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Biomedical subjects

J Heasman

Publications and source records attributed to J Heasman.

At least 55 records · Page 3Linked to original sources
PubMed1992

Involvement of a neutral glycolipid in differential cell adhesion in the Xenopus blastula.

Many different molecular species mediate cell adhesion during embryonic development. These can have either protein or carbohydrate functional groups, which can act in either a homophilic or a heterophilic manner, and often in concert. We report here that a monoclonal antibody, M4B, raised against Xenopus blastomere membranes, inhibits the calcium-dependent adhesion of dissociated blastomeres. M4B maintains its inhibitory effect on adhesion when converted into univalent fragments, and specifically affects calcium-dependent adhesion. The antigen is regulated in both space and time during early development. It is found on cell surfaces throughout the egg to blastula stages, but is more concentrated on cells in the animal and marginal zones of the blastula. It is dramatically downregulated during gastrulation, and becomes largely restricted to gut epithelium by the larval stages. We show also that M4B function is spatially differentiated at the blastula stage, since it inhibits the aggregation of dissociated animal cells to a greater extent than vegetal cells. This membrane antigen may therefore play a role in the differential adhesion observed between different regions of the blastula, and which we presume to underlie the segregation of the primary germ layers during gastrulation. M4B recognizes a complex of plasma membrane glycolipids. Periodate treatment destroys the ability of these glycolipids to react with the antibody, indicating that the epitope resides in the carbohydrate moiety of the glycolipids. Chemical characterization shows that it is a neutral glycolipid, and that the major component is of the glycoglycerolipid, rather than the more common glycosphingolipid class. Blocking experiments with oligosaccharides of defined structure, and antibody crossreactivity show that the M4B antibody does not recognize several known embryonic carbohydrate antigens. These results demonstrate that M4B antibody recognizes a novel group of developmentally regulated glycolipids which function in calcium-dependent cell--cell adhesion in the Xenopus blastula.

Animals↗
PubMed1992

Distinct distribution of vimentin and cytokeratin in Xenopus oocytes and early embryos.

We report the identity of a major component of Triton-insoluble extracts from Xenopus oocytes and early embryos. In a previous paper we showed that an antibody, Z9, cross-reacts with two polypeptides from such extracts (Mr 56,000 and 57,000) as well as Xenopus vimentin. Direct microsequencing of the Mr 57,000 protein shows near identity of three tryptic fragments with regions of the predicted amino acid sequence of XCK1(8), a basic cytokeratin whose mRNA is known to be expressed in Xenopus oocytes. We have raised an antibody, CK7, against a fusion protein generated from this cDNA. The specificity of this antibody has been tested using 1- and 2-dimensional immunoblotting, which show that it is specific for the Mr 56,000 and 57,000 proteins, suggesting that these two proteins may be the products of two non-allelic XCK1(8) genes. The antibody does not cross-react with vimentin. We have used CK7 to follow the distribution of XCK1(8) throughout development by immunoblotting and immunocytochemistry. In larval stages, strong staining is seen in the notocord, the apical epithelia of the gut, the mesentery, and a few cells in the spinal cord. In oocytes and early embryos, two distinct intermediate filament (IF) networks can be distinguished: a cortical cytokeratin network, and a deeper vimentin one. In addition, the oocyte germ plasm stains with Z9 but not CK7. We propose that such distinct distributions of each IF protein reflect functional differences during early development.

Amino Acid Sequence↗
PubMed1992

The role of intermediate filaments in early Xenopus development studied by antisense depletion of maternal mRNA.

The effects of depleting a maternal cytokeratin mRNA on the developing embryo are described. Cytokeratins are members of the intermediate filament family of cytoskeletal proteins, and are expressed in a cortical network of the superficial cytoplasm of the oocyte. After fertilisation, a new cortical network is built up, which comes to occupy only the most superficial cells of the blastula. The maternal cytokeratin mRNA is abundantly translated, both during oogenesis, and during oocyte maturation and after fertilisation. Depletion of the mRNA results in depletion of the cortical filaments at the blastula stage and leads to gastrulation abnormalities. We discuss the various possible control experiments required for antisense oligo depletion studies and the implications of these results for cytokeratin function.

Animals↗
PubMed1991

Fertilization of cultured Xenopus oocytes and use in studies of maternally inherited molecules.

