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Cell lineage and developmental potential of cells in the zebrafish embryo
Abstract
The zebrafish embryo is particularly well-suited for developmental studies in a vertebrate because of the small number and accessibility of its cells, its optical clarity and its rapid development to a stage where specific cell types can be recognized. Cell lineage analysis reveals considerable indeterminacy in the embryonic lineage, and there is no evidence that cell lineage restricts embryonic cell fate before the gastrula stage. However, cells in gastrula-stage embryos generate clones confined to particular tissues. This suggests that by this stage the morphogenetic behaviors, and perhaps the fates, of individual cells become developmentally programmed.
... Early lineage tracing experiments, where fluorescent dextran was injected into the early blastomeres, demonstrated that clones of these cells can be found spread across the entire embryo by the end of epiboly. It was also observed that large numbers of unlabelled cells had mixed in between these labelled clones (Kimmel and Warga, 1988). This demonstrated that the process of anteroposterior pattern formation during gastrulation must occur with temporal and spatial coordination, whilst the cells which make up the embryo mix readily with one another. ...
... In order to investigate the exact levels of cell mixing occurring in the gastrulating zebrafish embryo, the experiments of Kimmel and Warga (1988) were repeated using fluorescent dextran injections into the animal and vegetal blastomeres. From the current fate maps and understanding of gastrulation, the animal pole blastomeres would be expected to produce clones which contribute to the neural ectoderm, non-neural ectoderm and retina. ...
As embryos develop, they are required to generate organised patterns of gene expression across developing tissues. Previous hypotheses used to explain how patterns form in developing tissues have relied on the ideas of positional information and gradients of signalling across the developing tissue, which impart spatial coordinates to individual cells. These coordinates then inform the cell of its respective fate, and hence a pattern of gene expression with spatial organisation is generated. This way of thinking about the problem of pattern formation has been highly successful in explaining the patterning of tissues in vertebrate embryos. However, such an approach begins to fail when challenged with tissues where the cells which make it are highly motile. This thesis will examine the extent to which patterning is robust to cell mixing by disrupting early positional information prior to gastrulation, using whole embryonic explants which undergo widespread cell mixing. These explants elongate, form all three germ layers with spatial organisation and anteroposterior patterned neural tissue. This thesis will then examine the robustness of patterning within the presomitic mesoderm, where a posterior to anterior pattern of gene expression is observed despite high levels of cell rearrangement. Within this tissue, the pattern is observed to accurately scale to the shortening anterior to posterior length and is demonstrated to be highly robust to changes in the degree of cell mixing following experimental perturbation, including in vitro culture of individual cells and pharmacological treatment. Together, this thesis will argue that to understand pattern formation in tissues which are also undergoing extensive cell rearrangement, it is important to consider the gene regulatory network dynamics and how these are influenced by signalling. Rather than taking a positional information “bottom up” type view of cell fate decision making, where signalling gradients inform cells of their position and hence their identity, I will argue for a more holistic approach to understanding development. By considering how “top down” regulation of patterning, mediated via the movement of cells and tissues between domains of signalling, it will be demonstrated that pattern formation can be considered an emergent properly of cells, regulated by signalling as well as cell movements.
... In chick, bi-fated notochord-floor plate precursors have been identified in the node by single cell labelling [130]. In zebrafish, bi-fated notochord-ventral neural precursors were also found at blastula stages although it is unlikely that these cells are committed in any way [21,128,131]. By the early gastrula stage in zebrafish, notochord and floor plate precursors have segregated into distinct regions of the shield [107,127,128,131,132]. ...
... In zebrafish, bi-fated notochord-ventral neural precursors were also found at blastula stages although it is unlikely that these cells are committed in any way [21,128,131]. By the early gastrula stage in zebrafish, notochord and floor plate precursors have segregated into distinct regions of the shield [107,127,128,131,132]. Specification of floor plate identity begins early, during gastrulation, with floor plate character actively maintained in the tailbud at later stages [123,[133][134][135]. ...
Ascidians are invertebrate chordates and the closest living relative to vertebrates. In ascidian embryos a large part of the central nervous system arises from cells associated with mesoderm rather than ectoderm lineages. This seems at odds with the traditional view of vertebrate nervous system development which was thought to be induced from ectoderm cells, initially with anterior character and later transformed by posteriorizing signals, to generate the entire anterior-posterior axis of the central nervous system. Recent advances in vertebrate developmental biology, however, show that much of the posterior central nervous system, or spinal cord, in fact arises from cells that share a common origin with mesoderm. This indicates a conserved role for bi-potential neuromesoderm precursors in chordate CNS formation. However, the boundary between neural tissue arising from these distinct neural lineages does not appear to be fixed, which leads to the notion that anterior-posterior patterning and neural fate formation can evolve independently.
... In freshwater environments, the zebrafish (D. rerio) is the most commonly used fish species for genetic and environmental toxicology and lately it has also become one of the most popular laboratory model organisms due to several reasons such as their small size, easy husbandry, early morphology, high fecundity, and small and transparent embryos. Moreover, an advantage of the zebrafish embryo model is related to animal welfare and alternative in vitro methods for the testing of chemicals as the earliest life-stages of zebrafish are considered an in vitro test system [91][92][93][94][95][96]. Apart from using zebrafish as an in vivo model system, in addition to the zebrafish embryo model that can be regarded as an in vitro method, the use of zebrafish cultured cell lines in genetic toxicology is also on the rise [91,[97][98][99][100][101]. ...
... Besides their size, this species is valuable because of their high fecundity and transparent embryos. The rapid maturation of zebrafish also allows easy experimentation for transgenerational endpoints required for mutagenesis screening, establishing transgenic lines, and assessing chemicals for teratogenicity [91][92][93][94][95]. Moreover, another advantage of the zebrafish embryo model is related to animal welfare and alternative in vitro methods for the testing of chemicals (the ECVAM concept of 3″R″; replace, reduce and refine the experiments on animals) as the earliest lifestages of zebrafish are considered as an in vitro test system [96]. ...
