Biomedical subjects

A Ransick

Publications and source records attributed to A Ransick.

11 recordsLinked to original sources

PubMed2000

Recovery of developmentally defined gene sets from high-density cDNA macroarrays.

New technologies for isolating differentially expressed genes from large arrayed cDNA libraries are reported. These methods can be used to identify genes that lie downstream of developmentally important transcription factors and genes that are expressed in specific tissues, processes, or stages of embryonic development. Though developed for the study of gene expression during the early embryogenesis of the sea urchin Strongylocentrotus purpuratus, these technologies can be applied generally. Hybridization parameters were determined for the reaction of complex cDNA probes to cDNA libraries carried on six nylon filters, each containing duplicate spots from 18,432 bacterial clones (macroarrays). These libraries are of sufficient size to include nearly all genes expressed in the embryo. The screening strategy we have devised is designed to overcome inherent sensitivity limitations of macroarray hybridization and thus to isolate differentially expressed genes that are represented only by low-prevalence mRNAs. To this end, we have developed improved methods for the amplification of cDNA from small amounts of tissue (as little as approximately 300 sea urchin embryos, or 2 x 10(5) cells, or about 10 ng of mRNA) and for the differential enhancement of probe sequence concentration by subtractive hybridization. Quantitative analysis of macroarray hybridization shows that these probes now suffice for detection of differentially expressed mRNAs down to a level below five molecules per average embryo cell.

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PubMed1998

Disruption of primary mesenchyme cell patterning by misregulated ectodermal expression of SpMsx in sea urchin embryos.

The patterning of the mesoderm of the sea urchin embryo is a classical paradigm of epithelial mesenchymal interactions in organogenesis, yet little is known of its molecular basis. Here we address the role of the homeobox gene, SpMsx, a member of the highly conserved Msx gene family, in this process. Msx genes have been shown to function in the dorsoventral patterning of the central nervous system in Drosophila and in a variety epithelial-mesenchymal interactions in vertebrates. We showed previously that the SpMsx gene is expressed during embryogenesis in a complex and dynamic pattern consistent with roles in the development of subpopulations of endoderm, mesoderm, and oral ectoderm. To perturb this pattern of expression and thus probe the function of SpMsx, we injected SpMsx mRNA into single-cell zygotes and monitored development morphologically and with a series of territory-specific molecular markers. RT-PCR analysis revealed that injected SpMsx transcripts persisted at least until the gastrula stage in amounts comparable to endogenous levels. Injected embryos exhibited deficiencies in the organization of primary and secondary mesenchyme cells within the blastocoelic cavity, as well as abnormalities in spicule number and shape. Defects in the endoderm were also common, including reduced or absent archenterons. Micromere transplantation experiments revealed that the defects in skeletogenic mesenchyme patterning were non-cell autonomous, consistent with findings that cell-cell interactions between ectoderm and the progenitors of the skeletogenic mesenchyme, the primary mesenchyme cells (PMCs), are important both for PMC guidance and spicule morphogenesis. Our data, taken together with observations in other organisms on the role of Msx genes in embryonic signaling processes, particularly involving the BMP pathway, suggest that SpMsx may be a part of the mechanism by which the ectoderm influences both the arrangement of primary mesenchyme cells within the blastocoel and the shapes of the skeletal rods.

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PubMed1998

Late specification of Veg1 lineages to endodermal fate in the sea urchin embryo.

