytial layer; hence they do not contribute to its expansion. Prior to the onset of epiboly there is no such endocytosis at the surface of the egg. Since this endocytosis occurs only during epiboly and only at the surface of the external yolk syncytial layer just peripheral to the advancing margin of the blastoderm, and in the absence of large molecules in the medium, we conclude that it is programmed. We, therefore, present this as a case of programmed internalization of cell surface serving as the morphogenetic mechanism responsible for the disappearance of the surface of the yolk cytoplasmic layer during gastrulation of the teleost Fundulus heteroclitus
The fine structure of the chorion and the region of the unfertilized egg immediately beneath the micropylar apparatus of the zebra danio, Brachydanio rerio, were studied using Nomarski differential interference optics, and scanning and transmission electron microscopy. The chorion consisted of three distinct zones: an outer, electron-dense zone containing pore canal plugs (zona radiata externa), a middle fibrillar zone (superficial zona radiata interna), and an inner zone of 16 horizontal electrondense lamellae alternating with 15 interlamellae of lower electron density (deep zona radiata interna). The zona radiata interna was pierced by open pore canals. The single micropylar apparatus was regionalized into a cone-shaped vestibule and a tapered micropylar canal traversing the entire chorion. The outer diameter of the micropylar canal was 7.5–8.5 μn and the inner diameter about 2.3 μn. Since the diameter of the inner micropylar aperture was slightly larger than the size of the sperm head, the block to polyspermy in eggs of the zebra danio appears to be mechanical and guaranteed by the morphological design of the micropyle. The egg plasmalemma beneath the inner micropylar aperture was differentiated as a circular cluster of 15–20 microvilli-like projections. The cluster of surface projections, approximately 2.1–2.5 μn in diameter under scanning, electron microscopy, was distinguishable from the microplicae covering the rest of the egg surface and identified as the sperm entry site. The cortical cytoplasm subjacent to the sperm entry site was organized as a compact, electron-dense, and homogeneous band. The sperm entry site itself was circumscribed by an area of cytoplasm (approximately 100 μn in diameter) in which cortical granules were typically arranged as a single layer immediately beneath the plasmalemma. However, there was a complete absence of cortical granules in the cytoplasm directly below the sperm entry site. The single row arrangement contrasted with the multilayered rows of cortical granules found throughout the remainder of the egg cytoplasm. Based upon Nomarski and ultrastructural analyses, there was a significant polarity in the cortex created by the size distribution and volume density of the cortical granules layered just beneath the plasmalemma. The cortical granules in the vicinity of the sperm entry site were 2.7 to 2.8 μm in diameter and densely packed. From this region to the vegetal pole, the cortical granules appeared to progressively increase in size and become less densely packed. The polarity in granule distribution established a distinct gradient in the structural organization of the egg cortex from the site of sperm entry to the vegetal pole.
The last ten years have shown a dramatic revolution in our understanding of early animal development. This new edition of the successful first edition describes the result of this revolution and explains how the body plan of an embryo emerges from the newly fertilised egg. The book starts with a critical discussion of embryological concepts and explains in simple terms the mathematics of cell states, morphogen gradients and threshold responses. The experimental evidence on the mechanism of regional specification in Xenopus, molluscs, annelids, ascidians as well as Caenorhabditis, the mouse, the chick and Drosophila is then discussed. The whole chapter devoted to the exciting developments in Drosophila provides a clear guide to the subject, including a new table outlining the developmentally important genes. The emphasis throughout is on conceptual clarity and unity: bringing together the mathematical models, embryological experiments and molecular biology into a single, comprehensive coherent account.
A FATE map depicts how lineages from a defined blastomere will be restricted to particular tissues. In the amphibian Xenopus laevis, the early blastomeres of the embryo have traceable lineages1–4. However, it has been thought that mixing of the blastomeres of Brachydanio rerio prevents them from giving rise to a fate map5–10. For this reason the zebrafish has been used as an example of a class of embryos, including mammals, which apparently exhibit little fate until later in embryogenesis. The amphibia were the exception. We report here the use of fluorescent dyes on very-high-molecular-mass carriers to trace a fate map from the first three cleavages of the zebrafish embryo to the three principal adult body axes: the first cleavage defines dorsal–ventral; the second cleavage, left–right; and the third, anterior–posterior.