The comet assay has become one of the methods of choice for the evaluation and measurement of DNA damage. It is sensitive, quick to perform and relatively affordable for the evaluation of DNA damage and repair at the level of individual cells. The comet assay can be applied to virtually any cell type derived from different organs and tissues. Even though the comet assay is predominantly used on human cells, the application of the assay for the evaluation of DNA damage in yeast, plant and animal cells is also quite high, especially in terms of biomonitoring. The present extensive overview on the usage of the comet assay in animal models will cover both terrestrial and water environments. The first part of the review was focused on studies describing the comet assay applied in invertebrates. The second part of the review, (Part 2) will discuss the application of the comet assay in vertebrates covering cyclostomata, fishes, amphibians, reptiles, birds and mammals, in addition to chordates that are regarded as a transitional form towards vertebrates. Besides numerous vertebrate species, the assay is also performed on a range of cells, which includes blood, liver, kidney, brain, gill, bone marrow and sperm cells. These cells are readily used for the evaluation of a wide spectrum of genotoxic agents both in vitro and in vivo. Moreover, the use of vertebrate models and their role in environmental biomonitoring will also be discussed as well as the comparison of the use of the comet assay in vertebrate and human models in line with ethical principles. Although the comet assay in vertebrates is most commonly used in laboratory animals such as mice, rats and lately zebrafish, this paper will only briefly review its use regarding laboratory animal models and rather give special emphasis to the increasing usage of the assay in domestic and wildlife animals as well as in various ecotoxicological studies.
... They reported in 1986 and 1990 that predictable cell lineages arise at gastrula stage. In 1987, they found that detailed cell fates vary considerably before epiboly (for review, see also Kimmel and Warga 1988). The fact that cell fates were unpredictable was ascribed to uncoordinated movements of the undetermined blastomeres before gastrulation. ...
... Since Kimmel and Warga (1988) only described the appearance of label in somatic cell types, it remains possible that, in contrast to somatic cells, germ cells are determined early in development. Moreover, the variance in cell fate might be diminished by taking the orientation of the embryonic axis into account. ...
Lucifer Yellow-Dextran labelling of lower layer cells (LLC), sometimes together with upper layer cells (ULC), of the 64-cellBarbus conchonius embryo resulted in labelled primordial germ cells (PGCs) at 12 h after fertilization (a.f.) in about 25% of cases. The presence of labelled PGCs was independent of the location of the injected blastomere with respect to the later orientation of the embryonic axis. After injection of an ULC alone, however, labelled PGCs were never found. Also, the distribution of labelled somatic cells differed between the ULC- and LLC-injected embryos. When we found fluorescent PGCs, only a few of them were labelled, suggesting that either a single predecessor exists earlier than the 64-cell stage or that the formation of germ cells is a polyclonal process. Tracing the fluorescent cells at successive stages of development shows an extensive mixing with unlabelled cells during the epiboly stage, which might well be the cause of partly unpredictable cell lineages. The chance of being committed to a specific fate is different for the ULC and LLC descendants. This might be due to relatively limited cell mixing between these two cell populations.
... Since the fate map of zebrafish is well described, shifts in the fate map can be readily detected in mutants (26)(27)(28). Due to extensive cell mixing, the cell lineage of zebrafish is indeterminate during the cleavage and early blastula stages, and labeling at this stage does not lead to a reproducible fate map (29,30). This is in striking contrast to organisms like Xenopus laevis, in which the fate map can be determined as soon as the early cleavage stages (31). ...
... For example, when using a secondary antibody made in donkey, it is advisable to block embryos with normal donkey serum instead of normal sheep serum. 30. When visualized with epifluorescence, fluorescence from tissue layers above and below the focal plane contaminates the image and decreases the resolution. ...
Due to the powerful combination of genetic and embryological techniques, the teleost fish Danio rerio has emerged in the last decade as an important model organism for the study of embryonic development. It is relatively easy to inject material such as mRNA or synthetic oligonucleotides to reduce or increase the expression of a gene product. Changes in gene expression can be analyzed at the level of mRNA, by whole-mount in situ hybridization, or at the level of protein, by immunofluorescence. It is also possible to quantitatively analyze protein levels by Western and immunoprecipitation. Cell behavior can be analyzed in detail by cell transplantation and by fate mapping. Because a large number of mutations have been identified in recent years, these methods can be applied in a variety of contexts to provide a deep understanding of gene function that is often more difficult to achieve in other vertebrate model systems.
... Lineage studies in zebrafish also reveal a mesendoderm population of cells during epiboly (Warga and Nusslein-Volhard, 1999;Kikuchi et al., 2004;Kimmel and Warga, 1988) (Fig. 1). When single cells were labeled at the margin of the gastrulating zebrafish, the progeny of some of those cells gave rise to endoderm while other progeny of that labeled cell became mesoderm, and this was true if a single cell was initially labeled as late as 40% epiboly. ...
Early in animal development many cells are conditionally specified based on observations that those cells can be directed toward alternate fates. The endomesoderm is so named because early specification produces cells that often have been observed to simultaneously express both early endoderm and mesoderm transcription factors. Experiments with these cells demonstrate that their progeny can directed entirely toward endoderm or mesoderm, whereas normally they establish both germ layers. This review examines the mechanisms that initiate the conditional endomesoderm state, its metastability, and the mechanisms that resolve that state into definitive endoderm and mesoderm.
... 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)
... 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.
... • 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.
... 10 Similarly, the zebrafish embryo model has been extensively used as zebrafish are easy to raise and maintain, imaging-compatible due to their transparency, and straightforward to genetically modify. 9,11,12 While these systems are an improvement over previous animal models, moving toward human in vitro models would likely allow for more representative developmental outcomes. Currently, human cell-based assays are being developed and optimized in order to move away from these animal models. ...
Birth defects are a common occurrence in the United States and worldwide. Currently, evaluation of potential developmental toxicants (i.e., teratogens) relies heavily on animal-based models, which do not always adequately mimic human development. In order to address this, researchers are developing in vitro human-based models which utilize human pluripotent stem cells (hPSCs) to assess the teratogenic potential of chemical substances. The field of human developmental toxicity assays includes a variety of platforms including monolayer, micropattern, embryoid body, and 3D organoid cultures. In this review, we will overview the field of human teratogenic assays, detail the most recent advances, and discuss current limitations and future perspectives.
... Large-scale screening of mutants is possible because free-swimming 7 day old fish exhibit many behavioral and morphological traits of the parents but are only a few millimeters long. 21,22 These traits together with their transparency and availability and the ease of formation of transgenic animals, as well as their low chemical requirement for testing, have endeared the species to developmental biologists. The breadth and depth of research applications utilizing the zebrafish model are highly diverse and range from ototoxicity to oncology and plastic surgery (cranial suture) to population effects. ...