Single blastomeres of the sixth-cleavage veg1 and veg2 tiers of Strongylocentrotus purpuratus embryos were labeled with DiI lineage tracer, and the disposition of the progeny was followed through the blastula and gastrula stages in order to determine their respective endodermal and ectodermal contributions. In the endoderm of postgastrula embryos, veg1-derived cells constituted nearly all of the prospective hindgut and about half of the prospective midgut, while veg2-derived cells made up the prospective foregut and half the midgut. Oral veg1 clones consistently contributed more cells to endoderm than aboral veg1 clones. Oral veg1 clones extended along the archenteron up to the foregut region, while aboral veg1 clones contributed only small numbers of hindgut cells but large patches of ectoderm cells that extended out to the prospective larval vertex. The oral/aboral asymmetry in veg1 allocations was also demonstrated using chimeric embryos, the animal halves of which were labeled with a rhodamine-dextran. Lineages expressing the vegetal plate marker Endo16 were more precisely determined by combining lineage tracer injection with whole-mount in situ hybridization. Endo16 expression was found in all cells that are going to participate in gastrulation. Recruitment of new cells to the Endo16 domain occurs in advance of the actual invagination of those cells. During the blastula stages Endo16 expression expands radially until all cells in the veg2 lineages express this gene. The first phase of gastrulation, including the normal buckling of the vegetal plate and primary invagination of the archenteron, involves only the Endo16-expressing cells of the veg2 lineages. As the archenteron begins to elongate, marking the onset of the second phase of gastrulation, there is an asymmetric expansion of Endo16 into the veg1-derived cells that will contribute to the hindgut and midgut in accordance with lineage tracing observations. The results indicate a relatively late specification of veg1-derived cells, resulting in late recruitment to the periphery of the vegetal plate territory as gastrulation proceeds. Differential recruitment of veg1-derived cells on the oral side of the embryo introduces an oral bias to gastrulation by disproportionately increasing the number of cells on the oral side that are competent to participate in gastrulation.

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PubMed1998

Specification of cell fate in the sea urchin embryo: summary and some proposed mechanisms.

An early set of blastomere specifications occurs during cleavage in the sea urchin embryo, the result of both conditional and autonomous processes, as proposed in the model for this embryo set forth in 1989. Recent experimental results have greatly illuminated the mechanisms of specification in some early embryonic territories, though others remain obscure. We review the progressive process of specification within given lineage elements, and with reference to the early axial organization of the embryo. Evidence for the conditional specification of the veg2 lineage subelement of the endoderm and other potential interblastomere signaling interactions in the cleavage-stage embryo are summarized. Definitive boundaries between mesoderm and endoderm territories of the vegetal plate, and between endoderm and overlying ectoderm, are not established until later in development. These processes have been clarified by numerous observations on spatial expression of various genes, and by elegant lineage labeling studies. The early specification events depend on regional mobilization of maternal regulatory factors resulting at once in the zygotic expression of genes encoding transcription factors, as well as downstream genes encoding proteins characteristic of the cell types that will much later arise from the progeny of the specified blastomeres. This embryo displays a maximal form of indirect development. The gene regulatory network underlying the embryonic development reflects the relative simplicity of the completed larva and of the processes required for its formation. The requirements for postembryonic adult body plan formation in the larval rudiment include engagement of a new level of genetic regulatory apparatus, exemplified by the Hox gene complex.

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PubMed1996

Postembryonic segregation of the germ line in sea urchins in relation to indirect development.

The four small micromeres of the sea urchin embryo contribute only to the coelomic sacs, which produce major components of the adult body plan during postembryonic development. To test the proposition that the small micromeres are the definitive primordial germ cell lineage of the sea urchin, we deleted their 4th cleavage parents, and raised the deleted embryos through larval life and metamorphosis to sexual maturity. Almost all of the experimental animals produced functional gametes, excluding the possibility that the germ cell lineage arises exclusively and obligatorily from descendants of the small micromeres; rather, the germ cell lineage arises during the postembryonic development of the rudiment. A survey of the literature indicates that there is no known case of an embryonic primordial germ cell lineage in a bilaterian species that displays maximal indirect development.

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PubMed1995

Micromeres are required for normal vegetal plate specification in sea urchin embryos.

Vegetal plate specification was assessed in S. purpuratus embryos after micromere deletions at the 4th, 5th and 6th cleavages, by assaying expression of the early vegetal plate marker Endo 16, using whole-mount in situ hybridization. After 4th cleavage micromere deletions, the embryos typically displayed weak Endo16 expression in relatively few cells of the lineages that normally constitute the vegetal plate, while after 5th and 6th cleavage micromere deletions the embryos exhibited strong Endo16 expression in larger fractions of cells belonging to those lineages. When all four micromeres were deleted, the embryos were severely delayed in initiating gastrulation and sometimes failed to complete gastrulation. However, if only one micromere was allowed to remain in situ throughout development, the embryos exhibited strong Endo16 expression and gastrulation occurred normally, on schedule with controls. Additional measurements showed that these microsurgical manipulations do not alter cleavage rates or generally disrupt embryo organization. These results constitute direct evidence that the micromeres provide signals required by the macromere lineages for initiation of vegetal plate specification. The specification of the vegetal plate is completed in a normal manner only if micromere signaling is allowed to continue at least to the 6th cleavage stage.