The mechanisms controlling cell movements during vertebrate gastrulation are not known. Studies using the zebrafish embryo show promise at identifying these mechanisms, combining an embryo that is accessible and optically clear with mutations that affect early development. In this article we describe the movements of cells during the midblastula, early epiboly and gastrulation stages of the zebrafish, correlating 'domains of movement' with embryonic morphology. We suggest that these domains of movement may parallel the 'zones of movement' of Xenopus.
Chalk granules implanted among the cells of the stage 7 blasto-disc are carried along with the cells to final location in the differentiating tissues and organs. The results, more often than not, are inconsistent with predictions suggested by an earlier fate map for Salmo (Pasteels, (36). Granules vertically thrust into a given spot on the blastodisc are usually carried to various levels of the trunk and to a variety of organs, presumably because cells at different depths of one spot and at different distances from the center are moving at different rates. Data from hundreds of such implants are assembled into charts showing the areas from which cells are drawn, to contribute to formation of the endoderm, notochord, five separate zones of the central nervous system, the head mesoderm, anterior and posterior trunk somites, tail somites, lateral plate, and heart. Using evidence of the overlapping and intergradation of these areas, as well as evidence from vital dyes when allowed to penetrate to various depths of the pregastrular blastodisc, a three-dimensional fate map is synthesized for Salmo gairdneri, which is very different from the earlier map, but consistent with the previously reported patterns of morphogenetic movements in this species (Ballard, (73b). In contrast with the gastrulation of amphibia and birds, Salmo germ layers arise without any invagination, and there is at no time an anterior mesoderm-free area. Previous efforts to generalize upon vertebrate gastrulation and its evolution within the phylum seem to have been premature.
We describe a series of stages for development of the embryo of the zebrafish, Danio (Brachydanio) rerio. We define seven broad periods of embryogenesis—the zygote, cleavage, blastula, gastrula, segmentation, pharyngula, and hatching periods. These divisions highlight the changing spectrum of major developmental processes that occur during the first 3 days after fertilization, and we review some of what is known about morphogenesis and other significant events that occur during each of the periods. Stages subdivide the periods. Stages are named, not numbered as in most other series, providing for flexibility and continued evolution of the staging series as we learn more about development in this species. The stages, and their names, are based on morphological features, generally readily identified by examination of the live embryo with the dissecting stereomicroscope. The descriptions also fully utilize the optical transparancy of the live embryo, which provides for visibility of even very deep structures when the embryo is examined with the compound microscope and Nomarski interference contrast illumination. Photomicrographs and composite camera lucida line drawings characterize the stages pictorially. Other figures chart the development of distinctive characters used as staging aid signposts. ©1995 Wiley-Liss, Inc.
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.
The disposition of prospective areas and the course of morphogenetic movements during gastrulation and neurulation were investigated by vital staining. The prospective lining of the archenteron, the prospective neural area, and the prospective epidermal area are represented on the surface of the early gastrula. The prospective lining of the archenteron occupies the area within 65–70° of the vegetal pole and is divided into prospective archenteron roof and prospective archenteron floor by the blastopore pigment line which functions as the locus of invagination. A crescent-shaped neural area lies immediately above the prospective archenteron roof, rising from it at 125° lateral to the dorsal midline to a point 130° above the vegetal pole in the dorsal midline. In the early gastrula, most, if not all, mesoderm is deep to the surface layer and is mapped by the insertion of dyed agar spikes. Results thus far indicate that the prospective notochord lies in the dorsal deep marginal zone, followed laterally by the medial region of the somites, the lateral region of the somites, and the lateral plate.The morphogenetic significance of the comparative disposition of the anlagen in Xenopus is discussed.
Surface contour, contact relations, and cortical structure of marginal cells of the enveloping layer (EVL) and of the yolk syncytial layer (YSL) of Fundulus heteroclitus were studied before and during epiboly with transmission and scanning electron microscopy. The contacts of the marginal cells of the EVL with the underlying YSL involve only the most marginal part of each cell and consist of a mixture of tight and close junctions apically and wider appositions more proximally. This junctional complex is very extensive (2.1–2.4 μm) prior to the onset of epiboly, very restricted (0.5–0.8 μm) during early epiboly up to a mid‐gastrula, more extensive again (0.8–1.0 μm) at late midgastrula, and still more extensive (2.0–2.3 μm) at late gastrula toward the end of epiboly. At this time, the margin of each marginal cell is embedded in the YSL. Several lines of evidence suggest that these marginal contacts are stable and therefore that epiboly of the EVL occurs passively, in response to pull exerted by the independently expanding YSL.