There has been increasing focus on generation and assessment of in vitro developmental toxicology models for assessing teratogenic liability of chemicals. The driver for this focus has been to find reliable in vitro assays that will reduce or replace the use of in vivo tests for assessing teratogenicity. Such efforts may be eventually applied in testing pharmaceutical agents where a developmental toxicology assay or battery of assays may be incorporated into regulatory testing to replace one of the 2 species currently used in teratogenic assessment. Such assays may be eventually applied in testing broader spectrum of chemicals supporting efforts aligned with Tox21 strategies and responding to REACH legislation. This review describes the developmental toxicology assays that are of focus in these assessments: rodent whole embryo culture, zebrafish embryo assays and embryonic stem cell assays. Progress on assay development as well as future directions of how these assays are envisioned to be applied for broader safety testing of chemicals are discussed. Altogether, the developmental model systems described in this review provide rich biological systems that can be utilized in better understanding teratogenic mechanisms of actions of chemotypes and are promising in providing proactive safety assessment related to developmental toxicity. Continual advancements in refining/optimizing these in vitro assays is anticipated to provide a robust data set to provide thoughtful assessment of how whole animal teratogenicity evaluations can be reduced/refined in the future.
... Kimmel (1972) took advantage of the transparency of the embryos to study the development of Mauthner cell axons in living larvae. The appearance of a new generation of fluorescent probes, especially non-diffusible cell markers, the advent of epifluorescence and confocal microscopy, and the development of improved methods for intracellular injection of probes led to a series of studies of cell lineage, fate maps, and morphogenetic movements (Kimmel and Warga, 1988). Perhaps the most significant advantage of zebrafish derives from the development of an effective genetic system for this species. ...
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.
... Fluorescence labeling coupled with confocal microscopy can also enhance expression imagery and allow assessments in older, thicker larvae (MacDonald, 1999). Likewise, along with various other markers, fluorescent dyes can also be utilized for cell lineage tracing (Cooper et al., 1999a,b;Kimmel, 1989;Kimmel and Warga, 1988). ...
Zebrafish have been used predominantly in developmental biology and molecular genetics, but their value in toxicology as well as drug discovery has been recognized. To evaluate the toxicity of a chemical, it is essential to identify the endpoints of toxicity and their dose-response relationships, elucidate the mechanisms of toxicity, and determine the toxicodynamics of the chemical. In addition to detailed toxicological investigations of a single chemical, there also is a need for high-throughput large- scale screening for toxicity of several hundreds of chemicals at a time. In both cases, the zebrafish has numerous attributes. More is probably known about ''what is normal'' in the zebrafish than any other fish species. This includes morpho- logical, biochemical, and physiological information at all stages of early development and in juveniles and adults of both sexes. This makes using the zebrafish ideal for toxicology research where the objective is to identify adverse effects of chemical exposure.
... At the same time Streisinger was perfecting genetic methods for zebrafish, several of his University of Oregon colleagues were developing techniques for imaging and manipulating cells within the living zebrafish embryo. A series of publications from the mid-1980s to early 1990s demonstrated the ability to fate map cells at a variety of different stages of embryogenesis (Kimmel and Warga, 1988), to label and transplant cells individually and follow their devel-opmental fates (Westerfield and Eisen, 1988;Eisen, 1991;Ho and Kimmel, 1993), and to test the requirement for gene function in chimeras by transplanting specific cells between embryos of different genotypes (Ho and Kane, 1990;Eisen and Pike, 1991). The ability to couple genetics and superb embryology in zebrafish propelled this model into the limelight. ...
... They are also suitable for developmental analysis because they are transparent throughout the entire embryonic period (Fig. 1B). This characteristic makes possible the precise time-lapse imaging of a single embryonic cell for lineage tracing, or a single neuron for analyses of axonal pathfinding in live embryos [17,33]. Zebrafish were the first vertebrate for which a large scale muta-genesis study and screening were performed to the saturation level with a chemical mutangen, ethyl nitrosourea (ENU) [20,34]. ...
The zebrafish is the simplest model vertebrate amenable to genetics, and genome information and methods of embryo manipulation have been accumulated worldwide. The numbers of mutant and transgenic zebrafish strains are rapidly increasing, and these strains will play important roles in the basic biology research and as model systems of the human diseases in the future. Although researchers who had established zebrafish strains, were distributing the fish on a discretionary basis, a well-established system for distributing the strains did not exit in Japan prior to 2003. Due to these circumstances, a system to collect, preserve, and provide zebrafish strains was established as part of the National BioResource Project useful model vertebrates in Japan and to the world.
... 1). Comparisons of hybridizing band patterns between the offspring pools of F1 transgenic fish (lanes[1][2][3][4][5][6] and30 pg of the pRSV-,3Gal plasmid (lane 7), equivalent to -1.34 copies per diploid genome. Genomic zebrafish DNA in lanes: 1, F2-45; 2, F9-45; 3, F17-114; 4, M14-5; 5, M9-51; 6, M9-37; 8, offspring pool from a nontransgenic F1 fish derived from transgenic fish F17. ...
With the goal of developing techniques for DNA insertional mutagenesis in zebrafish, we established procedures for rapidly obtaining and injecting large numbers of fertilized eggs. Using either of two plasmid constructs, we injected uncut DNA into fertilized eggs at the one- or two-cell stage. Fish hatched from injected eggs were raised to sexual maturity, and the frequency of transgenic founder fish was determined by pair-mating the fish and testing DNA extracted from pools of their 16-hr-old offspring by the polymerase chain reaction (PCR) and then Southern analysis. Eggs injected with one of two different plasmids yielded no transgenic fish, but 7-25% (19 of 115 overall) of the eggs injected with the other plasmid transmitted the injected sequences to their offspring (F1). Of seven lines studied further, all were able to pass the foreign DNA sequences to the next (F2) generation. Inheritance in the F2 generation was Mendelian in the five lines tested. PCR and Southern analysis indicated that the plasmid sequences were present in multiple copies, probably tandemly arranged. Two founder fish carried more than one independent integration of the plasmid sequences. The line studied in more detail was a mosaic carrying two independently segregating copies of the transgene in one germ cell and a third copy in another germ-line precursor cell. The ability to obtain and inject large numbers of zebrafish eggs combined with a high frequency of germ-line integration may be steps toward the goal of being able to perform insertional mutagenesis with this organism.
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.
Early in animal development many cells are conditionally specified based on observations that those cells can be directed toward alternate fates. The endomesoderm is so named because early specification produces cells that often have been observed to simultaneously express both early endoderm and mesoderm transcription factors. Experiments with these cells demonstrate that their progeny can directed entirely toward endoderm or mesoderm, whereas normally they establish both germ layers. This review examines the mechanisms that initiate the conditional endomesoderm state, its metastability, and the mechanisms that resolve that state into definitive endoderm and mesoderm.