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PubMed1994

Complexity and organization of DNA-protein interactions in the 5'-regulatory region of an endoderm-specific marker gene in the sea urchin embryo.

This study concerns the organization of sites of specific DNA/protein interaction within the regulatory domain of the Endo16 gene of Strongylocentrotus purpuratus. Earlier work had displayed a complex pattern of expression of this gene during embryogenesis. Endo16 transcripts are confined to the definitive vegetal plate in blastula stage embryos; at gastrula stage this gene is expressed throughout the archenteron, but later only in the midgut. In this work we exploited the exceptional experimental accessibility of the sea urchin embryo, with respect to both functional assays of gene regulatory systems and to characterization of transcription factors, in order to approach a complete description of potential Endo16 regulatory interactions. Accurate expression of an Endo16 fusion gene was obtained with a 2200-nucleotide (nt) upstream fragment of the gene. We present a map locating high specificity target sites for DNA-binding proteins within the 2200-nt Endo16 regulatory domain, and an assessment of the complexity of the set of putative Endo16 transcription factors that we have been able to recover from 24-h (blastula stage) nuclear extract. Protein binding sites were initially mapped by gel shift reactions carried out on nested sets of end-labeled restriction fragments, and then to finer resolution by oligonucleotide gel shift competitions. Thirty-eight sites of high specificity DNA-protein interaction were thus identified. Appropriate oligonucleotides were then used for partial purification of the DNA-binding proteins by affinity chromatography. DNA-binding proteins specific for each target site were identified by molecular weight, using southwestern blotting procedures and two-dimensional gel shift separations, and by directly renaturing and reacting with oligonucleotide probes specific proteins that had been resolved by SDS-PAGE from selected affinity column fractions. A complete series of gel shift cross-competitions amongst the target sites was carried out. We conclude that nine different protein factors are bound at unique sites within the Endo16 regulatory domain. Multiple target sites for five other proteins account for the remaining binding site locations. The target sites appear to be organized in a sequence of clusters, focused on the unique factors. The high complexity of the Endo16 gene regulatory system may be characteristic for genes that are spatially regulated in early embryonic development.

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PubMed1993

A complete second gut induced by transplanted micromeres in the sea urchin embryo.

Founder cells for most early lineages of the sea urchin embryo are probably specified through inductive intercellular interactions. It is shown here that a complete respecification of cell fate occurs when 16-cell stage micromeres from the vegetal pole of a donor embryo are implanted into the animal pole of an intact recipient embryo. Animal pole cells adjacent to the transplanted micromeres are respecified from presumptive ectoderm into vegetal plate founder cells. These induced vegetal plate cells express the entire battery of genes characteristic of the endogenous vegetal plate cells. The ectopic vegetal plate invaginates during gastrulation to form a second archenteron which differentiates properly into a tripartite gut, as shown by the spatial pattern of expression of an endoderm-specific marker gene. Thus, transplanted micromeres can signal neighboring cells to induce them to change their fate.

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PubMed1993

Whole mount in situ hybridization shows Endo 16 to be a marker for the vegetal plate territory in sea urchin embryos.

We have used whole mount in situ hybridization to analyze the pattern of expression of the gene Endo 16 in S. purpuratus embryos. The mRNA is first detectable at 18 h post-fertilization in the cytoplasm of blastomeres derived from the Veg2 6th cleavage tier. The number of Endo 16 positive cells increases gradually through the beginning of gastrulation, and these cell numbers are in agreement with estimates of the number of cells that should be in the vegetal plate at these stages. We conclude that Endo 16 expression is indeed an early vegetal plate marker and that this gene is expressed by all Veg2 tier derivatives while they are part of the vegetal plate. The progressive regionalization of Endo 16 expression that occurs in normal embryos is also seen in lithium chloride induced exogastrulae, leading to the conclusion that genetic regulation of endoderm differentiation is programmed into the vegetal plate cells once they have been specified. Finally, we report a reproducible phenomenon seen in cultures of LiCl exogastrulae, in which the tips of the everted archenterons fuse, followed by the induction of supernumerary pigment cells.