As the external YSL (E‐YSL) narrows during the earliest phase of epiboly, its surface becomes more and more convoluted. Since the network of 4–6 nm microfilaments in the cortical cytoplasm of the E‐YSL thickens with increasing convolution of its surface, it seems possible that a contractile force resides in the E‐YSL cortex which simultaneously throws the surface of the E‐YSL into folds, narrows the E‐YSL, and exerts tension on the attached margin of the EVL. Networks of thin microfilaments are also found in the cortex of EVL cells, especially in the leading edge of the marginal cells (as in fibroblasts and epithelial cells in vitro) and in the cortex of the yolk cytoplasmic layer (YCL), where they are presumably responsible for the contractile tension of this layer; 10‐nm microfilaments are also present but have a different distribution. They are arranged in bundles in both the marginal cytoplasm of each marginal cell and in the YSL beneath the marginal contact, running parallel to the contact and circumferentially relative to the whole egg. This arrangement in this location coincides with constriction of the egg in this marginal region and suggests that these thick filaments might provide the contractile force for the constriction, along with the thin filaments with which they are associated. The morphological relationship between the E‐YSL and the YCL during epiboly is also described.
SEM reveals that the surface of the internal YSL (I‐YSL) is covered with long microvilli at the beginning of gastrulation and that they disappear and are replaced by shorter microvilli as epiboly progresses. Estimation of the amount of surface in the long microvilli at the beginning of epiboly indicates that there is enough membrane on the surface of the I‐YSL at this time to account for epibolic expansion up to a late gastrula. Although it is not known how the surface membrane in these microvilli might be redistributed, the presence within them of abundant microfilaments that appear to insert in the plasma membrane suggests that this process might be accomplished by these presumed contractile elements.
Finally, measurements of cell surface expansion and calculations of cell number show that the number of cells in the EVL remains approximately constant during epiboly. Clearly, cell division is not a factor in the epibolic expansion of the enveloping layer. Instead, there is a marked thinning of individual cells.
The prospective areas in the deep layer of the early gastrula were mapped and their morphogenetic movements during gastrulation and neurulation were followed by vital dye marking of cells. It was found that the deep layer of the early gastrula consists of prospective mesoderm and prospective neural and epidermal ectoderm. At Stage 10+ the prospective mesoderm lies deep to the suprablastoporal endoderm in the form of a thick collar which has already begun involution in the dorsal sector. The prospective notochord is located middorsally and is followed laterally, in turn, by prospective somite and lateral mesoderm. Prospective anterior mesoderm involutes first, followed in succession by prospective mesoderm of more posterior regions. The relative movements of the neural and epidermal ectoderm and the underlying mesodermal mantle during neurulation were mapped by simultaneous marking of both layers. These movements show the highly coordinated nature of the thickening of somite mesoderm and folding of the neural plate. Strong dorsal convergence and ventral divergence occur in both the prospective mesoderm and ectoderm. In the ectoderm, dorsal convergence of cells results in anteroposterior extension; however, such dorsal movement in the mesoderm is absorbed in the thickening of the somite region. The extension of the dorsal mesoderm is brought about during neurulation by addition of cells posteriorly from the uninvoluted circumblastoporal mesoderm. The significance of the location and movements of the mesoderm in Xenopus laevis is discussed.
The increase in surface area that occurs as cells spread from the rounded to the flattened state has been examined in synchronized BHK 21 cells in the scanning electron microscope. Rounded cells, whether in mitosis or dissociated and freshly seeded in culture, are covered with a mixture of folds, blebs, and microvilli. As cells spread, these protuberances disappear, first in the flattening marginal region and progressively submarginally until the entire cell surface is virtually smooth. The estimated surface area of rounded post-mitotic daughter cells, taking microvilli into account, is close to that of fully spread cells 4 h after mitosis. Likewise, rounded early mitotic mother cells, which are also covered with microvilli, have approximately the same surface as fully spread cells just prior to mitosis. These findings suggest that cells possess a membrane reserve in their microvilli and other protuberances which can be utilized for spreading and initiating cell locomotion.