The methods described here of fertilizing stage VI oocytes are lengthy and quite difficult techniques. They would become more attractive if the success rate (i.e., the number of fertilizations compared to the numbers of matured oocytes) could be improved. An important step toward this for the host transfer technique would be to monitor carefully the status of mature Xenopus females ovaries in relation to cyclical HCG stimulation, so that we could predict more accurately whether stage VI oocytes are fertilizable. The in vitro technique would obviously be improved if oocytes could be fertilized without removing their membranes, perhaps by using oviduct extracts. So far, this approach has had only limited success. It seems that the rewards of using these techniques could be great, in terms of understanding the maternal contribution to development. Although our experiments have not yet shown that oocyte injection of DNA has any advantage over egg injection, it is clear that it is possible to make "mRNA-minus mutants" by this approach. In the message depletion experiments mentioned here, we targeted the cleavage of an mRNA which is of low abundance in the full grown oocyte, but preliminary experiments have shown that we can deplete more abundant messages and produce specific phenotypes. Of course such experiments need to be controlled to show that the effect is specific, and the best proof that this is the case is to rescue the effect with injection of the appropriate mRNA. Finally, it seems likely that the method can be used to study the function of both localized molecules, such as the putative primordial germ cell (PGC) or dorsal determinants, and more ubiquitous molecules such as cytoskeletal elements.

Animals↗
PubMed1991

Response to fibronectin of mouse primordial germ cells before, during and after migration.

The adhesive extracellular matrix glycoprotein fibronectin is thought to play a central role in cell migration during embryogenesis. In order to define this role, we have examined the response to fibronectin in cell culture of mouse primordial germ cells (PGCs) before, during and after their migration from the hindgut into their target tissue, the genital ridges. Using an explant culture system, we show that PGCs will emigrate from tissue fragments containing hindgut, and that fibronectin stimulates this migration. Adhesion assays show that the start of PGC migration is associated with a fall in adhesion to fibronectin. Double-labelling studies using in situ hybridization and histochemistry demonstrate that migrating PGCs do not contain detectable fibronectin mRNA, suggesting that they do not synthesize and secrete the fibronectin within their migratory substratum. Taken together, these findings are consistent with an important role for fibronectin in stimulating PGC migration. In addition, however, they suggest that the interaction between PGCs and fibronectin may be important in timing the start of migration, with the fall in adhesion allowing the PGCs to commence their migration towards the genital ridges.

Animals↗
PubMed1990

Genital ridges exert long-range effects on mouse primordial germ cell numbers and direction of migration in culture.

The functional gametes of all vertebrates first arise in the early embryo as a migratory population of cells, the primordial germ cells (PGCs). These migrate to, and colonise, the genital ridges (GR) during the early organogenesis period, giving rise to the complete differentiating gonad. PGCs first become visible by alkaline phosphatase staining in the root of the developing allantois at 8.5 days post coitum (dpc). At 9.5 dpc they are found in the wall of the hind-gut and, during the following three days, they migrate along the hind-gut mesentery to the dorsal body wall, and then to the genital ridges. By 12.5 dpc, the great majority of PGCs have colonised the genital ridges. During this period the number of PGCs increases from less than 100 to approximately 4000. In a previous paper (Donovan et al. 1986), we showed that 10.5 dpc PGCs can be explanted from the hind-gut mesentery, and will spread and migrate on feeder cell layers. We showed also that the intrinsic ability of PGCs to spread and migrate changes as they colonise the genital ridges. In this paper, we examine extrinsic factors that control PGC behaviour in vitro. Using PGCs taken from 8.5 dpc embryos, at the beginning of their migratory phase, we show that culture medium conditioned by 10.5 dpc genital ridges causes an increase in the number of PGCs in these cultures. We also show that PGCs migrate towards 10.5 dpc genital ridges in preference to other explanted organs. These experiments show that genital ridges exert long-range effects on the migrating population of PGCs.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Identification of vimentin and novel vimentin-related proteins in Xenopus oocytes and early embryos.

