The endoderm is the inner germ layer of the vertebrate embryo from which the respiratory and digestive systems are derived. These include organs such as the liver, pancreas, stomach, lungs and intestine. Recent research has helped our understanding of early vertebrate endoderm specification and terminal differentiation of specific endodermal lineages. However, very little is known about the molecular mechanisms that control endoderm patterning and morphogenesis during vertebrate development. As a way to identify genes involved in these elusive steps of development, I performed a differential hybridisation screen in a macroarray tailbud ventral foregut cDNA library coupled with <i>in situ</i> hybridisation analysis. My aim was to identify and characterise new regionally expressed endodermal genes in <i>Xenopus laevis</i>, a classical embryologic model organism. Here, I report the identification and characterisation of a dozen novel regionally expressed endoderm genes. At tailbud stages their expression patterns fall into three re-occurring domains; anterior ventral midgut endoderm, posterior endoderm and dorsal endoderm. In addition, regional expression of some of these genes is observable at gastrula stages, endoderm specification. These are the first early stable endodermal markers for different regions of the gastrula endoderm. This suggests that the earliest steps in endoderm patterning are concurrent with endoderm specification. Furthermore I describe the identification of a mesodermal transcription factor, which appears to be expressed in ‘early embryonic macrophages’ - and a poorly characterised embryonic cell population. I present an overview of endoderm development together with the results from my screen. Overall, these results reveal an unexpected degree of early endodermal patterning and assist our understanding of the link between early and late events of vertebrate endoderm development. In addition, this work provides us with new and very useful markers for endodermal patterning, and potentially some key developmental regulators of endodermal formation.

No description available.

This work concerns the roles of neuron glia interactions in the control of neuronal survival during Central Nervous System (CNS) development in <I>Drosophila. </I>The question of whether glia are required to maintain neuronal survival in insects was addressed. Firstly, the GAL4 system was used to achieve in vivo targeted genetic ablation of glia. Secondly, plasmid rescue and P-element excision were exploited to locate and mutate genes which might participate in neuron glia interactions. Targeted glial ablation did not affect pioneer neuron survival. However, increased apoptosis was observed among the FasII and 22C10 expressing subsets of the follower neurons. Targeted ablation only of the interface glia was sufficient to induce follower neuron apoptosis. This difference in the survival requirements of pioneer and follower neurons may be instructive in patterning of the CNS. Neuronal apoptosis was rescued by ablating glia in an apoptosis deficient genetic background, and by expressing the <I>p35</I> apoptosis inhibitor under the neural <I>elav</I> promoter. Hence the loss of neighbouring glia induces nonautonomous neuronal apoptosis. The <I>Mz1131</I> enhancer-trap line, expressed in the interface glia, was used to examine the effects of mutating a putative glial gene upon CNS development. Lethal <I>Mz1131</I> mutants displayed increased apoptosis of lateral glia and FasII expressing neurons, but not of 22C10 expressing neurons. The fasciculation and defasciculation of the longitudinal axon tracts as also affected. Both lethality and the characteristic GAL4 expression pattern of <I>Mz1131</I> were lost upon P-element excision. Nonradioactive southern blotting revealed that line <I>Mz1131</I> contains multiple P-insertions. Identification of sequences neighbouring P-insertions, and complementation tests revealed a mutation at the <I>sin3A </I>locus.

Here I show that the so-called core components of NMD are not all essential genes, since flies that lack the function of Upf3 are viable, in contrast to flies that lack the function of Upf1 and Upf2. By removing the <i>upf1 </i>and <i>upf2 </i>maternal contribution during oogenesis, I demonstrate that Upf1 and Upf2 are required during embryogenesis, as was the case in mice and zebrafish, exemplifying the fly as a valuable NMD model system for vertebrates. In addition, <i>upf1 </i>and <i>upf2­</i> mutant eggs exhibit a specific dorsal appendage phenotype, possibly the result of developmental perturbation. I have observed that <i>upf1 </i>and <i>upf2,</i> but not <i>upf3</i>, mutant cell clones are unable to grow, suggesting that <i>upf1 </i>and <i>upf2 </i>mutant cells do not proliferate and, in addition, undergo apoptosis. This supported the hypothesis that Upf1 and Upf2 have Upf3-independent functions that are essential for cell and organism viability, and that NMD is not required for the development of an organism. NMD target gene expression levels were analysed using qPCR. My results suggested that potential NMD targets can be separated into distinct groups depending on which NMD factors are most important for mediating their degradation. In adult flies, genes with known PTCs required Upf2 and Upf3, but not Smg1 for their full degradation by NMD, whereas targets with as yet unidentified NMD-inducing features were fully stabilised only in <i>upf2 </i>mutants. These results suggest that Smg1 is a weak inducer of the pathway, rather than a core component, and that Upf3 is only essential for NMD of specific target mRNAs. In embryos, Upf1 and Upf2 were essential for NMD whereas Smg1 and Upf3 had only moderate effects.