In Xenopus, convergence and extension are produced by active intercalation of the deep mesodermal cells between one another along the mediolateral axis (mediolateral cell intercalation), to form a narrower, longer array. The cell motility driving this intercalation is poorly understood. A companion paper shows that the endodermal epithelium organizes the outermost mesodermal cells immediately beneath it to undergo convergence and extension, and other evidence suggests that these deep cells are the most active participants in mediolateral intercalation (Shih, J. and Keller, R. (1992) Development 116, 887-899). In this paper, we shave off the deeper layers of mesodermal cells, which allows us to observe the protrusive activity of the mesodermal cells next to the organizing epithelium with high resolution video microscopy. These mesodermal cells divide in the early gastrula and show rapid, randomly directed protrusive activity. At the early midgastrula stage, they begin to express a characteristic sequence of behaviors, called mediolateral intercalation behavior (MIB): (1) large, stable, filiform and lamelliform protrusions form in the lateral and medial directions, thus making the cells bipolar; (2) these protrusions are applied directly to adjacent cell surfaces and exert traction on them, without contact inhibition; (3) as a result, the cells elongate and align parallel to the mediolateral axis and perpendicular to the axis of extension; (4) the elongate, aligned cells intercalate between one another along the mediolateral axis, thus producing a longer, narrower array. Explants of essentially a single layer of deep mesodermal cells, made at stage 10.5, converge and extend by mediolateral intercalation. Thus by stage 10.5 (early midgastrula), expression of MIB among deep mesodermal cells is physiologically and mechanically independent of the organizing influence of the endodermal epithelium, described previously (Shih, J. and Keller, R. (1992) Development 116 887-899), and is the fundamental cell motility underlying mediolateral intercalation and convergence and extension of the body axis.
The first signs of cell motility appear in Fundulus toward the end of cleavage, after cleavages 11 and 12. When blastomeres cease cleaving, their surfaces undulate and form blebs. At first, these blebbing cells remain in place. Gradually thereafter they begin movement, with blebs and fllolamellipodia serving as organs of locomotion. Non-motile cleaving blastomeres have thus differentiated into motile blastula cells. This transformation corresponds to the midblastula transition of amphibian embryos.
Gastrulation in Fundulus begins with vegetalward contraction of the external yolk syncytial layer. This causes narrowing of the E- YSL and initiates the epibolic expansion of the blastoderm. Convergent movements of deep cells within the blastoderm begin toward the end of this contraction. The YSL forms as a result of invasion of the yolk cell cytoplasm by nuclei from open marginal blastomeres during cleavage. These YSL nuclei then undergo five metachronous divisions. After this, they divide no more. YSL contraction begins approximately 1.5 hours after cessation of these divisions (21–22°C). This cessation of nuclear divisions is preceded by a gradual decrease in rate. (1) The duration of each succeeding mitosis increases steadily and often some nuclei do not divide at mitosis V. (2) The duration of interphases between succeeding mitoses also increases, but to a much greater degree, and the longest interphase by far is the last one, I-FV, between mitoses FV and V. (3) The mitotic waves responsible for mitosis V move much more slowly than those for the first four mitoses and invariably decelerate. This gradual cessation of YSL nuclear divisions clearly sets the stage for the contraction of YSL cytoplasm and thus the beginning of gastrulation. We call this the YSL transition. It is not to be confused with the midblastula transition, which occurs 3–4 hours earlier. The MBT commences cytodifferentiation; the YSL transition commences morphogenesis.
At the midblastula transition in the zebrafish, three, and only three, spatially separate mitotic domains arise with distinctive cycle lengths and rhythms. As in Drosophila and at about the equivalent stage, the mitotic domains reflect the fate map, but they do so only very crudely: two are extraembryonic and the third forms the entire embryo. The domains appear not to subdivide during gastrulation, when the germ layers form and when cells probably commit to their eventual fates. The domains may signal specification of morphogenesis rather than cell fate, because, shortly after they appear, each assumes a different role during epiboly, the first morphogenetic movement of the embryo. During meroblastic cleavage, and continuing in the early blastula, zebrafish blastomeres divide rapidly and synchronously. At the time of the tenth cleavage, the beginning of the midblastula transition, the cell cycle lengthens, and, as in Xenopus and Drosophila, cycle length comes under nucleocytoplasmic control (D.A.K. and C.B.K., manuscript in preparation). This nucleocytoplasmic control seems to be maintained during cycle lengthening in the next 2 or 3 cycles, comprising a midblastula transition period. We now show that functionally distinct subsets of cells that arise during this period have reproducibly different mitotic cycle lengths.