A number of teleost eggs present outstanding advantages for the study of vertebrate development. Teleosts occur all over the world in both fresh and sea water of all degrees of salinity and often have long spawning seasons. Indeed, some species, such as the zebrafish (Brachydanio rerio), the medaka (Oryzias latipes), and the rosy barb (Barbus conchonius), can be induced to spawn year-round in the laboratory. Moreover, the eggs and embryos of teleosts are often unusually resistant to small environmental changes and thus will withstand much experimental manipulation. Although removal of the chorion is difficult in certain species (like Fundulus heteroclitus), it is easy in many others, such as the zebrafish, rosy barb, blennies, and gobies. Further, many species produce eggs of striking lucidity, so that cytokinesis, cell movement and form changes of individual cells can be observed directly and followed in the living embryo. And now, with new powerful means of marking cells by injecting non-toxic fluorescent macro-molecules, the lineage of cells may also be followed readily during early embryogenesis in these transparent embryos. Finally, because of the ease of propagation of some fish embryos throughout the year and their rapidity of development, sophisticated genetic studies of development can be pursued.
In order to contribute developmental technology, the early embryonic stages during the pre-gastrula were studied by cytological, histological or biochemical viewpoints in goldfish Carassius auratus. Synchronous cleavages occur 9 times every about 30 min from 50 min to 6 h post-fertilization at 20°C, and, thereafter, transpose asynchronous phase, suggesting mid-blastula transition (MBT). This timing is staged as the mid-blastula. Genomic expression of mesodermal markers, goosecoid and no tail, are detected with in situ hybridization assay from 8 h post-fertilization. This timing is staged as the late-blastula. Cytoplasmic protrusions as cell movement are ovserved after MBT, and autonomous mixing of blastomeres from the late-blastula stage. Deep cells were produced from the surface part of blastomeres and central part of yolk cell during the late-blastula stage. Marginal yolk syncytial layer forms during the mid-blastula stage, but central yolk syncytial layer forms after the late-blastula stage. This result suggests that each yolk syncytial layer has a different origin of nuclei.
Because of the genetic and developmental information available, Danio rerio stands out as a vertebrate model system in which significant progress in the areas of development and evolution can be made. Despite its increasing popularity, little research has been done on skeletal development. In this report, we provide developmental information on the structure and composition of the zebrafish skull, pectoral, and pelvic girdle. We describe the sequence of ossification of the skull and paired fins from a large series of cleared and Alizarin red-stained specimens at larval and adult stages. The most commonly followed developmental sequence in Danio rerio is described. Chondrocranial development is noted from Alcian blue-stained specimens. General trends in ossification patterns are examined from developmental, phylogenetic, and functional contexts. No clear pattern in ossification order of dermal versus cartilage bones is evident. Ossification sequence conforms to functional need in a general way, but there are inconsistencies in the details of order. Selected phylogenetic comparisons of ossification sequence within cranial regions are made among Danio rerio, Betta splendens, Oryzias latipes, and Barbus barbus. Greater sequence conservation is apparent between D. rerio and Barbus barbus, the ostariophysans, than among other taxon pairs. Intraspecific variation in ossification order is apparent, most of which involves small adjustments in timing. © 1996 Wiley-Liss, Inc.
The zebrafish is rapidly increasing in popularity with developmental biologists. Driving this interest are the elegant methods for in vivo observations and recovery of early developmental mutations. The past year has seen the introduction of additional methods for in vivo manipulation of identified cells and the application of these methods to mutant analysis.
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 1997. Includes bibliographical references. by Christina Migyung Yoon. Ph.D.
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 1994. Includes bibliographical references (leaves 184-207).
One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps.
We have isolated a POU domain-containing cDNA (ZFPOU1) from a cDNA library of zebrafish (Brachydanio rerio). The ZFPOU1 cDNA contained an open reading frame encoding a 425 amino acid peptide. The conserved POU domain was located near the carboxy terminus. The deduced amino acid sequence of the reading frame was most similar to that of the mouse class III POU-domain gene, Brain-1. Northern blot analysis revealed that the ZFPOU1 transcripts first appeared at the early neurula stage of embryogenesis and transiently increased thereafter. A significant level of expression, however, was not found in adult tissues except in the brain. In situ hybridization analysis demonstrated that the ZFPOU1 transcripts were localized in the neural tissues of embryos, but not in mesodermal, endodermal or ectodermal tissues. In adult zebrafish, the ZFPOU1 transcripts were detected in the restricted regions of the brain. Spatial and temporal expression patterns suggest that ZFPOU1 has distinct roles in the early neural development of zebrafish.
The zebrafish (Brachydanio rerio), a small fresh water fish native to rivers of northeast India, has long been a favorite of tropical fish fanciers. Within the past few years, it has also become an organism of great interest to vertebrate embryologists. The potential of the zebrafish as an effective experimental system can be traced to the work of G. Streisinger who recognized that the organism was highly suited to genetic analysis (Streisinger et al. 1981). After his untimely death, the development of the system was continued at the University of Oregon, and more recently, at many laboratories in the United States and Europe. The result has been a productive investigation of the embryology, genetics, neurobiology, and molecular biology of the zebrafish embryo (reviewed most recently in Kimmel and Warga 1988; Kimmel 1989; Ekker and Akimenko 1991; Fulwiler and Gilbert 1991). In this review, I will attempt to summarize what is known about the early development, genetics, and molecular biology of the zebrafish. Development of the nervous system has been more intensively investigated than any other aspect of embryogenesis in this organism. This subject has been reviewed recently (Eisen 1991) and will not be covered in detail here.
Ependymins are secretory products of meningeal cells and represent the predominant glycoproteins in the cerebrospinal fluid from various orders of teleost fish. In the zebrafish, their expression starts between 48 and 72 h post-fertilization. Generally, they share characteristics with proteins involved in cell-contact phenomena. Here, we characterize the ependymin gene from Brachydanio rerio and its flanking regions. The sequence was obtained from clones generated using the polymerase chain reaction (PCR), including a variation of an "anchored" PCR. Also, clones from a conventional phage library were analyzed. We found that the transcribed portion is arranged in six exons. Transient expression of an ependymin-promoter-lacZ gene fusion in zebrafish embryos revealed that the 2.0-kb upstream regulatory region used is sufficient to direct the ependymin-specific correct temporal and spatial expression pattern of the lacZ reporter gene.
Recessive lethal germline and specific locus somatic mutations were induced efficiently in the zebrafish by exposure of mature sperm to UV light. Mutagenesis of sperm yielded mosaic individuals: clones bearing novel mutations represented approximately 12-25% of the haploid germ cells and 25-50% of the somatic tissue. Simple methods are described for the reliable identification and propagation of newly arising developmental mutations in zebrafish.
