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Wallis H Clark - One of the best experts on this subject based on the ideXlab platform.
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mesendoderm cell and archenteron formation in isolated Blastomeres from the shrimp sicyonia ingentis
Developmental Biology, 1994Co-Authors: Philip L Hertzler, Steven W Wang, Wallis H ClarkAbstract:The fate map of 2- and 4-cell-stage Sicyonia ingentis embryos was determined by microinjection of lysyl-rhodamine-dextran into single Blastomeres. Microinjected embryos were cultured to the limb bud stage, when the body plan of the nauplius larva was evident. The animal Blastomere, AB, gave rise to anterior ectoderm, while the vegetal Blastomere, CD, gave rise to posterior structures, including the invagination site during gastrulation. The A Blastomere gave rise to mirror-image patterns of dorsal-lateral ectoderm, while the B Blastomere gave rise to anterior, ventral ectoderm. The C Blastomere gave rise to posterior, dorsal-lateral ectoderm, complementary to the A pattern, as well as some naupliar mesoderm. The D Blastomere gave rise to mesendoderm, naupliar mesoderm, and some posterior ectoderm. To study the specification of the early Blastomeres, they were microsurgically separated and cultured in isolation. Two mesendoderm cells formed in 1/2, 1/4, 1/8, and 1/16 Blastomeres in embryos dissociated at the 2-, 4-, 8-, and 16-cell stages, respectively. CD and D Blastomeres could be distin-Swished by their larger size and gave rise to the mesendoderm cells. Archenteron formation and elongation of the embryo occurred in CD but not in AB isolates. Isolated Blastomeres were recombined in various ways to determine whether their state of commitment could be altered in different cellular environments. Duplicated mesendoderm cells and archenterons formed in CD + CD recombinations, while AB + AB recombinations formed blastulae but did not produce mesendoderm cells and did not invaginate. The normal number of mesendoderm cells and a single archenteron formed in D + AB recombinations, while C + AB recombinations remained as blastulae and did not form mesndoderm cells. The results suggest that the mesendoderm cells are autonomously specified, possibly by cytoplasmic localization at the vegetal pole. The mesendoderm may also function as a signaling region to organize other developmental events.
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cleavage and gastrulation in the shrimp sicyonia ingentis invagination is accompanied by oriented cell division
Development, 1992Co-Authors: Philip L Hertzler, Wallis H ClarkAbstract:Embryos of the penaeoidean shrimp Sicyonia ingentis were examined at intervals during cleavage and gastrulation using antibodies to beta-tubulin and DNA and laser scanning confocal microscopy. Cleavage occurred in a regular pattern within four domains corresponding to the 4-cell-stage Blastomeres and resulted in two interlocking bands of cells, each with similar spindle orientations, around a central blastocoel. Right-left asymmetry was evident at the 32-cell-stage, and mirror-image embryos occurred in a 50:50 ratio. Gastrulation was initiated by invagination into the blastocoel at the 62-cell-stage of two mesendoderm cells, which arrested at the 32-cell-stage. Further invagination and expansion of the archenteron during gastrulation was accompanied by rapid and oriented cell division. The archenteron was composed of presumptive naupliar mesoderm and the blastopore was located at the site of the future anus of the nauplius larva. In order to trace cell lineages and determine axial relationships, single 2- and 4-cell-stage Blastomeres were microinjected with rhodamine-dextran. The results showed that the mesendoderm cells which initiated gastrulation were derived from the vegetal 2-cell-stage Blastomere, which could be distinguished by its slightly larger size and the location of the polar bodies. The mesendoderm cells descended from a single vegetal Blastomere of the 4-cell-stage. This investigation provides the first evidence for oriented cell division during gastrulation in a simple invertebrate system. Oriented cell division has previously been discounted as a potential morphogenetic force, and may be a common mechanism of invagination in embryos that begin gastrulation with a relatively small number of cells.
Philip L Hertzler - One of the best experts on this subject based on the ideXlab platform.