Firstly I examined pRb, which is a key negative regulator of cell proliferation. It prevents cells from progressing into S phase by inactivating transcription transcription factors essential for DNA synthesis until it is appropriate for the cell to divide. In addition to implementing cell cycle arrest during differentiation, and independently of this function, pRb is required during myogenesis to promote the full transcriptional activity of the muscle differentiation factor MEF2. However, I have shown by overexpression and depletion studies that it is not required for neural differentiation during early <i>Xenopus</i> embryogenesis. Subsequently I have investigated the roles of cyclins, CDKs and CDK1s in regulating differentiation. I have demonstrated by overexpression studies that cyclin A/CDK2 regulates both cell cycle progression and differentiation in a tissue-specific manner. In contrast, overexpression of another G1 cyclin, cyclin E, with CDK2 causes loss of nuclear DNA and apoptosis. The <i>Xenopus</i> CDKI p27^Xic1 regulates the activity of CDK2 and has been shown to play two distinct roles in regulating the cell cycle and in promoting muscle and neural differentiation. In contrast to cyclin A/CDK2, I have demonstrated by overexpression and depletion studies that, while p27^{Xic1<i> </i>}regulates proliferation in the epidermis, its effect on epidermal differentiation is minimal. Finally, I studied further potential regulators of the cell cycle in the ectoderm. I have shown that both p63, a p53 family member, and members of the myc family inhibit proliferation in the epidermis. Moreover, I have shown that c-myc and N-myc regulate the size of the neural plate. In conclusion, I demonstrate that differentiation cell cycle molecules have the potential to regulate both cell cycle progression and differentiation, but that each achieves this in a distinct manner.

The antero-posterior and dorso-ventral axes of the <i>Drosophila</i> embryo are set up during oogenesis. These axes of polarity are the results of several symmetry-breaking steps that characterise the development of the <i>Drosophila</i> oocyte. During my PhD, I have studied two of these steps. Oocyte determination. During early oogenesis, one cell is selected to become the oocyte within a cyst of 16 germ cells. These 16 germ cells are all siblings and share the same cytoplasm. By using several oocyte-specific markers, I have found that there at least three different pathways to restrict the oocyte fate to one cell. The restriction of cytoplasmic proteins depends on the microtubules and on the activity of the Egl/BicD complex. The restriction of meiosis also depends on the same complex but is independent of the microtubules. Finally, the restriction of the centrosomes is independent of both the microtubules and the Egl/BicD complex. From this analysis, I also concluded that the selection of the oocyte does not depend on the microtubules and that the oocyte is probably selected while the cyst is still dividing, as shown by the asymmetric inheritance of the fusome. By homology with the polarisation of the C. elegans one cell embryo, I have shown that the <i>Drosophila</i> homologues of <i>par-1, par-3 </i>and <i>par-6</i> are required for the first polarisation of the oocyte. This polarisation is then required to maintain the oocyte fate. Oocyte polarisation. During mid-oogenesis, the A/P and D/V axes of the oocyte are defined by the asymmetric localisation of several mRNAs. I have conducted a germline clone screen to find lethal genes involved in this polarisation. In particular, I report here that one of the major <i>Drosophila</i> hnRNP, <i>hrp48</i>, is required for the localisation of <i>oskar</i> mRNA within the oocyte.