This chapter discusses the patterning of body segments of the zebrafish embryo. Descriptive studies in zebrafish have provided exceptionally clear information, often at the level of individual cells, about the structure, extent in the body, and development of the segments. Experimental analyses, including the use of mutations, reveal cellular interactions required for segmentation. Information from zebrafish provides a useful background for understanding how genes make vertebrate segments, an issue for the future, and for which this species also holds some promise. The developmental studies of zebrafish strengthen the notion that vertebrates seem to be metameric creatures. Attempts to dispel this notion in the past have pointed up the limited extent of vertebrate body segmentation. Segmentation does indeed seem to be limited, but it is even limited in annelids that are certainly segmented animals and that show no hint of segmental organization in lineages that produce the gut or the skin, organs that also appear unsegmented in vertebrates. Accordingly, the presence of nonsegmented tissues within a segmented body plan does not seem particularly problematic for the common segmented ancestor hypothesis; a most interesting question for both development and evolution is how the apparently unsegmented axial sets of cells, the notochord and the floor plate, come to be insinuated into the metameres. These axial tissues have dominant developmental roles and certainly have also been extremely important in chordate evolution.
This chapter discusses the evolutionary genetics of fish to demonstrate how studies on fish provide critical links in understanding the evolution of other vertebrate classes. By using selected examples, the chapter points out where genetic studies on fish requires a re-evaluation of evolutionary models that is previously based on the data obtained from higher vertebrates. The chapter begins with the discussion of general methods that are used to detect genetic variation in fish systems, along with the presentation of current fish genetic nomenclature for genes encoding proteins. The evolution of higher order genomic structures including genome size, karyotype, isochore structures, and linkage relationships is described in the chapter. Fish have a fossil record that is relatively well characterized, and there are a number of “living fossils” that are particularly useful in comparative biochemical and physiological studies. Fish are also excellent experimental models for genetic and evolutionary studies. Many fish species are amenable to both laboratory and field experiments and are easily raised and bred under laboratory conditions. The chapter concludes with a discussion of gene duplication and multigene families and provides some examples of fish genes that respond to environmental parameters.
The present report describes Lucifer Yellow (LY) transfer between the syncytial layer of the yolk cell (YSL) and blastodermal cells during epiboly in the teleost fish Barbus conchonius. The fate of a group of labeled cells is described until germ layer formation.
At the onset of epiboly, LY seems to be transferred from the YSL to all blastodermal cells. Between 10 % and 40 % epiboly, dye-coupling appears to be restricted to the marginal region. Within 60 min individually labeled cells are distributed among unlabeled cells within the blastoderm. Between 40 % and 60 % epiboly, we observed a ring-shaped group of labeled cells, which probably have involuted during early gastrulation. Consequently, this cell group may correlate with the leading edge of the hypoblast layer within the germ ring. At 60 % epiboly and later, the blastodermal cells are dye-uncoupled from the YSL.
A gradual translocation of the ring-shaped hypoblast towards a dorsally located bar-like structure is observed between 50 % and 100 % epiboly. At 100 % epiboly, fluorescent cells were located in contact with the YSL within the embryo proper, with the brightest fluorescence in the future head region. The translocation is due to dorsalwards convergent cell movements during the gastrulation process. The appearance of the hypoblast as a dye-coupled cell layer may correlate with some restriction in cell fate since the hypoblast differs in fate from the epiblast.
Classical developmental biology has distinguished two major modes of embryogenesis, determinate and indeterminate. Nematodes have been considered the chief paradigm for determinate and cell-autonomous development, but recent experiments on the early development of Caenorhabditis elegans suggest that most blastomeres of this nematode are, in fact, determined by interactions.
Species of small fish are becoming useful tools for studies on vertebrate development. We have investigated the developing embryo of the Japanese medaka for its application as a transient expression system for the in vivo analysis of gene regulation and function. The temporal and spatial expression patterns of bacterial chloramphenicol acetyltransferase and galactosidase reporter genes injected in supercoiled plasmid form into the cytoplasm of one cell of the two-cell stage embryo was promoter-specific. The transient expression was found to be mosaic within the tissue and organs reflecting the unequal distribution of extrachromosomal foreign DNA and the intensive cell mixing movements that occur in fish embryogenesis. The expression data are consistent with data on DNA fate. Foreign DNA persisted during embryogenesis and was still detectable in some 3- and 9-month-old adult fish; it was found in high molecular weight form as well as in circular plasmid conformations. The DNA was replicated during early and late embryogenesis. Our data indicate that the developing medaka embryo is a powerful in vivo assay system for studies of gene regulation and function.
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.
The ability to mark a cell and its descendants genetically so that the resulting cell clone can be distinguished from neighboring
cells facilitates studies in animal biology and development. A method of generating clones by inducing homologous mitotic
recombination in Drosophila with a site-specific yeast recombinase is described. This method allows for frequent mosaicism
after mitotic exchange is induced at predefined sites in the genome.
The floor plate is a specialized group of midline neuroepithelial cells that appears to regulate cell differentiation and axonal growth in the developing vertebrate nervous system. A floor plate-specific chemoattractant was used as a marker to examine the role of the notochord in avian floor plate development. Expression of this chemoattractant in lateral cells of the neural plate and neural tube was induced by an ectopic notochord, and midline neural tube cells did not express the chemoattractant after removal of the notochord early in development. These results provide evidence that a local signal from the notochord induces the functional properties of the floor plate.
Ependymins are unique, brain specific glycoproteins, which are major constituents of the cerebrospinal fluid. Originally, they were discovered in goldfish and are thought to be involved in synaptic plasticity. In the present study two transcripts were characterized in Brachydanio rerio originating from a single gene possibly by alternative splicing. These transcripts differ only in the length of their 3'-non-coding-regions and the encoded protein shares 90 and 88% homology with the two corresponding goldfish proteins, respectively. In situ hybridization revealed the expression of ependymins exclusively in the leptomeninx including its invaginations but not at all in the ependymal layer surrounding the ventricles. An initial developmental profile showed that ependymins first appear before hatching, i.e. between 48 and 72 h postfertilization.
