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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.
Craig P Hunter - One of the best experts on this subject based on the ideXlab platform.
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the homeodomain protein pal 1 specifies a lineage specific regulatory network in the c elegans embryo
Development, 2005Co-Authors: Ryan L Baugh, Andrew A Hill, Julia M Claggett, Kate Hillharfe, Joanne C Wen, Donna K Slonim, Eugene L Brown, Craig P HunterAbstract:Maternal and zygotic activities of the homeodomain protein PAL-1 specify the identity and maintain the development of the multipotent C blastomere lineage in the C. elegans embryo. To identify PAL-1 regulatory target genes, we used microarrays to compare transcript abundance in wild-type embryos with mutant embryos lacking a C blastomere and to mutant embryos with extra C Blastomeres. pal-1-dependent C-lineage expression was verified for select candidate target genes by reporter gene analysis, though many of the target genes are expressed in additional lineages as well. The set of validated target genes includes 12 transcription factors, an uncharacterized wingless ligand and five uncharacterized genes. Phenotypic analysis demonstrates that the identified PAL-1 target genes affect specification, differentiation and morphogenesis of C-lineage cells. In particular, we show that cell fate-specific genes (or tissue identity genes) and a posterior HOX gene are activated in lineage-specific fashion. Transcription of targets is initiated in four temporal phases, which together with their spatial expression patterns leads to a model of the regulatory network specified by PAL-1.
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spatial and temporal controls target pal 1 blastomere specification activity to a single blastomere lineage in c elegans embryos
Cell, 1996Co-Authors: Craig P Hunter, Cynthia KenyonAbstract:The early asymmetric cleavages of Caenorhabditis elegans embryos produce Blastomeres with distinct developmental potentials. Here, we show that the caudal-like homeodomain protein PAL-1 is required to specify the somatic identity of one posterior blastomere in the 4 cell embryo. We find that pal-1 activity is sequentially restricted to this blastomere. First, at the 4 cell stage, it is translated only in the two posterior Blastomeres. Then, its function is restricted to one of these Blastomeres. This second targeting step is dependent on the activities of the posteriorly localized SKN-1 and asymmetrically segregated PIE-1 proteins. We propose that the segregation of PIE-1, combined with the temporal decay of SKN-1, targets pal-1 activity to this posterior lineage, thus coupling the regulation of this conserved posterior patterning gene to asymmetric cell cleavages.
Rueyling Lin - One of the best experts on this subject based on the ideXlab platform.
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multiple rna binding proteins function combinatorially to control the soma restricted expression pattern of the e3 ligase subunit zif 1
Developmental Biology, 2012Co-Authors: Marieke Oldenbroek, Scott M Robertson, Tugba Guvenozkan, Steven Gore, Yuichi Nishi, Rueyling LinAbstract:In C. elegans embryos, transcriptional repression in germline Blastomeres requires PIE-1 protein. Germline blastomere-specific localization of PIE-1 depends, in part, upon regulated degradation of PIE-1 in somatic cells. We and others have shown that the temporal and spatial regulation of PIE-1 degradation is controlled by translation of the substrate-binding subunit, ZIF-1, of an E3 ligase. We now show that ZIF-1 expression in embryos is regulated by five maternally-supplied RNA-binding proteins. POS-1, MEX-3, and SPN-4 function as repressors of ZIF-1 expression, whereas MEX-5 and MEX-6 antagonize this repression. All five proteins bind directly to the zif-1 3′ UTR in vitro. We show that, in vivo, POS-1 and MEX-5/6 have antagonistic roles in ZIF-1 expression. In vitro, they bind to a common region of the zif-1 3′ UTR, with MEX-5 binding impeding that by POS-1. The region of the zif-1 3′ UTR bound by MEX-5/6 also partially overlaps with that bound by MEX-3, consistent with their antagonistic functions on ZIF-1 expression in vivo. Whereas both MEX-3 and SPN-4 repress ZIF-1 expression, neither protein alone appears to be sufficient, suggesting that they function together in ZIF-1 repression. We propose that MEX-3 and SPN-4 repress ZIF-1 expression exclusively in 1- and 2-cell embryos, the only period during embryogenesis when these two proteins co-localize. As the embryo divides, ZIF-1 continues to be repressed in germline Blastomeres by POS-1, a germline blastomere-specific protein. MEX-5/6 antagonize repression by POS-1 and MEX-3, enabling ZIF-1 expression in somatic Blastomeres. We propose that ZIF-1 expression results from a net summation of complex positive and negative translational regulation by 3′ UTR-binding proteins, with expression in a specific blastomere dependent upon the precise combination of these proteins in that cell.
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wnt signaling and an apc related gene specify endoderm in early c elegans embryos
Cell, 1997Co-Authors: Christian E Rocheleau, Rueyling Lin, William D Downs, Claudia Wittmann, Yanxia Bei, Yoon Hee Cha, Mussa AliAbstract:In a 4-cell stage C. elegans embryo, signaling by the P2 blastomere induces anterior-posterior polarity in the adjacent EMS blastomere, leading to endoderm formation. We have taken genetic and reverse genetic approaches toward understanding the molecular basis for this induction. These studies have identified a set of genes with sequence similarity to genes that have been shown to be, or are implicated in, Wnt/Wingless signaling pathways in other systems. The C. elegans genes described here are related to wnt/wingless, porcupine, frizzled, beta-catenin/armadillo, and the human adenomatous polyposis coli gene, APC. We present evidence that there may be partially redundant inputs into endoderm specification and that a subset of these genes appear also to function in determining cytoskeletal polarity in certain early Blastomeres.
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
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.