Search PubMed⌕ Search

Biomedical subjects

C Wylie

Publications and source records attributed to C Wylie.

At least 37 records · Page 2Linked to original sources
PubMed1998

The role of maternal VegT in establishing the primary germ layers in Xenopus embryos.

VegT is a T-box transcription factor whose mRNA is synthesized during oogenesis and localized in the vegetal hemisphere of the egg and early embryo. We show that maternally expressed VegT controls the pattern of primary germ layer specification in Xenopus embryos. Reduction of the maternal store completely alters the fates of different regions of the blastula so that animal cell fate is changed from epidermis and nervous system to epidermis only, equatorial cell fate is changed from mesoderm to ectoderm, and vegetal cell fate is changed from endoderm to mesoderm and ectoderm. Vegetal cells lose their capacity both to form endoderm and to release mesoderm-inducing signals. These results show that a single maternally expressed gene controls the patterning of the Xenopus blastula.

Animals↗
PubMed1998

The origin and migration of primordial germ cells in the mouse.

Primordial germ cells (PGCs) are the founders of the gametes. They migrate from their origin to combine with the somatic component of the gonad. Progenitors in mice have been recognized in the proximal epiblast. When and how these cells are allocated to the germ line has not been elucidated fully. Numerous growth factors have been described which affect PGC survival, proliferation and directional control during their migration. The extracellular matrix also may play a role in PGC migration, particularly fibronectin and laminin. Once they have arrived at the gonad, PGCs aggregate with one another. The molecules governing this PGC-PGC interaction have yet to be identified.

Animals↗
PubMed1998

Early stages in male germ cell differentiation in the mouse. Review article.

Primordial germ cells arise during gastrulation and migrate from the hindgut into the gonad primordium during early organogenesis. In this article, we discuss factors that control migration, proliferation and targeting of the PGCs. In particular we discuss how changes in adhesiveness control germ cell positioning in the gonad, and the molecules involved.

Animals↗
PubMed1997

Interactions between germ cells and extracellular matrix glycoproteins during migration and gonad assembly in the mouse embryo.

Cells are known to bind to individual extracellular matrix glycoproteins in a complex and poorly understood way. Overall strength of adhesion is thought to be mediated by a combinatorial mechanism, involving adhesion of a cell to a variety of binding sites on the target glycoproteins. During migration in embryos, cells must alter their overall adhesiveness to the substrate to allow locomotion. The mechanism by which this is accomplished is not well understood. During early development, the cells destined to form the gametes, the primordial germ cells (PGCs), migrate from the developing hind gut to the site where the gonad will form. We have used whole-mount immunocytochemistry to study the changing distribution of three extracellular matrix glycoproteins, collagen IV, fibronectin, and laminin, during PGC migration and correlated this with quantitative assays of adhesiveness of PGCs to each of these. We show that PGCs change their strength of adhesion to each glycoprotein differentially during these stages. Furthermore, we show that PGCs interact with a discrete tract of laminin at the end of migration. Closer analysis of the adhesion of PGCs to laminin revealed that PGCs adhere particularly strongly to the E3 domain of laminin, and blocking experiments in vitro suggest that they adhere to this domain using a cell surface proteoglycan.

Animals↗
PubMed1997

Modified mRNA rescue of maternal CK1/8 mRNA depletion in Xenopus oocytes.

This work addresses two issues, the use of antisense oligodeoxynucleotides to deplete specific mRNAs in Xenopus oocytes to analyze their functions during development and the role of cytokeratin filaments in cells of the early Xenopus embryo. We have shown previously that depletion of cytokeratin CK1/8 mRNA causes defects in the early embryo. In this study, we show that the oligos, modified with phosphoramidate linkages to improve stability, are capable of degrading exogenous mRNA up to 27 hours after injection in the oocyte. For this reason, the phenotype could not be rescued by injection of a synthetic CK1/8 mRNA. However, modification of the synthetic CK1/8 mRNA, which prevents annealing of the antisense oligonucleotide used for depleting the endogenous CK1/8 mRNA, did result in the rescue of the CK1/8 depletion phenotype. These results demonstrate that the phenotype observed after depletion of the CK1/8 mRNA is truly caused by the lack of CK1/8 protein. Injection of the closely related type II cytokeratin (CK55) did not result in the same level of rescue of the CK1/8 depletion phenotype, suggesting that structurally similar members of the cytokeratin family, expressed at different stages of development, cannot substitute for each other in the early embryo.