We have made antibodies against fusion proteins of Xenopus vimentin. We show for the first time the distribution of vimentin in larval stages, where it is found in cells of mesenchymal origin, and in radial glial cells. In sections of Xenopus oocytes and early embryos, immunocytochemistry reveals the presence of an extensive cytoplasmic network, distributed in an animal-vegetal gradient. Germ plasm stains particularly strongly. The form of the IF proteins in this network is unusual. In immunoblot experiments the anti-vimentin antibodies detect a number of distinct proteins. We have identified those that are the products of the two known vimentin genes, by injection of synthetic mRNA transcribed from cloned vimentin cDNAs into oocytes, followed by two-dimensional Western blotting. This has demonstrated unambiguously that one Xenopus vimentin, Vim1, is present in oocytes and early embryos. However, two other immunoreactive proteins detected in Triton extracts of oocytes and early embryos are not the products of Vim1, since depletion of vimentin mRNA by antisense oligonucleotide injection has no effect on the synthesis of these proteins. These results suggest that novel IF-like proteins are expressed in Xenopus oocytes and early embryos.

Animals↗
PubMed1989

The establishment of regional identity in the Xenopus blastula.

The molecules involved in the commitment of Xenopus cells to particular germ layers are unknown. The question has been investigated for the cells of the blastula in in vivo cell transplantation assays and in vitro aggregation assays. Using the former technique, we have shown that vegetal cells become committed before gastrulation, even when placed in inappropriate sites. We could find no evidence of regional determination within the endoderm germ layer at the early gastrula stage. In aggregation assays, animal and vegetal cells sorted incompletely at the mid-blastula stage and more efficiently at the gastrula stage. Their behaviour may be mediated by differential expression of adhesion molecules, which so far remain unidentified.

Animals↗
PubMed1989

Regional identity is established before gastrulation in the Xenopus embryo.

Regional differences in cell recognition properties along the animal-vegetal axis of Xenopus laevis embryos were investigated by using an in vitro cell sorting assay. Dissociated cells were obtained from defined regions of blastula- and early gastrula-stage embryos. Binary combinations of cells from different regions, or from the same region at different ages, were aggregated in stationary culture. Labelling of one population in each pair with a cell-autonomous dye allowed the degree of sorting out in the resulting aggregates to be scored. In combinations of cells from animal caps (prospective ectoderm) and vegetal masses (prospective endoderm), sorting was detectable at the equivalent of late blastula stage and increased with developmental age to the gastrula stage. In addition, cells from the same region but different stages sorted from each other, indicating temporal changes in regional identity. Marginal cells (prospective mesoderm) sorted strongly from vegetal cells but only weakly from animal cells. These results indicate the presence of regional identity in the form of specific recognition properties in cells of the Xenopus blastula. We suggest that these properties act to establish and maintain coherent cell populations, corresponding to the primary germ layers, prior to gastrulation. These results account for the gradual restriction in developmental capacity of blastomeres seen previously in single-cell transplantation experiments from this laboratory.

Animals↗
PubMed1989

Cloned mouse melanocyte lines carrying the germline mutations albino and brown: complementation in culture.

We have established two new immortal lines of mouse melanocytes, melan-b and melan-c, from mice homozygous for the brown (b) and albino (c) mutations respectively. Both lines were derived through differentiation in vitro of embryonic epidermal melanoblasts. The brown melanocytes are visibly brown by light microscopy, and centrifuged cell suspensions form brown pellets. The albino melanocytes form white pellets and contain abundant unpigmented premelanosomes as shown by transmission electron microscopy. Like normal, non-immortal melanocytes and like the immortal black melanocyte line melan-a, both lines show little or no growth in a standard, serum-supplemented medium, but proliferate well in the presence of 12-o-tetradecanoyl phorbol-13-acetate (TPA). Sustained growth of the albino cells also requires either keratinocyte feeder cells or 2-mercaptoethanol (2-ME). The modal chromosome numbers are 39 for melan-b and 40 (diploid) for melan-c. Neither line is tumorigenic in nude mice. Heterokaryons between the two lines can be constructed and form wild-type, black pigment. Melanocyte lines can now be reproducibly generated from mice of different strains, and provide tools for molecular studies of germline coat-colour mutations. These two lines provide elegant means to study the developmentally controlled expression of the two complementary genes, B and C, with black melanin pigment as a readily detectable natural marker.

Animals↗
PubMed1987

Vegetal pole cells and commitment to form endoderm in Xenopus laevis.