The sperm entry site (SES) of zebrafish (Brachydanio rerio) eggs was studied before and during fertilization by fluorescence, scanning, and transmission electron microscopy. Rhodamine phalloidin (RhPh), used to detect polymerized filamentous actin, was localized to microvilli of the SES and to cytoplasm subjacent to the plasma membrane in the unfertilized egg. The distribution of RhPh staining at the SES correlated with the ultrastructural localization of a submembranous electrondense layer of cortical cytoplasm approximately 500 nm thick and containing 5- to 6-nm filaments. Actin, therefore, was organized at the SES as a tightly knit meshwork of filaments prior to fertilization. Contact between the fertilizing sperm and the filamentous actin network was observed by 15-20 sec postinsemination or just before the onset of fertilization cone formation. Growing fertilization cones of either artificially activated or inseminated eggs exhibited intense RhPh staining and substantial increase in thickness of the actin meshwork. Collectively, TEM and RhPh fluorescence images of inseminated eggs demonstrated that the submembranous actin became rearranged in fertilization cones to form a thickened meshwork around the sperm nucleus during incorporation. The results reported here suggest that activation of the egg triggers a dramatic polymerization of actin beneath the plasma membrane of the fertilization cone. Furthermore, the actin involved in sperm incorporation is sensitive to the action of cytochalasins.
Mainly because of its transparency, the Fundulus gastrula constitutes ideal material for direct study of morphogenetic cell movements in vivo. Marking studies show that deep cells of the germ ring converge toward and enter the embryonic shield, where they undergo extension. Those close to the shield move faster. Analysis of videotapes reveals that all deep cells of the dorsal germ ring move toward the shield. But none moves in a direct line. All meander considerably. Germ ring cells nearer the shield move toward it at a higher net rate than those farther away because they meander less. This suggests that exogenous factors promote their directionality. Cells in the prospective yolk sac adjacent to the germ ring also show net convergence, but they meander more. Directional forces are apparently stronger in the germ ring.
Converging deep cells move both by filolamellipodia and, less frequently, by blebs. However, there is very little individual cell movement; all cells are almost always in adhesive contact with other cells in moving cell clusters. Clusters vary constantly in size, continually aggregating with other cells and other clusters and splitting. Filolamellipodial cells show contact inhibition of cell movement. Nevertheless, they move and do so directionally, presumably in part because, as members of cell clusters, much of their movement is passive. They also show intercalation or invasive activity, but, consistent with their contact-inhibiting properties, only when neighboring cells separate and provide free space. Cells moving by blebbing locomotion are non-contact inhibiting and intercalate readily. Cell division continues during convergence. Although this temporarily arrests their movement, the daughter cells soon join in the mass convergent movement.
We have analyzed cell behavior in the organizer region of the Xenopus laevis gastrula by making high resolution time-lapse recordings of cultured explants. The dorsal marginal zone, comprising among other tissues prospective notochord and somitic mesoderm, was cut from early gastrulae and cultured in a way that permits high resolution microscopy of the deep mesodermal cells, whose organized intercalation produces the dramatic movements of convergent extension. At first, the explants extend without much convergence. This initial expansion results from rapid radial intercalation, or exchange of cells between layers. During the second half of gastrulation, the explants begin to converge strongly toward the midline while continuing to extend vigorously. This second phase of extension is driven by mediolateral cell intercalation, the rearrangement of cells within each layer to lengthen and narrow the array. Toward the end of gastrulation, fissures separate the central notochord from the somitic mesoderm on each side, and cells in both tissues elongate mediolaterally as they intercalate. A detailed analysis of the spatial and temporal pattern of these behaviors shows that both radial and mediolateral intercalation begin first in anterior tissue, demonstrating that the anterior-posterior timing gradient so evident in the mesoderm of the neurula is already forming in the gastrula. Finally, time-lapse recordings of intact embryos reveal that radial intercalation takes places primarily before involution, while mediolateral intercalation begins as the mesoderm goes around the lip. We discuss the significance of these findings to our understanding of both the mechanics of gastrulation and the patterning of the dorsal axis.