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mesendoderm cell and archenteron formation in isolated Blastomeres from the shrimp sicyonia ingentis
Developmental Biology, 1994Co-Authors: Philip L Hertzler, Steven W Wang, Wallis H ClarkAbstract:The fate map of 2- and 4-cell-stage Sicyonia ingentis embryos was determined by microinjection of lysyl-rhodamine-dextran into single Blastomeres. Microinjected embryos were cultured to the limb bud stage, when the body plan of the nauplius larva was evident. The animal Blastomere, AB, gave rise to anterior ectoderm, while the vegetal Blastomere, CD, gave rise to posterior structures, including the invagination site during gastrulation. The A Blastomere gave rise to mirror-image patterns of dorsal-lateral ectoderm, while the B Blastomere gave rise to anterior, ventral ectoderm. The C Blastomere gave rise to posterior, dorsal-lateral ectoderm, complementary to the A pattern, as well as some naupliar mesoderm. The D Blastomere gave rise to mesendoderm, naupliar mesoderm, and some posterior ectoderm. To study the specification of the early Blastomeres, they were microsurgically separated and cultured in isolation. Two mesendoderm cells formed in 1/2, 1/4, 1/8, and 1/16 Blastomeres in embryos dissociated at the 2-, 4-, 8-, and 16-cell stages, respectively. CD and D Blastomeres could be distin-Swished by their larger size and gave rise to the mesendoderm cells. Archenteron formation and elongation of the embryo occurred in CD but not in AB isolates. Isolated Blastomeres were recombined in various ways to determine whether their state of commitment could be altered in different cellular environments. Duplicated mesendoderm cells and archenterons formed in CD + CD recombinations, while AB + AB recombinations formed blastulae but did not produce mesendoderm cells and did not invaginate. The normal number of mesendoderm cells and a single archenteron formed in D + AB recombinations, while C + AB recombinations remained as blastulae and did not form mesndoderm cells. The results suggest that the mesendoderm cells are autonomously specified, possibly by cytoplasmic localization at the vegetal pole. The mesendoderm may also function as a signaling region to organize other developmental events.
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cleavage and gastrulation in the shrimp sicyonia ingentis invagination is accompanied by oriented cell division
Development, 1992Co-Authors: Philip L Hertzler, Wallis H ClarkAbstract:Embryos of the penaeoidean shrimp Sicyonia ingentis were examined at intervals during cleavage and gastrulation using antibodies to beta-tubulin and DNA and laser scanning confocal microscopy. Cleavage occurred in a regular pattern within four domains corresponding to the 4-cell-stage Blastomeres and resulted in two interlocking bands of cells, each with similar spindle orientations, around a central blastocoel. Right-left asymmetry was evident at the 32-cell-stage, and mirror-image embryos occurred in a 50:50 ratio. Gastrulation was initiated by invagination into the blastocoel at the 62-cell-stage of two mesendoderm cells, which arrested at the 32-cell-stage. Further invagination and expansion of the archenteron during gastrulation was accompanied by rapid and oriented cell division. The archenteron was composed of presumptive naupliar mesoderm and the blastopore was located at the site of the future anus of the nauplius larva. In order to trace cell lineages and determine axial relationships, single 2- and 4-cell-stage Blastomeres were microinjected with rhodamine-dextran. The results showed that the mesendoderm cells which initiated gastrulation were derived from the vegetal 2-cell-stage Blastomere, which could be distinguished by its slightly larger size and the location of the polar bodies. The mesendoderm cells descended from a single vegetal Blastomere of the 4-cell-stage. This investigation provides the first evidence for oriented cell division during gastrulation in a simple invertebrate system. Oriented cell division has previously been discounted as a potential morphogenetic force, and may be a common mechanism of invagination in embryos that begin gastrulation with a relatively small number of cells.
Norman J Dovichi - One of the best experts on this subject based on the ideXlab platform.