A yeast two-hybrid screen searched for novel interactions using a C-terminal fragment of Miranda as bait. This region (Mira 4) has been implicated in mutant studies as important for Miranda degradation in the GMC. The first screens yielded suggestive results but it was not possible to reproduce the interactions, probably due to a combination of low bait expression and high system background. Modifications to the system have produced a more robust screening protocol. <i>In vivo, </i>Mira4 confers instability on GFP, with expression levels of GFP-Mira4 much lower in the GMC than the neuroblast. Mira4 contains a KEN motif, a sequence recognised by APC/C^Cdh1, and its mutation partially stabilises the construct. Full length GFP-tagged Miranda competes with endogenous Miranda, slowing both its degradation and also, interestingly, Pros release. Mutation of the KEN-box both relieves the competition, and increases the half-life of the GFP-fusion. Immuno-staining of wild-type embryos with a new Miranda antibody revealed some GMCs which retained cortical Miranda while Pros was entirely nuclear, showing Pros can be released without Mira degradation. Instead a model is proposed in which one signal is upstream of both events but that they lie in different pathways. The contribution of the KEN-box to Mira instability suggests the involvement of the APC/C although other E3 ligases cannot yet be ruled out. Biochemical assays to establish the ubiquitylation state of Miranda – by double immunoprecipitation and by affinity-binding to isolate ubiquitylated proteins – have also been developed.

The renal system of <i>Drosophila</i> consists of 4 single cell-layered epithelial tubes. Organogenesis of these tubes involves both cellular rearrangement, as they elongate, and the integration of an additional cell population from the surrounding mesoderm. To understand how polarity is established and maintained during these processes, I have characterised the activity of cell polarity genes throughout development. My data suggest that there is a temporal difference in the requirement for one of the key apical proteins, Crumbs, between the epidermis and the tubules. In the epidermis, Crumbs is essential for the formation of the Zonula Adherens junctions (ZAs) so that in Crumbs mutants, the epidermis falls apart at the onset of germ band elongation. The tubules however, form ZAs and localise polarity markers during early stages of development, but by the end of embryogenesis, they lose their tubular organisation and mislocalise both polarity and junctional proteins. The requirement for Crumbs in the tubules occurs at the onset of cellular rearrangement and cell integration. I have used genetics to stall both these morphogenetic movements and find that Crumbs is not longer required to maintain polarity in the tubules. These data suggest that the temporal difference in the requirement for Crumbs between the two tissues is due to the timing of their major morphogenetic movements. This suggests that Crumbs is required to maintain polarity when tissues are morphogenetically active. A key event that could trigger the requirement for Crumbs is the integration of the second mesenchymal cell population, the stellate cells. I have analysed how polarity is established in the stellate cells and how this relates to tubule polarity. I have shown that cell polarity genes become localised in the stellate cells once they have integrated into the tubule epithelium.

The aim of this project is to understand how germ cells are specified in the grasshopper <I>Schistocerca gregaria</I>. I made expression constructs of the <I>Schistocerca </I>Vasa protein and the <I>Drosophila</I> Vasa protein, and produced them through bacteria hosts. Affinity purified <I>Schistocerca</I> and <I>Drosophila vasa </I>fusion proteins were sequentially injected into rabbits to induce antibodies cross reacting to conserved <I>vasa</I>-specific motifs. I affinity purified antibodies from two rabbits and named the antibodies <I>Formosa</I> 1 (for most vasa) and <I>Formosa</I> 2. Western blots demonstrate that both of the purified <I>Formosa</I> antibodies recognise protein specifically expressed in the <I>Schistocerca </I>ovary and testis. Both antibodies also recognise germ cells in the pea aphid. <I>Formosa </I>2 antibody recognises <I>Drosophila</I> pole cells. Furthermore, like the anti-mouse vasa protein antibody, both <I>Formosa </I>antibodies specifically detect a protein in the mouse testis. The size of the protein is very similar to that of the mouse Vasa protein. My immunostaining results show that putative germ cells lie in the outer germ band layer at a dorsal site in embryos from 15% to 30% of development. After 35% of development, they are guided into the bilateral gonadal mesoderm, which migrates dorsally toward the midline of the body. The bilateral gonad primordia coalesce around 70% of development. I conclude that the <I>Schistocerca</I> Vasa protein is a germ-cell marker. Using this marker I have traced the germline lineage through all developmental stages, though the identification of germ cells at early stages remains to be confirmed.

No description available.