Amino Acid Sequence↗
PubMed1997

The roles of maternal alpha-catenin and plakoglobin in the early Xenopus embryo.

Catenins (alpha-, beta- and gamma- or plakoglobin) are cytoplasmic cadherin-associated proteins. Studies on cultured cells have suggested that both alpha-catenin and plakoglobin are important for the adhesive function of cadherins. alpha-catenin binds to both beta-catenin and plakoglobin and may link the cadherin/catenin complex to actin filaments. Separate domains of plakoglobin bind to cadherin and alpha-catenin, suggesting it may act as a bridge between these molecules. However, plakoglobin may have other activities: it is expressed in both desmosomal junctions in association with desmogleins and the cytoplasm in conjunction with APC, and previous work suggests it may act in a dorsal signalling pathway when overexpressed in Xenopus embryos. Here, we have studied the roles of alpha-catenin and plakoglobin directly, by depleting the maternal mRNAs coding for each of them in developing Xenopus embryos. We find that depletion of maternal alpha-catenin causes the loss of intercellular adhesion at the blastula stage, similar to that reported previously for EP cadherin. Depletion of plakoglobin results in a partial loss of adhesion, and a loss of embryonic shape, but does not affect dorsal signalling.

Animals↗
PubMed1996

A kinesin-like protein is required for germ plasm aggregation in Xenopus.

In embryos of X. laevis, and many other species, early development requires targeted movements of molecules and molecular aggregates within the oocyte or egg cytoplasm. One well-known example in Xenopus is the aggregation of germ plasm, a group of cytoplasmic islands that become distributed during the first few cell cycles to cells that will give rise to the germ line. Nothing is known about the cytoskeletal motor proteins that may drive these movements. We show here that a recently identified Xenopus kinesin-like protein, Xklp1, is required for the aggregation of germ plasm in early Xenopus embryos, thus assigning this protein a role in a developmentally important cytoplasmic localization.

Animals↗
PubMed1996

The passing score in the objective structured clinical examination.

The objective structured clinical examination (OSCE) now has an established place in the assessment of the medical undergraduate. While much has been written about the reliability of the OSCE, empirical work on the determination of the passing score which represents competence on the OSCE is rarely encountered. If the OSCE is to play its role in the 'high stakes' testing of clinical competence, it is important that this passing score be set reliably and defensibly. This article illustrates how a two-session modified Angoff standard-setting procedure is used to set the passing score on a 14 station Obstetrics and Gynaecology OSCE used to assess final year students at The Queen's University of Belfast. The Angoff methodology harnesses the professional judgement of expert judges to establish defensible standards. Four university teachers, five non-academic consultants and six junior clinical staff took part in a two-session Angoff standard-setting procedure. In the first session, the judges (individually and in silence) used their professional judgement to estimate the score which a minimally competent final year obstetrics and gynaecology student should achieve on each tested element of the OSCE. In the second session they revised their session 1 judgements in the light of the OSCE scores of real students and the opportunity for structured discussion. The passing score for the OSCE is reported together with the statistical measures which assure its reliability.

Clinical Competence↗
PubMed1996

Maternal beta-catenin establishes a 'dorsal signal' in early Xenopus embryos.

In previous work, we demonstrated that maternally encoded beta-catenin, the vertebrate homolog of armadillo, is required for formation of dorsal axial structures in early Xenopus embryos (Heasman, J., Crawford, A., Goldstone, K., Garner-Hamrick, P., Gumbiner, B., Kintner, C., Yoshida-Noro, C. and Wylie, C. (1994). Cell 79, 791-803). Here we investigated, firstly, the role(s) of beta-catenin in spatial terms, in different regions of the embryo, by injecting beta-catenin mRNA into individual blastomeres of beta-catenin-depleted embryos at the 32 cell stage. The results indicate that beta-catenin can rescue the dorsal axial structures in a non-cell-autonomous way and without changing the fates of the injected cells. This suggests that cells overexpressing beta-catenin send a 'dorsal signal' to other cells. This was confirmed by showing that beta-catenin overexpressing animal caps did not cause wild-type caps to form mesoderm, but did cause isolated beta-catenin-deficient marginal zones to form dorsal mesoderm. Furthermore beta-catenin-deficient vegetal masses treated with overexpressing caps regained their ability to act as Nieuwkoop Centers. Secondly, we studied the temporal activity of beta-catenin. We showed that zygotic transcription of beta-catenin starts after the midblastula transition (MBT), but does not rescue dorsal axial structures. We further demonstrated that the vegetal mass does not release a dorsal signal until after the onset of transcription, at the midblastula stage, suggesting that maternal beta-catenin protein is required at or before this time. Thirdly we investigated where, in relationship to other gene products known to be active in axis formation, beta-catenin is placed. We find that BVg1, bFGF, tBR (the truncated form of BMP2/4R), siamois and noggin activities are all downstream of beta-catenin, as shown by the fact that injection of their mRNAs rescues the effect of depleting maternally encoded beta-catenin. Interference with the action of glycogen synthase kinase (GSK), a vertebrate homolog of the Drosophila gene product, zeste white 3 kinase, does not rescue the effect, suggesting that it is upstream.