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single cell proteomics using frog xenopus laevis Blastomeres isolated from early stage embryos which form a geometric progression in protein content
Analytical Chemistry, 2016Co-Authors: Liangliang Sun, Kyle M Dubiak, Elizabeth H Peuchen, Zhenbin Zhang, Guijie Zhu, Paul W Huber, Norman J DovichiAbstract:Single cell analysis is required to understand cellular heterogeneity in biological systems. We propose that single cells (Blastomeres) isolated from early stage invertebrate, amphibian, or fish embryos are ideal model systems for the development of technologies for single cell analysis. For these embryos, although cell cleavage is not exactly symmetric, the content per Blastomere decreases roughly by half with each cell division, creating a geometric progression in cellular content. This progression forms a ladder of single-cell targets for the development of successively higher sensitivity instruments. In this manuscript, we performed bottom-up proteomics on single Blastomeres isolated by microdissection from 2-, 4-, 8-, 16-, 32-, and 50-cell Xenopus laevis (African clawed frog) embryos. Over 1 400 protein groups were identified in single-run reversed-phase liquid chromatography–electrospray ionization-tandem mass spectrometry from single balstomeres isolated from a 16-cell embryo. When the mass of yolk...
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Single Cell Proteomics Using Frog (Xenopus laevis) Blastomeres Isolated from Early Stage Embryos, Which Form a Geometric Progression in Protein Content
2016Co-Authors: Liangliang Sun, Kyle M Dubiak, Elizabeth H Peuchen, Zhenbin Zhang, Guijie Zhu, Paul W Huber, Norman J DovichiAbstract:Single cell analysis is required to understand cellular heterogeneity in biological systems. We propose that single cells (Blastomeres) isolated from early stage invertebrate, amphibian, or fish embryos are ideal model systems for the development of technologies for single cell analysis. For these embryos, although cell cleavage is not exactly symmetric, the content per Blastomere decreases roughly by half with each cell division, creating a geometric progression in cellular content. This progression forms a ladder of single-cell targets for the development of successively higher sensitivity instruments. In this manuscript, we performed bottom-up proteomics on single Blastomeres isolated by microdissection from 2-, 4-, 8-, 16-, 32-, and 50-cell Xenopus laevis (African clawed frog) embryos. Over 1 400 protein groups were identified in single-run reversed-phase liquid chromatography–electrospray ionization-tandem mass spectrometry from single balstomeres isolated from a 16-cell embryo. When the mass of yolk-free proteins in single Blastomeres decreased from ∼0.8 μg (16-cell embryo) to ∼0.2 μg (50-cell embryo), the number of protein group identifications declined from 1 466 to 644. Around 800 protein groups were quantified across four Blastomeres isolated from a 16-cell embryo. By comparing the protein expression among different Blastomeres, we observed that the Blastomere-to-Blastomere heterogeneity in 8-, 16-, 32-, and 50-cell embryos increases with development stage, presumably due to cellular differentiation. These results suggest that comprehensive quantitative proteomics on single Blastomeres isolated from these early stage embryos can provide valuable insights into cellular differentiation and organ development
Makoto Asashima - One of the best experts on this subject based on the ideXlab platform.