The times of origin (birthdays) of sensory and motor neurons that innervate the triceps brachii muscles of the bullfrog (Rana catesbeiana) were determined to learn whether neurons innervating a specific target are generated at a particular developmental time. 3H-thymidine (3H-TdR) was made available continuously throughout a specific developmental period. All neurons that innervated the triceps muscle in juvenile frogs (identified by filling cells retrogradely with HRP) were generated prior to metamorphosis. Triceps motoneurons were all postmitotic by early limb bud stage V. Triceps sensory neurons were generated over a protracted period of larval development, from stage V through early pre-metamorphic stage XV. Most large triceps sensory neurons were generated before the majority of the small cells. However, there was considerable overlap in the times of origin of the two populations; both large and small cells were generated at all stages of sensory neurogenesis. There was thus no strict relationship between sensory soma size and birthdate. Late-generated sensory neurons tended to be located in clusters within ganglia, whereas HRP-filled triceps neurons were not. These 3H-labeled clusters may represent clones of neurons which would indicate that late stage neuroblasts give rise to neurons that supply different peripheral targets. The time course of triceps neuronal generation paralleled that of all other brachial sensory neurons implying that the time of last cell division does not in itself determine either the target a neuron will innervate or the sensory modality to which it will respond.
In order to construct fish specific expression vectors for studies on gene regulation in vitro and In vivo a variety of heterologous
enhancers and promoters from mammals and from viruses of higher vertebrate cells were tested for expression of the bacterial
chloramphenicol acetyl transferase reporter gene In three teleost fish cell lines. Several viral enhancers were found to be
constitutively active at high levels. The human metallothionein promoter showed induclble expression in the presence of heavy
metal ions. A fish sequence was isolated that can be used as a homologous constitutively active promoter for expression of
foreign genes. Using the human growth hormone gene with an active promoter in fish cells for transient expression insufficient
splicing and lack of
translation were observed, pointing to limitations in the use of heterologous genes In gene transfer experiments. On the contrary,
some heterologous promoters and enhancers functioned in fish cells as well as In their cell type of origin, indicating that
corresponding transcription factors are sufficiently conserved between fish and human over a period of 900 million years of
independent evolution.
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.
We have studied the lineage history of the progenitors of the somite mesoderm and of the neural tube in the chick embryo by injecting single cells with the fluorescent tracer, rhodamine-lysine-dextran. We find that, although single cells within the segmental plate give rise to discrete clones in the somites to which they contribute, neither the somites nor their component parts (sclerotome, dermatome, myotome or their rostral and caudal halves) are `compartments' in the sense defined in insects. Cells in the rostral two thirds or so of the segmental plate contribute only to somite tissue and divide about every 10 h, while those in the caudal portions of this structure contribute both to the somites and to intermediate and lateral plate mesoderm derivatives. In the neural tube, the descendants of individual prospective ventral horn cells remain together within the horn, with a cycle time of 10 h.
We have also investigated the role of the cell division cycle in the formation and subsequent development of somites. A single treatment of 2-day chick embryos with heat shock or a variety of drugs that affect the cell cycle all produce repeated anomalies in the pattern of somites and vertebrae that develop subsequent to the treatment. The interval between anomalies is 6-7 somites (or a multiple of this distance), which corresponds to 10 h. This interval is identical to that measured for the cell division cycle. Given that cell division synchrony is seen in the presomitic mesoderm, we suggest that the cell division cycle plays a role in somite formation.
Finally, we consider the mechanisms responsible for regionalization of derivatives of the somite, and conclude that it is likely that both cell interactions and cell lineage history are important in the determination of cell fates.
Somites represent the first visual evidence of segmentation in the developing vertebrate embryo and it is becoming clear that this segmental pattern of the somites is used in the initial stages of development of other segmented systems such as the peripheral nervous system. However, it is not known whether the somites continue to contribute to the maintenance of the segmental pattern after the dispersal of the somitic cells. In particular, the extent to which cells from a single somite contribute to all of the tissues of a single body segment and the extent to which they mix with cells from adjacent segments during their migration is not known. In this study, we have replaced single somites in the future cervical region of 2-day-old chick embryos with equivalent, similarly staged quail somites. The chimerae were then allowed to develop for a further 6 days when they were killed. The cervical region was dissected and serially sectioned. The sections were stained with the Feulgen reaction for DNA to differentiate between the chick and quail cells. The results showed that the cells from a single somite remained as a clearly delimited group throughout their migration. Furthermore, the sclerotome, dermatome and myotome portions from the single somites could always be recognised as being separate from similar cells from other somites. The somitic cells formed all of the tissues within a body segment excluding the epidermis, notochord and neural tissue. There was very little mixing of the somitic cells between adjacent segments. The segmental pattern of the somites is therefore maintained during the migration of the somitic cells and this might be fundamental to a mechanism whereby the segmentation of structures, such as the peripheral nervous system, is also maintained during development.
Cell lineages during development of leeches can be ascertained by injection of horseradish peroxidase as a tracer into identified
cells at early stages of embryogenesis. The injected embryos continue their normal development, in the course of which horseradish
peroxidase is passed on in catalytically active form to the descendants of the injected cell. The distribution of the tracer
enzyme and hence of the progeny of the injected cell can then be observed at a later stage of development by staining the
preparation for horseradish peroxidase.
Horseradish peroxidase injected into individual blastomeres of 128-, 256-, and 512-cell embyros of Xenopus laevis was identified in cells of the central nervous system (CNS) at early to middle larval stages. Labeled cells were dispersed, mingled with unlabeled cells. Four boundaries in the CNS could be defined by the behavior of clones of labeled cells: in the transverse plane at the level of the isthmus; in the horizontal plane between dorsal and ventral regions extending the entire length of the CNS; in the dorsal midline extending the entire length; and in the ventral midline of rhombencephalon and spinal cord but absent more rostrally. Cells injected with HRP at the 512-cell stage produced clones that, with rare exceptions, did not cross any boundary, whereas labeled clones initiated at earlier stages frequently crossed boundaries. Axons and dendrites were not restricted by these boundaries. These boundaries subdivided the CNS into seven compartments, each of which was occupied exclusively by the descendants of a group of 14 to 26 blastomeres in the 512-cell embryo. These groups of blastomeres formed a bilaterally symmetrical pattern composed of a single anterior median group straddling the dorsal midline near the animal pole and three groups on each side. Because cells mingled in each compartment but not across compartmental boundaries, there was a one-to-one relationship between individual blastomeres and CNS compartments but one-many and many-one relationships between individual blastomeres and neuroanatomical subdivisions smaller than a compartment. There was no constant relationship between phenotypes of nerve cells and their ancestry from individual blastomeres of the 512-cell or earlier stages.