The floor plate is a set of epithelial cells present in the ventral midline of the neural tube in vertebrates that seems to have an important role in the developmental patterning of central nervous system fibre pathways, and arrangements of specific neurons. The floor plate arises from dorsal ectodermal cells closely associated with the mesoderm that forms notochord, and it may depend on interactions from the notochord for its specification. To learn the nature of these interactions we have analysed mutations in zebrafish (Brachydanio rerio). We report here that in wild-type embryos the floor plate develops as a simply organized single cell row, but that its development fails in embryos bearing the newly discovered zygotic lethal 'cyclops' mutation, cyc-1(b16). Mosaic analysis establishes that cyc-1 blocks floor plate development autonomously and reveals the presence of homeogenetic induction between floor plate cells.
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.
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.
The study of developmental patterning has been facilitated by the availability of mutations that produce changes in cell fate, in animals such as Caenorhabditis elegans and Drosophila melanogaster. We now describe a zygotic lethal mutation in the zebrafish, Brachydanio rerio, that also changes how particular embryonic cells develop. Severe pattern deficiencies are observed that are restricted to a single body region, the trunk. The mutation may directly affect mesoderm, as somites do not form in the trunk. Head and tail structures, including tail somites, are relatively undisturbed. The earliest detected expression of the mutation is during gastrulation, when movements of mesodermal cells occur incorrectly. We injected prospective trunk mesodermal cells with lineage tracer dye and observed that in mutants these cells may enter a new body region, the tail, and there may express a new fate appropriate for the changed position.
Cell lineage may contribute to determining the numbers, positions and types of cells formed during embryogenesis. In vitro clonal analyses show that vertebrate cells can autonomously maintain lineage commitments to single fates and that terminal development may include an invariant sequence of cell divisions. In addition, in vivo studies with Xenopus led to the proposal that clonal restrictions to spatial 'compartmental' domains arise during early development, analogous to what is observed in insects. In the zebrafish, individual gastrula cells generate clones of progeny that are confined within single tissues, but spatial restrictions have not been described. We now have examined the in vivo terminal cell lineages of zebrafish axial muscles. We obtained no evidence either for strict developmental regulation of division pattern or for spatial compartmentation within muscle lineages.
We have examined the clonal progeny descended from individual blastomeres injected with lineage-tracer dye in the zebrafish embryo. Blastomeres arising by the same cleavages in different embryos generated clones in which the types and positions of cells were highly variable. Several features of early development were correlated with this diversity in cell fate. There was no fixed relationship between the plane of the first cleavage and the eventual plane of bilateral symmetry of the embryo. By blastula stages the cleavages of identified blastomeres were variable in pattern. Moreover, cell fate was not easily related to the longitudinal and dorsoventral position of the clone in the gastrula. These results establish that single blastomeres can potentially generate a highly diverse array of cell types and that the cell lineage is indeterminate.
Silver nitrate staining of blastoderms of Fundulus heteroclitus gastrulae shows that the number of marginal cells of the enveloping layer (EVL) is reduced from 160 to 25 during epiboly. To determine whether this decrease in the number of marginal cells was due to ingression, cell death, or rearrangement of cells, marginal and submarginal regions of the late gastrula were observed directly by time-lapse cinemicrography. Marginal cells rearrange to occupy submarginal positions by first narrowing their boundary with the external yolk syncytial layer (E-YSL), thus becoming tapered in shape. Then, the narrowed marginal boundary retracts from the E-YSL and moves submarginally in the plane of the epithelium. Concurrently, the marginal cells on both sides come into apposition; no gap or break appears in the circum-apical continuity of the epithelial sheet. Marginal cells leave the margin of the EVL during epiboly at a rate of about six per hour. The rate of movement of the EVL cells with respect to one another is about 0.5 to 1.0 micron/min at 21 degrees C. Submarginal cells rearrange in a similar fashion. Although no protrusive activity was seen at the lateral aspects of rearranging cells, the tapering or narrowing associated with rearrangement was accompanied by formation of microfolds on their apical surfaces, and separating or recently separated submarginal cells form "flowers" of microfolds on their apices adjacent to the site of separation. Morphometric analysis shows that about half the narrowing of the margin of the EVL during epiboly is accounted for by cell rearrangement and the other half by the associated tapering and narrowing. These results suggest that epiboly of the EVL may have an active component as well as a passive one.