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effect of activin and lithium on isolated xenopus animal Blastomeres and response alteration at the midblastula transition
Development, 1995Co-Authors: Kei Kinoshita, Makoto AsashimaAbstract:Dorsoventral mesoderm patterning in the amphibian embryo involves a series of interactions mediated by several peptide growth factors. Animal Blastomeres isolated at the 8-cell stage are useful for studying mesoderm patterning, since they contain the prospective (uninduced) mesoderm region and allow examination of the default state of animal cells. When activin is applied to these dorsal and ventral animal half explants, a competence prepattern for responding to activin is observed. In order to investigate the characteristics of prepatterning, we treated animal Blastomeres with the embryo dorsalizing agent LiCl. Treatment with lithium alone did not induce normal trunk mesoderm in either Blastomere. Lithium did, however, alter the competence of animal Blastomeres to activin. Dorsal mesoderm was formed in the ventral Blastomeres, as well as in the dorsal Blastomeres. This result reveals that the early dorsoventral polarity in the animal hemisphere is not fixed. Using goosecoid(gsc) and Xwnt-8 genes as dorsal and ventral mesoderm markers, it was verified that lithium modifies the competence to activin. Unexpectedly, lithium treatment on its own resulted in gsc expression in the animal half explants. This suggests that embryo goosecoid expression may be induced by the effect of dorsal determination activity, but not by mesoderm induction. However, lithium induced also the expression of brachyury (Xbra) gene at very low levels. This would indicate the formation of dorsal-anterior mesoderm, which was not identified by the tissue observations. Expression of Xwnt-8, a ventral mesoderm marker usually induced in blastula animal caps by activin, was hardly induced in the Blastomere explants. We isolated whole animal half explants at the 8-cell stage and exposed to activin at different stages. It was found that the same concentration of activin induces gsc before the midblastula stage, and induces Xwnt-8 at later stages. This suggests that the response of animal Blastomeres alters depending on the stage of activin signaling.
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competence prepattern in the animal hemisphere of the 8 cell stage xenopus embryo
Developmental Biology, 1993Co-Authors: Kei Kinoshita, Tomoko Bessho, Makoto AsashimaAbstract:Abstract Activins are known to be potentially important regulators in Xenopus developmental processes. It has been shown that activins exist maternally in the egg and can induce mesodermal tissues in blastula animal cap explants. However, the blastula ectoderm is known to possess a predisposed local response pattern to activin, and the process of the prepatterning is not understood. We isolated animal hemispheres from late 8-cell-stage embryos and treated them briefly with activin A. Expression of the muscle-specific actin gene was induced after a 30-min activin treatment, even when it was followed by treatment with follistatin, an activin-specific binding protein. This suggests that the animal-half Blastomeres become competent to activin A before the 16-cell stage. In the normal embryo, the 8-cell stage animal dorsal Blastomeres populate neural ectoderm and most of the dorsal lip of the gastrula blastopore, the region of Spemann's organizer, and are the major progenitor for dorsal mesodermal tissues. When the dorsal and ventral animal-half Blastomeres of the 8-cell stage were isolated and treated with activin independently, significant differences in tissue differentiation were observed. Dorsal Blastomeres gave rise to trunk and tail structures containing dorsal mesoderm, whereas the ventral Blastomere explants formed spheres containing solely ventral mesoderm. Further, both muscle actin transcription and goosecoid transcription were induced primarily in dorsal Blastomeres. Our results suggest that a competence prepattern of response to activin exists as early as the 8-cell stage.
Sally A. Moody - One of the best experts on this subject based on the ideXlab platform.
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cleavage Blastomere deletion and transplantation to test cell fate commitment in xenopus
CSH Protocols, 2019Co-Authors: Sally A. MoodyAbstract:Fate maps identify the precursors of an organ, and tracing the members of a Blastomere lineage over time shows how its descendants come to populate that organ. The fates of the individual Blastomeres of the two- to 32-cell Xenopus embryo have been fully mapped to reveal which cells are the major contributors to various cell types, tissues, and organs. However, because these fate maps were produced in the normal embryo, they do not reveal whether a precursor Blastomere is competent to give rise to additional tissues or is already committed to its fate-mapped repertoire of descendants. To identify the mechanisms by which a cell's fate is committed, one needs to expose the cell to different experimental environments. If the cell's fate is determined, it will express its normal fate or gene expression profile in novel environments, whereas if it is not yet determined it will express different fates or gene expression profiles when exposed to novel external factors or neighboring cells. This protocol describes two techniques for testing cell fate commitment: single cell deletion and single cell transplantation. Deleting a Blastomere allows one to test whether the deleted cell is required for the remaining cells to produce their normal, specific cell fates. Transplanting a Blastomere to a novel location in a host embryo allows one to test whether the transplanted cell is committed to produce its normal fate-mapped repertoire, or whether it is still competent to respond to novel cell-cell interactions.