The compartmental and clonal relationships between primary motoneurons and the myotubes they innervate have been studied in Xenopus laevis embryos by initiating clones at blastula stages (32 to 512 cells) with intracellular injections of horseradish peroxidase (HRP). Primary motoneurons and ventral myotome belong to the posterior-ventral compartment, whereas sensory neurons and dorsal myotome belong to the posterior-dorsal compartment (Jacobson, M. (1983) J. Neurosci. 3: 1019-1038). The pathways of HRP-labeled primary motor axons, which pioneer the peripheral pathway to the adjacent myotome beginning at stage 21/22, were traced. Within the spinal cord, the axons remained in the posterior-ventral compartment. Upon leaving the cord they were confronted with both dorsal and ventral myotome; they remained almost exclusively in the ventral myotome. At late embryonic stages (40/42) primary motoneurons could be retrogradely labeled by applying HRP to the dorsal myotome, indicating that their axons or a branch had crossed the compartmental boundary. The primary motor axon also displayed a highly significant preferred association with clonally related myotubes within the ventral myotome. Axon growth only in the compartmentally related myotome and preferred association with clonally related myotubes suggest that the guidance of pioneer axons in the periphery may be based upon factors derived from common ancestry and lineage.
Cell lineages during embryogenesis of the ascidian Halocynthia roretzi were analyzed up until the stage where each blastomere was fated to be only a single tissue type (i.e., the tissue restricted stage) by intracellular injection of horseradish peroxidase using the iontophoretic injection method. Initially, the developmental fates of all blastomeres of the 64-cell stage embryo were examined, and thereafter, only the fates of daughter blastomeres of those blastomeres that were not tissue restricted at the 64-cell stage were traced. The developmental fates of blastomeres were highly invariant except for two candidates for “equivalence groups” (J. Kimble, J. Sulston, and J. White (1979). In “Cell Lineage, Stem Cells and Cell Determination,” pp. 59–68. Elsevier, Amsterdam/New York), in which cellular interaction is suggested to be involved in the specification of the fates. The right and left a8.25 cells gave rise to the otolith and ocellus, and the right and left b8.17 cells gave rise to the spinal cord and endodermal strand in a complementary manner. No fixed relationship existed between the position of the blastomere and its derivative. Most restrictions of cell fates occurred early in cleavage. The numbers of blastomeres which generated a single type of tissue were 44 at the 64-cell stage and 94 at the 110-cell stage. Eight pairs of blastomeres had not yet become tissue restricted by the 110-cell stage. Almost complete lineages of epidermis, nervous system, muscle, mesenchyme, notochord, and endodermal tissues were described, and a fate map was constructed for the blastula. For certain tissues, the primordial cells occupied two different regions. Supplementary investigations of the lineage of muscle cells were also performed on embryos of another species, Ciona intestinalis.
A homeobox sequence has been used to isolate a new Xenopus cDNA, named XIHbox6. A short probe from this gene serves as an early marker of posterior neural differentiation in the Xenopus nervous system. The gene recognized by this cDNA sequence is first transcribed at the late gastrula stage and solely in the posterior neural cells. The gene is expressed when ectodermal and mesodermal tissues of an early gastrula are placed in contact, but not by either tissue cultured on its own. However, gene expression is most easily inducible in ectoderm from the dorsal region, i.e., in ectoderm normally destined to form neural structures. This establishes the principle, in contrast to previous belief, that the induction of the embryonic nervous system involves a predisposition of the ectoderm and does not depend entirely on an interaction with inducing mesoderm.
Study of cell lineage in the mammalian embryo has relied heavily on the use of chimeras to follow the fate of genetically marked cells in later development. Such studies have often been limited by the types of genetic markers available; there are very few markers that allow analysis of the spatial distribution of individual cells at all stages of development. We have developed a marker system that is based on the identification of cells of Mus musculus origin in M. musculus-M. caroli chimeras by in situ DNA-DNA hybridization using a cloned probe to M. musculus satellite DNA. This provides the first ubiquitous in situ cell marker system for mammalian chimeras. We have recently refined the system by the use of biotin-labelled probes and detection of hybridization by streptavidin-peroxidase binding. This increases both the speed and the resolution of the assay. We have used the marker for cell lineage analysis in both embryonic and adult chimeras and results from analysis of the derivatives of early cell lineages in later development and study of coherent growth versus cell mixing in the postimplantation embryo are presented. The importance of understanding embryonic cell lineages as a prelude to molecular studies is emphasized.
Studies of the role of cell lineage in development began in the latter part of the 19th century, fell into decline in the early part of the 20th, and were revived about 20 years ago. This recent revival was accompanied by the introduction of new and powerful analytical techniques. Concepts of importance for cell lineage studies include the principal division modes by which a cell may give rise to its descendant clone (proliferative, stem cell and diversifying); developmental determinacy, or indeterminacy, which refer to the degree to which the normal cleavage pattern of the early embryo and the developmental fate of its individual cells is, or is not, the same in specimen after specimen; commitment, which refers to the restriction of the developmental potential of a pluripotent embryonic cell; and equivalence group, which refers to two or more equivalently pluripotent cell clones that normally take on different fates but of which under abnormal conditions one clone can take on the fate of another. Cell lineage can be inferred to have a causative role in developmental cell fate in embryos in which induced changes in cell division patterns lead to changes in cell fate. Moreover, such a causative role of cell lineage is suggested by cases where homologous cell types characteristic of a symmetrical and longitudinally metameric body plan arise via homologous cell lineages. The developmental pathways of commitment to particular cell fates proceed according to a mixed typologic and topographic hierarchy, which appears to reflect an evolutionary compromise between maximizing the ease of ordering the spatial distribution of the determinants of commitment and minimizing the need for migration of differentially committed embryonic cells. Comparison of the developmental cell lineages in leeches and insects indicates that the early course of embryogenesis is radically different in these phyletically related taxa. This evolutionary divergence of the course of early embryogenesis appears to be attributable to an increasing prevalence of polyclonal rather than monoclonal commitment in the phylogenetic line leading from an annelid-like ancestor to insects.
This chapter discusses the cellular basis of a particular instance of positional specification—the divergence of the O and P cell lines that occur during the embryonic development of the leech. Positional specification requires that cells be able to detect and respond to positional cues by choosing one of two or more alternative developmental pathways. The means by which cells detect and respond to position is a central issue in the field of pattern formation, and by studying this process within a context of defined cell lineages, it may be possible to resolve positional cues in terms of specific interactions occurring among a manageable number of cells. The chapter describes the way in which position-dependent cell interactions become converted into cell-intrinsic states of commitment, leading to a discussion of the ways in which developmental commitment of an embryonic progenitor cell may influence the composition of its descendant clone. Position-dependent cell interactions are a fundamental part of vertebrate neurogenesis and an understanding of pattern formation in invertebrate nervous systems may lend insights there as well.
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.