Aspects of the early lineages of blastomeres in the embryo of the zebrafish, Brachydanio rerio have been described. Because of the optical clarity of the embryo, lineages of selected cells can be followed directly by microscopy through many cell divisions. Also, it is shown here that the fluorescent molecules fluorescein-dextran and rhodamine-horseradish peroxidase can be used as cell lineage tracers, marking the clonal progeny of founding blastomeres. The labeled cells can be easily visualized in the live embryo, and utilizing a sensitive video camera to amplify fluorescence, the same clone may be examined repeatedly while the cells divide and migrate. Cells that descend from a single blastomere remain closely associated together through the end of the blastula stage. At the time when epiboly begins (early gastrula) cells in the labeled clone scatter and become dispersed among unlabeled cells. It has been observed that there is no invariant mapping of the embryo's midline (determined by the position of the embryonic shield in the gastrula) with respect to the early planes of cleavage. This finding shows that in the zebrafish the region of the embryo that a cell will occupy is not specified by the cell's early ancestory.
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.
Patterns of cleavage and cytoplasmic connections between blastomeres in the embryo of the zebrafish, Brachydanio rerio have been described. The cell division pattern is often very regular; in many embryos a blastomere's lineage may be ascertained from its position in the cluster through the 64-cell stage. At the 5th cleavage, however, significant variability in pattern is observed, and alternative patterns of the 5th cleavage are described. The early cleavages are partial, incompletely separating blastomeres from the giant yolk cell. The tracer fluorescein-dextran (FD) was injected into blastomeres to learn the extent of the cytoplasmic bridging. It was observed that until the 10th cleavage, blastomeres located along the blastoderm margin maintain cytoplasmic bridges to the yolk cell. Beginning with the 5th cleavage, FD injected into a nonmarginal blastomere either remains confined to the injected cell, or if the injection was early in the cell cycle, the tracer spreads to the cell's sibling, through a bridge persisting from the previous cleavage. On the other hand, injected Lucifer yellow spreads, presumably via gap junctions, widely among blastomeres in a pattern unrelated to lineage.
Measurements of several indices of shape, contact, position and arrangement of deep cells in the late blastula and gastrula were made from scanning electron micrographs of carefully staged, fractured embryos in order to describe the cellular processes which account for the increased area of the deep region of the gastrula during extension of the dorsal marginal zone and epiboly of the animal region. At the onset of gastrulation, the deep cells of the dorsal marginal zone become elongated, extend protrusions between one another along radii of the embryo and interdigitate to form fewer layers of cells of greater area in a process of radial interdigitation. When interdigitation, is complete, the deep region consists of one layer of columnar cells which then flatten and spread and thus account for additional increase in area of the deep region. During epiboly of the animal region, interdigitation occurs and the number of cell layers decreases without the changes in cell shape seen in the dorsal marginal zone. These differences may be related to the anisotropy of expansion (extension and convergence) in the dorsal marginal done as opposed to uniform spreading in the animals region, or they may reflect an active cell motility in the dorsal marginal zone as opposed to a passive behavior in the animal region. A cellular and mechanical model is presented in which active (autonomous) spreading is brought about by active, force-producing interdigitation and subsequent flattening of deep cells. A model of passive spreading (stretching) is also presented. These observations suggest experiments that would determine the relationship of cell behavior to the mechanics of gastrulation.
Descendants of early blastomeres in the zebrafish come to populate distinctive regions of the fate map. We present a model suggesting that the distribution of cells in the early gastrula (the fate map stage) results from the passive response of cells to reproducible forces that change the overall shape of the blastoderm just prior to gastrulation. We suggest that one of the morphogenetic changes that accompanies epiboly, the upward doming of the yolk cell into the overlying blastoderm, could be responsible for cell mixing. In support of the model, we show that the timing, extent, and directions of cell mixing in the embryo accurately reflect the expectations of the model. Finally, we show that one portion of the gastrula, a marginal region that later gives rise to many of the mesendodermal derivatives, experiences little cell mixing during the doming process. As a result, this region in the gastrula is populated by the descendants of the subset of the early blastomeres that were originally at the margin. The finding that cytoplasm initially at the edge of the 1-celled blastodisc is transmitted specifically to mesendodermal precursors at the fate map stage raises the possibility that maternal determinants may contribute to initiation of embryonic patterning in the zebrafish embryo.