Animals↗
PubMed1995

Xklp1, a chromosomal Xenopus kinesin-like protein essential for spindle organization and chromosome positioning.

Xklp1 is a novel Xenopus kinesin-like protein with a motor domain at the amino terminus, nuclear localization sequences in the stalk, and a putative zinc finger-like sequence in the tail. It is nuclear during interphase and chromosomal during mitosis. During late anaphase, a fraction of the protein relocalizes to the spindle interzone and accumulates in the midbody during telophase. Depletion of Xklp1 protein by antisense oligo knockout in oocytes leads to defective mitosis during the first cell cycles following fertilization. The bipolarity of spindles assembled in vitro in the presence of anti-Xklp1 antibodies is unstable, and the chromosomes fail to congress on the metaphase plate.

Amino Acid Sequence↗
PubMed1994

Overexpression of cadherins and underexpression of beta-catenin inhibit dorsal mesoderm induction in early Xenopus embryos.

The cadherin-catenin complex has an important role in cell-cell adhesion and may also function in signaling pathways. We report that overexpression of three cadherin types in Xenopus embryos causes them to develop with reduced dorsal axial structures. The same phenotype is produced in embryos that have been depleted of maternal beta-catenin protein by an antisense oligodeoxynucleotide complementary to beta-catenin mRNA. They show an inhibition in the expression of dorsal mesodermal markers MyoD and goosecoid, but not of ventral and general mesodermal markers. They lack notochords, somites, and neural tubes and are defective in dorsal mesodermal signaling in Nieuwkoop assays. The phenotype can be rescued by the injection of beta-catenin mRNA and not by the injection of Xwnt-8 mRNA. These results show that beta-catenin has an important role in dorsal mesoderm induction. They directly demonstrate the activity of a maternal mRNA in axis specification.

Animals↗
PubMed1994

Primordial germ cell migration.

Primordial germ cells are migratory cells. They arise very early in embryogenesis and have a similar pattern of migration in Drosophila, Xenopus, chick and mouse. In each case the primordial germ cells associate with the developing gut from which they migrate to the gonads during organogenesis. Germ cells proliferate mitotically from the time they begin to migrate to the time they colonize the genital ridges. From the study of mouse primordial germ cells, we now know of a number of agents which affect primordial germ cell proliferation, migration and adhesion in vitro. More recently, we have studied the interactions between primordial germ cells and the cells and extracellular matrix molecules on their migratory route. By labelling germ cells in whole-mount preparations with an antibody to the germ cell marker SSEA-1, we have studied the spatial distribution of germ cells in situ using confocal microscopy. This study has revealed that germ cells link up with each other forming extensive networks during migration.

Animals↗
PubMed1994

Interactions between primordial germ cells play a role in their migration in mouse embryos.

Primordial germ cells (PGCs) are the founder cell population of the gametes which form during the sexually mature stage of the life cycle. In the mouse, they arise early in embryogenesis, first becoming visible in the extraembryonic mesoderm, posterior to the primitive streak, at 7.5 days post coitum (d.p.c.). They subsequently become incorporated into the epithelium of the hind gut, from which they emigrate (9.5 d.p.c.) and move first into the dorsal mesentery (10.5 d.p.c.), and then into the genital ridges that lie on the dorsal body wall (11.5 d.p.c.). We have used confocal microscopy to study PGCs stained with an antibody that reacts with a carbohydrate antigen (Stage-Specific Embryonic Antigen-1, SSEA-1) carried on the PGC surface. This allows the study of the whole PGC surface, at different stages of their migration. The appearance of PGCs in tissue sections has given rise to the conventional view that they migrate as individuals, each arriving in turn at the genital ridge. In this paper, we show that PGCs leave the hind gut independently, but then extend long (up to 40 microns) processes, with which they link up to each other to form extensive networks. During the 10.5-11.5 d.p.c. period, these networks of PGCs aggregate into groups of tightly apposed cells in the genital ridges. As this occurs, their processes are lost, and their appearance suggests they are now non-motile. Furthermore, we find that PGCs taken from the dorsal mesentery at 10.5 d.p.c. perform the same sequence of movements in culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