It has been proposed that clonal restriction boundaries develop in Xenopus embryos between clones initiated at the 512-cell
stage, and that these boundaries result in formation of morphological compartments, each populated by progeny of a group of
ancestral cells. Although this hypothesis has gained some acceptance, it has also been criticized because the use of only
one cell lineage tracer was not a conclusive test of the hypothesis. However, the critical experiment, an assessment of the
extent of mingling between two labeled clones in the same embryo, has now been performed. A model of the proposed arrangement
of the ancestral cell groups in the 512-cell embryo predicted that the two clones would remain separate in 49% of cases and
intermingle in 51% of cases. In fact, there was a bimodal distribution, in which separation of the clones occurred in 46%
of embryos and extensive interclonal mingling was observed in 54%. These results are not compatible with hypotheses in which
a unimodal distribution of mingling would be predicted but are consistent with the compartment hypothesis.
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.
The fate of each of the blastomeres in the 16-cell stage Xenopus embryo which had been carefully selected for stereotypic cleavages was determined by intracellularly marking a single blastomere with horseradish peroxidase and identifying the labeled progeny in the tailbud embryo by histochemistry. Each blastomere populated all three primary germ layers. The progeny of each blastomere were distributed characteristically both in phenotype and in location. For example, most organs were populated by the descendants of particular sets of blastomeres. Furthermore, within an organ the progeny of a single blastomere were restricted to defined spatial addresses. This study describes the fates of identified 16-cell stage blastomeres and demonstrates that they are distinct and predictable if embryos are preselected for stereotypic cleavages.
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One of the first steps in segmentation of the Drosophila embryo seems to be not the formation of segments, but instead the definition of 14 domains, each of which encroaches into adjacent segments. We call these domains parasegments and discuss their developmental significance.
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.
Cell lineages during development of the leech are revealed by injection of a fluorescent peptide, rhodamine-D-peptide, into
identified embryonic cells. Use of this peptide together with a nuclear stain showed a stereotypic cleavage pattern of stem
cells and their progeny. Combined injection of rhodamine-D-peptide and pronase demonstrated the arrest of stem cell production
in the pronase-injected teloblast.
Zebra fish (Brachydanio rerio) are particularly suited for carcinogenicity testing and for the establishment of quantitative dose-response relationships. They are small, prolific, have short generation times, and produce eggs which develop synchronously and can be treated at identical developmental stages. Clones of homozygotes with minimal genetic variability provide a reproducible biological test system. Rapid measurements of the frequency of induced somatic mosaicism (demonstrated with gamma radiation, ethyl methanesulfonate, and ethylnitrosourea) and induced germ-line recessive-lethal mutation provide indexes of exposure.
The embryonic cell lineage of Caenorhabditis elegans has been traced from zygote to newly hatched larva, with the result that the entire cell lineage of this organism is now known. During embryogenesis 671 cells are generated; in the hermaphrodite 113 of these (in the male 111) undergo programmed death and the remainder either differentiate terminally or become postembryonic blast cells. The embryonic lineage is highly invariant, as are the fates of the cells to which it gives rise. In spite of the fixed relationship between cell ancestry and cell fate, the correlation between them lacks much obvious pattern. Thus, although most neurons arise from the embryonic ectoderm, some are produced by the mesoderm and a few are sisters to muscles; again, lineal boundaries do not necessarily coincide with functional boundaries. Nevertheless, cell ablation experiments (as well as previous cell isolation experiments) demonstrate substantial cell autonomy in at least some sections of embryogenesis. We conclude that the cell lineage itself, complex as it is, plays an important role in determining cell fate. We discuss the origin of the repeat units (partial segments) in the body wall, the generation of the various orders of symmetry, the analysis of the lineage in terms of sublineages, and evolutionary implications.
To ascertain the embryonic origins of the cells in various tissues of the leech Helobdella triserialis, horseradish peroxidase (HRP) was injected as a cell lineage tracer into all identified blastomeres of the early embryo in turn, except for a few of the micromeres, and the resulting distribution of HRP-labeled cells was then examined in the late embryo. In this way it was found that in every body segment a topographically characteristic set of neurons in the ganglion and body wall and a characteristic territory of the epidermis is derived from each of the four paired ectodermal teloblasts N, O/P, O/P, and Q, whereas the muscles, nephridia, and connective tissue, as well as a few presumptive neurons in each segmental ganglion, are derived from the paired mesodermal teloblast, M. Each topographically characteristic, segmentally iterated set of neurons descended from a given teloblast is designated as a kinship group. However, the prostomial (nonsegmental) epidermis and the neurons of the supraesophageal ganglion were found to be derived from the a, b, c, and d micromere quartet to which the A, B, C, and D blastomeres give rise at the dorsal pole of the embryo. The superficial epithelium of the provisional integument, which covers the surface of the embryo midway through development and is sloughed off at the time of body closure, was found to be derived from the a, b, c, and d micromere quartet, as well as from other micromeres produced in the course of teloblast formation. The contractile fibers of the provisional integument were found to be derived from the paired M teloblast. These results demonstrate that development of the leech embryo proceeds according to a highly stereotyped pattern, in the sense that a particular identifiable blastomere of the early embryo regularly gives rise to a particular set of cells of the adult (or provisional embryonic) tissues.
Recent analysis of cell lineages in ascidian embryos by the intracellular injection of a tracer enzyme has clearly demonstrated that muscle cells are derived not only from the B4.1-cell pair of the eight-cell stage embryo, as has hitherto been believed, but also from both the b4.2- and A4.1-cell pairs (H. Nishida and N. Satoh, 1983, Dev. Biol. 99, 382-394). In order to reexamine the developmental autonomy in muscle lineage cells, the B4.1 pair was isolated from the eight-cell stage embryo. The progeny cells of the B4.1 pair, as well as those of the six other blastomeres, were then allowed to develop in isolation into partial embryos. Autonomous muscle cell differentiation not only in partial embryos originating from the B4.1 cells but also in those from the six other blastomeres was substantiated by (a) occurrence of localized histospecific muscle acetylcholinesterase and (b) development of myofibrils. These results support the validity of the recent cell lineage study and confirmed the self-differentiation potency of muscle lineage cells in ascidian embryos according to the newly verified cell lineages.
In the zebrafish, cells of a clone derived from a single blastomere migrate away from one another during gastrulation. Later in development their descendants are usually found scattered within several different types of tissues of embryo. The divisions and migrations of individual cells were monitored during early development, revealing that in most cases the lineal descendants of single cells present at gastrula stage exclusively populate only single tissues, and may have stereotyped positional relationships within these tissues. Thus the gastrula stage is the first stage when heritable restrictions in cell type might arise in the zebrafish.