In order to compare their states of commitment with their normal developmental fate, single vegetal pole cells from early Xenopus embryos were labeled and transplanted into the blastocoels of host embryos. In a previous study we showed, using this single cell transplantation assay, that vegetal pole cells become committed to endoderm by the early gastrula stage. In this paper we examine some properties of the commitment process. First, we show that it is gradual. When vegetal blastomeres are taken from progressively older embryos an increasing number of them enter only the endoderm, until by the early gastrula stage they all do. Second, we show that commitment can continue in vitro when an appropriate tissue mass is present. We suggest that commitment to form endoderm may be, in the right conditions, a cell autonomous process.

Animals↗
PubMed1987

Changes in states of commitment of single animal pole blastomeres of Xenopus laevis.

The experiments described in this paper were designed to compare the normal fates of animal pole blastomeres of Xenopus laevis with their state of commitment. Single animal pole blastomeres were labeled with a lineage marker and transplanted into the blastocoels of host embryos of different stages. The distribution of labeled daughter cells in the tadpole reflects the state of commitment of the parent cell at the time of transplantation. It is known that cells from the animal pole of the early blastula normally contribute predominantly to ectoderm with a small, but significant, contribution to the mesoderm. We show that on transplantation to the blastocoels of late blastula host embryos these blastomeres are pluripotent, contributing to all three germ layers. At later stages the normal fate of these cells becomes restricted solely to ectoderm and concomitantly the proportion of pluripotent cells is reduced, although the results depend upon the stage of the host embryo. Blastomeres from late blastula donors transplanted to mid gastrulae contribute solely to ectoderm in 34% of cases; however, in earlier hosts, when the vegetal hemisphere cells have "mesoderm inducing" or "vegetalizing" activity, late blastula animal pole blastomeres contribute to mesoderm and endoderm rather than ectoderm. Thus during the blastula stage animal pole cells pass from pluripotency to a labile state of commitment to ectoderm.

Animals↗
PubMed1987

Studies on the migration of mouse germ cells.

Primordial germ cells in the mouse embryo migrate from their site of origin to the gonad where they differentiate, giving rise eventually to the gametes of the mature adult animal. The migratory phase is transient and therefore permits analysis of factors regulating the motile activity of cells in tissues. Germ cells can be isolated during migration and cultured on feeder cells of an established cell line (STO). In this system the molecular interactions mediating germ cell adhesion can be probed using antibodies to both germ cell surface determinants and to extracellular matrix components.

Animals↗
PubMed1986

Migratory and postmigratory mouse primordial germ cells behave differently in culture.

In all vertebrate groups, the progenitors of the germ line, the primordial germ cells (PGCs) arise extragonadally and move to the developing gonad early in embryonic development. We have examined the behavior of isolated pregonadal and gonadal PGCs in vitro on feeder layers of an embryo-derived cell line. Histochemically and serologically identified pregonadal germ cells are found to be actively motile in vitro and, furthermore, show behavior characteristic of invasive cells. PGCs isolated from the developing gonad, however, show little locomotory activity and are not invasive on the same cellular substrate. These observations suggest that PGCs undergo a major change in phenotype at the time of their entry into the gonad anlagen.

Alkaline Phosphatase↗
PubMed1986

Macrophages in the urogenital ridge of the mid-gestation mouse fetus.

Isolated urogenital ridges from mouse embryos 11 1/2-16 1/2 days post coitum were separated into genital ridge and mesonephric areas; the latter were then cut into small fragments and cultured as explants on glass or plastic. The explants soon became surrounded by flat outgrowths of cells, followed by a population of large round granular cells. The behaviour of these cells, first on the outgrowth and then on the glass is described. Evidence is presented that they are fetal macrophages.

Animals↗
PubMed1986

Cytoskeletal changes during oogenesis and early development of Xenopus laevis.

The frog oocyte is well known for studies on the control of gene expression, but has been used much less in studies on the cytoskeleton. However, frog oocytes are very large single cells, whose cytoplasmic movements and asymmetries are fundamental to the correct development of the subsequent embryo. One particular example of asymmetrically distributed cytoplasm is germ plasm, thought to be important in the formation of the germ line. Data are presented that show that germ plasm is highly concentrated mass of cytoskeletal elements, which include tubulin, and an intermediate filament protein of molecular weight 55 X 10(3). The distribution of these molecules has been studied during oogenesis and during early post-fertilization development. The implications of these findings are discussed.

Animals↗