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microinjection of mrnas and oligonucleotides
CSH Protocols, 2018Co-Authors: Sally A. MoodyAbstract:: Microinjecting lineage tracers into a single Blastomere in the normal, intact embryo identifies the repertoire of cell types derived from it. In order to reveal the full developmental potential of that Blastomere or identify the mechanisms by which its fate is determined, one needs to modify its gene expression under controlled experimental conditions. One method by which this is easily accomplished in Xenopus is by microinjecting synthetic mRNAs or antisense oligonucleotides into an identified Blastomere to target altered gene expression specifically to its lineage. Xenopus Blastomeres are robust and tolerate pressure-driven microinjection up to a few hundred cells, and they efficiently translate exogenously supplied mRNAs. Targeted microinjections, described here, significantly reduce off-target effects of the mRNAs or oligonucleotides. Many types of constructs can be synthesized to provide specific information about gene function. For example, microinjecting mRNA encoding the wild-type gene in its normal expression domain or in an ectopic site tests whether it promotes or represses target genes or alters the formation of tissues of interest. Mutant forms of a gene transcript can illuminate the function of different domains of the encoded protein or show the developmental consequences of a mutation found in a human disease. mRNAs encoding dominant-negative forms of a protein can elicit a functional knockdown and thereby establish the necessity for that gene in a developmental process. Microinjecting antisense morpholino oligonucleotides (MOs) that are designed to block either endogenous mRNA translation or splicing is an effective method to reduce the levels of endogenous protein.
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neural transcription factors bias cleavage stage Blastomeres to give rise to neural ectoderm
Genesis, 2016Co-Authors: Shailly Gaur, Max Mandelbaum, Mona Herold, Himani D Majumdar, Karen M Neilson, Thomas M Maynard, Kathy Mood, Ira O Daar, Sally A. MoodyAbstract:Summary The decision by embryonic ectoderm to give rise to epidermal versus neural derivatives is the result of signaling events during blastula and gastrula stages. However, there also is evidence in Xenopus that cleavage stage Blastomeres contain maternally derived molecules that bias them toward a neural fate. We used a Blastomere explant culture assay to test whether maternally deposited transcription factors bias 16-cell Blastomere precursors of epidermal or neural ectoderm to express early zygotic neural genes in the absence of gastrulation interactions or exogenously supplied signaling factors. We found that Foxd4l1, Zic2, Gmnn, and Sox11 each induced explants made from ventral, epidermis-producing Blastomeres to express early neural genes, and that at least some of the Foxd4l1 and Zic2 activities are required at cleavage stages. Similarly, providing extra Foxd4l1 or Zic2 to explants made from dorsal, neural plate-producing Blastomeres significantly increased the expression of early neural genes, whereas knocking down either significantly reduced them. These results show that maternally delivered transcription factors bias cleavage stage Blastomeres to a neural fate. We demonstrate that mouse and human homologs of Foxd4l1 have similar functional domains compared to the frog protein, as well as conserved transcriptional activities when expressed in Xenopus embryos and Blastomere explants. genesis 54:334–349, 2016. © 2016 Wiley Periodicals, Inc.
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testing retina fate commitment in xenopus by Blastomere deletion transplantation and explant culture
Methods of Molecular Biology, 2012Co-Authors: Sally A. MoodyAbstract:The lineages of individual cells of the Xenopus cleavage-stage embryo have been fate-mapped to reveal the subset of Blastomeres that are the major and minor precursors of the retina. Using this retina fate map, one can test the commitment of each of these cells to various retinal cell fates by manipulating the environment in which they develop. This chapter presents the techniques for identifying specific retina Blastomere precursor cells, deleting them to test whether they are required for producing specific kinds of retinal cells, transplanting them to novel embryonic locations in host embryos to test whether they are committed to produce specific kinds of retinal cells, and growing them in explant culture to determine if their ability to produce specific kinds of retinal cells is autonomous.