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PubMed1993

The relationship between cell size and cell fate in Volvox carteri.

In Volvox carteri development, visibly asymmetric cleavage divisions set apart large embryonic cells that will become asexual reproductive cells (gonidia) from smaller cells that will produce terminally differentiated somatic cells. Three mechanisms have been proposed to explain how asymmetric division leads to cell specification in Volvox: (a) by a direct effect of cell size (or a property derived from it) on cell specification, (b) by segregation of a cytoplasmic factor resembling germ plasm into large cells, and (c) by a combined effect of differences in cytoplasmic quality and cytoplasmic quantity. In this study a variety of V. carteri embryos with genetically and experimentally altered patterns of development were examined in an attempt to distinguish among these hypotheses. No evidence was found for regionally specialized cytoplasm that is essential for gonidial specification. In all cases studied, cells with a diameter > approximately 8 microns at the end of cleavage--no matter where or how these cells had been produced in the embryo--developed as gonidia. Instructive observations in this regard were obtained by three different experimental interventions. (a) When heat shock was used to interrupt cleavage prematurely, so that presumptive somatic cells were left much larger than they normally would be at the end of cleavage, most cells differentiated as gonidia. This result was obtained both with wild-type embryos that had already divided asymmetrically (and should have segregated any cytoplasmic determinants involved in cell specification) and with embryos of a mutant that normally produces only somatic cells. (b) When individual wild-type blastomeres were isolated at the 16-cell stage, both the anterior blastomeres that normally produce two gonidia each and the posterior blastomeres that normally produce no gonidia underwent modified cleavage patterns and each produced an average of one large cell that developed as a gonidium. (c) When large cells were created microsurgically in a region of the embryo that normally makes only somatic cells, these large cells became gonidia. These data argue strongly for a central role of cell size in germ/soma specification in Volvox carteri, but leave open the question of how differences in cell size are actually transduced into differences in gene expression.

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PubMed1991

Reproductive cell specification during Volvox obversus development.

Asexual spheroids of the genus Volvox contain only two cell types: flagellated somatic cells and immotile asexual reproductive cells known as gonidia. During each round of embryogenesis in Volvox obversus, eight large gonidial precursors are produced at the anterior extremity of the embryo. These cells arise as a consequence of polarized, asymmetric divisions of the anteriormost blastomeres at the fourth through nine cleavage cycles, while all other blastomeres cleave symmetrically to yield somatic cell precursors. Blastomeres isolated from embryos at any point between the 2-cell and the 32-cell stage cleaved in the normal pattern and produced the same complement and spatial distribution of cell types as they would have in an intact embryo. This result indicates that intrinsic features control the cleavage patterns and developmental potentials of blastomeres, and rules out any significant role for cell-cell interactions in gonidial specification. When substantial quantities of anterolateral cytoplasm were deleted from uncleaved gonidia or 4-cell stage blastomeres, the cell fragments frequently regulated and embryos were produced with the expected number of asymmetrically cleaving cells and gonidial precursors at their anterior ends. However, when anterior cytoplasm was deleted from 8-cell stage blastomeres, the depleted cells frequently failed to cleave asymmetrically and produced no gonidial precursors. Furthermore, when compression was used to reorient cleavage planes at the fourth division cycle, so that anterior cytoplasm was transmitted to more than the normal number of cells, those cells receiving a significant amount of such cytoplasm cleaved asymmetrically to produce supernumerary gonidial precursors. Together, these last two experiments indicate that blastomeres in the V. obversus embryo acquire (at least by the end of the third cleavage cycle) a polarized organization in which anterior cytoplasm plays a causal role in the process of reproductive-cell specification.

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