The zebrafish has emerged as an important model system for the experimental analysis of vertebrate development because it is amenable to genetic analysis and because its optical clarity allows the movements and the differentiation of individual cells to be followed in vivo. In this paper, we have sought to characterize the spatial distribution of tissue progenitors within the outer cell layers of the embryonic shield region of the early gastrula. Single cells were labeled by iontophoretic injection of fluorescent dextrans. Subsequently, we documented their position with respect to the embryonic shield and their eventual fates. Our data show that progenitor cells of the neural, notochordal, somitic and endodermal lineages were all present within the embryonic shield region, and that these progenitors were arranged as intermingled populations. Moreover, close to the midline, there was evidence for significant biases in the distribution of neural and notochord progenitors between the layers, suggesting some degree of radial organization within the zebrafish embryonic shield region. The distributions of tissue progenitors in the zebrafish gastrula differ significantly from those in amphibians; this bears not only on interpretations of mutant phenotypes and in situ staining patterns, but also on our understanding of morphogenetic movements during gastrulation and of neural induction in the zebrafish.
We have examined the zebrafish embryo to ascertain the location of endocardial and myocardial progenitors prior to gastrulation, in an attempt to define the earliest stages of cardiac patterning. Currently there is uncertainty as to the spatial and lineage relationship of the progenitors for these two phenotypically distinct cell types that form the two concentric layers of the primitive heart tube. By single-cell injection and tracking, we distinguish a region in the early and midblastula which has the properties of a heart field, in that it defines a zone of cardiac progenitors within which there is a spatial gradient of propensity to generate heart cells, and which regulates, in the sense of adapting to the transplantation of pluripotential cells. This zone extends from the future ventral axis dorsally along the margin, with cardiogenic propensity tapering off laterally and dorsally. Myocardial progenitors are spread throughout this region, but endocardial precursors are restricted to the ventral marginal region. The cardiovascular progeny of the ventral cells include, in addition to endocardium and myocardium, cells in the endothelium and blood.
Previously, a tissue-specific fate map was compiled for the gastrula stage of the zebrafish embryo, indicating that development
subsequent to this stage follows a reproducible pattern. Here it is shown that each early zebrafish blastomere normally contributes
to a subset of the gastrula and thus gives rise to a limited array of tissues. However, the final contribution that any early
blastomere makes to the fate map in the gastrula cannot be predicted because of variability in both the position of the future
dorsoventral axis with respect to the early cleavage blastomeres and the scattering of daughter cells as the gastrula is formed.
Therefore, early cell divisions of the zebrafish embryo cannot reproducibly segregate determinants of tissue fates.
THE mechanisms underlying the formation of dorsoventral polarity in the zebrafish Danio rerio are unknown. Here we describe the zebrafish recessive maternal-effect mutation janusm55. The mutant phenotype is a division of the blastoderm along the first cleavage plane into two detached half-sized blastoderms. Partial-axis bifurcation occurs in a subset of mutants. Analysis of goosecoid expression in the mutant embryos indicates that only one organizer region is present in each embryo. Furthermore, the position of this organizer region is random with respect to the first cleavage plane bisecting the two blastoderms. Finally, cell tracing in wild-type embryos demonstrates that there is no strict correlation of the dorsoventral axis with early cleavage planes in zebrafish. These findings support the notion that the establishment of the dorsoventral axis and the first cleavage planes are determined by separate mechanisms in the zebrafish embryo.
The zebrafish dorsoventral axis can first be distinguished at gastrulation, upon formation of the embryonic shield, the site of the organizer. We have asked whether the shield is specified before gastrulation. First, we show that brief exposure of premidblastula embryos to lithium, which is known to shut down the phospho-inositol signaling pathway, produces excessive shield formation and extreme hyper-dorsal development. Second, we show that the zebrafish goosecoid homeobox gene is activated at or just after the midblastula in a localized domain of cells that subsequently populate the most anterior region of the incipient shield and axial hypoblast, goosecoid expression is elevated and radialized by early lithium treatment, suggesting that goosecoid plays a role in establishing the organizer and shield. Our results demonstrate that the zebrafish dorsal axis is signaled by a pathway initiated in the cleavage-stage embryo. Furthermore, they provide novel insights into anterior morphogenesis.