No description available.

In order to study the behaviour and normal potential of axial progenitors and their differentiated descendants, transgenic mice carrying a 4-hydroxytamoxifen (4OHT) inducible form of <i>cre </i>recombinase, under the control of a fragment of the <i>T (Brachyury)</i> regulatory sequence known to confer expression in the primitive streak and tail bud, but not in the node or notochord, was generated. On crossing these ‘TcreER^T2, mice with a Cre-dependent lacZ reporter line, embryos carrying both transgenes exhibited inducible primitive streak/tail bud-specific Cre activity upon administration of 4OHT to pregnant mothers. In the absence of 4OHT, Cre activity was not detected. Labelled descendants of streak cells populated somites, lateral/intermediate mesoderm and neurectoderm. The tetracycline-controlled transcriptional activation system (tet-on) was developed and tested as a means to reversibly manipulate gene function in axial progenitors. The reverse tetracycline transactivator (rtTA) was placed under the control of the primitive streak/tail bud-specific <i>T</i> promoter. T-rtTA was targeted to the Rosa26 locus in ES cells to provide a stable transcriptionally active genomic environment, and its function tested by inducing expression of a DsRed reporter gene from the tetracycline response element (TRE), which was integrated at the hprt locus. After allowing the cells to differentiate in the presence of doxycycline, induction was detected by fluorescence microscopy, FACS analysis and RT-PCR. However, DsRed levels were very low compared to a T-rtTA cell line generated in parallel in the lab by random integration. Therefore, the tet system is usable for manipulation of gene expression, but targeted integration provides no benefits over random integration. Thirdly, whole-mount embryo culture-electroporation (WEC-EP) in embryos that were lineage marked upon expression of the electroporated construct, was developed as an alternative experimental approach.

Cell polarity plays a key role in the development multicellular organisms. Several mechanisms exist to ensure that cytoplasmic determinants are distributed heterogeneously within cells. mRNA localisation, is one such process that targets proteins to their site of function. Although the function of mRNA localisation has been widely studied in various organisms ranging from yeast to mammals, since its discovery 20 years ago the mechanism by which mRNAs are transported to their destination is poorly understood. This thesis studies the mechanism by which <i>grk</i> mRNA localisation, crucial for axis of specification of Drosophila embryos, is transported in oogenesis from the cell where it is transcribed to its final destination in the oocyte. Two in vivo approaches were utilised, to study <i>grk </i>RNA movement in Drosophila oogenesis. Molecular beacons, probes that fluoresce upon hybridisation, were not successful in faithfully detecting endogenous <i>grk</i> mRNA. On the other hand, an injection assay of in vitro transcribed and fluorescently labelled <i>grk </i>RNA in living egg chambers, revealed a microtubule and dynein dependence throughout the entire movement of <i>grk </i>RNA transport from the nurse cells to its final destination in the ooycte. We propose, that other key maternal transcripts are transported and localised in Drosophila oogeoeuia via a similar motor driven mechanism.

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No description available.

Microtubule plus ends are occupied and controlled by a special group of proteins. The first microtubule plus end tracking protein to be described was the human CLIP- 170, a member of a conserved protein family. Despite of the large number of studies devoted to the CLIP proteins their function is still poorly understood, especially in the context of a developing metazoan organism. In order to study the function of CLIP-190 in <i>Drosophila </i>development, two P-element insertion alleles were characterised. They were found to be hypomorphic, decreasing the expression of the gene, which however, had no effect on fly viability or development. Other efforts to disrupt CLIP-190 demonstrated that flies are tolerant to substantial variations from the normal levels of the protein. Studies of the subcellular behaviour of CLIP-190 in <i>Drosophila </i>cell culture revealed its distinct localisation during the cell cycle. Its interphase microtubule plus end binding ability was found to depend on the presence of another microtubule regulator, EB1 and a direct interaction between the two proteins was demonstrated. In prometaphase CLIP-190 localised to unattached kinetochores, which depended on the dynein-dynactin complex and associated proteins. The cell cycle regulation on CLIP-190 was investigated and several possible regulators were tested. An RNAi screen in <i>Drosophila </i>cell culture was undertaken to identify new microtubule regulators. Proteins were selected on the basis of their ability to bind to microtubules or tubulin. Their systematic depletion followed by immunofluorescence examination of the microtubule cytoskeleton identified 3 proteins required for normal microtubule organisation. This study revealed the molecular mechanisms that control the localisation of CLIP-190 during the cell cycle and identified 3 new microtubule regulators. It will therefore increase our understanding of the microtubule regulation in <i>Drosophila </i>cells.

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No description available.

No description available.

I have generated an extremely sensitive reporter cell line that unveils a new domain of Hex expression in murine embryonic stem (ES) cells. the introduction of a cDNA encoding a tagged version of Hex upstream of an internal ribosomal entry site and a variant of the Yellow Fluorescent Protein, Venus, into the first exon of <i>Hex</i>, has revealed a heterogeneous expression pattern among ES cell cultures. Manipulation of fibroblast growth factor signalling alters the percentage of venus positive cells and suggests that this subpopulation maybe failed to become endoderm, the earliest domain of <i>Hex </i>expression in the mouse embryo. Although there is an equivalence of <i>Oct3/4 </i>in both venus positive and negative subpopulations, <i>Nanog </i>and venus appear mutually exclusive. Microarray and quantitative PCR analyses show an enrichment of primitive endoderm specific genes in venus positive ES cells while markers of pluripotency are comparatively reduced. While clonal density plating of these subpopulations demonstrate interconvertability, venus positive ES cells have a reduced ability for clonal growth and contribution to the embryo in chimera analyses. I also attempt to overexpress <i>Hex </i>in ES cell cultures. Establishment of stable clones overexpressing <i>Hex </i>in ES and other cell types is difficult, suggesting intolerance. Construction of an inducible system to characterize the phenotype of ectopic <i>Hex </i>expression in ES cells reveals the onset of apoptosis upon induction by a mechanism that depends on its ability to bind DNA. These observations reflect previous studies which suggest that <i>Hex </i>is a key regulator in maintaining a balance between immediate early cell lineage decisions and proliferation. These studies suggest that <i>Hex </i>may be an important marker of early cell fate decisions, but is probably not the primary mediator of early blastocyst/ES cell asymmetry.