A functional test for maternally inherited cadherin in Xenopus shows its importance in cell adhesion at the blastula stage.

We report here on the consequences of reducing the expression of EP-cadherin at the earliest stages of Xenopus development. Injection of oligodeoxynucleotides antisense to maternal EP-cadherin mRNA into full-grown oocytes reduced the mRNA level in oocytes, and the protein level in blastulae. Adhesion between blastomeres was significantly reduced, as seen in whole embryos, and in assays of the ability of blastomeres to reaggregate in culture. This effect was especially conspicuous in the inner cells of the blastula and included the disruption of the blastocoel. The severity of the EP-cadherin mRNA depletion and of the disaggregation phenotype was dose dependent. This phenotype was rescued by the injection into EP-cadherin mRNA-depleted oocytes of the mRNA coding for a related cadherin, E-cadherin, that is normally expressed at the gastrula stage in the embryonic ectoderm.

Animals↗
PubMed1993

XLPOU-60, a Xenopus POU-domain mRNA, is oocyte-specific from very early stages of oogenesis, and localised to presumptive mesoderm and ectoderm in the blastula.

POU-domain proteins are a large family of transcriptional regulatory proteins, related to the homeodomain proteins, many of which are implicated in the control of gene expression during early development. We describe here the isolation of a cDNA encoding a Xenopus POU-domain protein, XLPOU-60. The predicted protein sequence of this cDNA is most closely related to the mouse germ line-specific transcription factor Oct-3/4. The XLPOU-60 gene is specifically expressed in oocytes of newly metamorphosed frogs, from the earliest stages at which transcription is known to occur. The mRNA is concentrated in the animal half of fully grown oocytes and is inherited maternally by the embryo, where it remains localised to animal cap and marginal zone cells of the blastula. Transcripts decline abruptly to a low level during gastrulation, but remain detectable throughout larval stages. However, unlike Oct-3/4, the transcript is not detectable in primordial germ cells, and XLPOU-60 is therefore probably not the functional homologue of the murine gene. We suggest that XLPOU-60 is one of the earliest genes to be transcribed in oocyte development, and that the XLPOU-60 protein may therefore be involved in initiating oocyte-specific patterns of transcription. Localisation of the transcript in the embryo may indicate that XLPOU-60 is also required for the initiation of mesoderm- and ectoderm-specific patterns of transcription in the embryo.

Amino Acid Sequence↗
PubMed1993

Multiple kinesin-like transcripts in Xenopus oocytes.

Recent evidence shows that kinesin-like proteins (Klps) form a very large multigene family. A recent study using the polymerase chain reaction (PCR) identified six new candidate Klps in Drosophila, making the total number of members of this family in Drosophila at least 11 (Stewart et al., 1991, Proc. Natl. Acad. Sci. USA 88, 4424-4427). The functional basis of this diversity is not clear. Different Klps could have cell type-specific functions, or they could perform different functions within the same cell type, or a mixture of both. To investigate the degree to which different Klps are expressed in the same cell, we chose the Xenopus oocyte. During oocyte differentiation, and in the egg, different types of microtubule-based motility occur; all are important to the normal development of the embryo after fertilization. Using PCR we identified and partially sequenced four novel Klp mRNAs from the Xenopus oocyte (denoted XKlps 1-4). Multialign sequence comparison suggests that one of them, XKlp3, may be the Xenopus counterpart of Drosophila Klp4. Similarly Xenopus Eg5 is closely related to Drosophila Klp2. Northern blot analysis reveals that the Xenopus XKlps have different patterns of expression during embryogenesis. These data show that at least four Klps can exist in the same cell and that they can be differentially regulated during early development, and suggest their differential function in oogenesis and 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↗