Ubiquitination provides a means of rapidly and irreversibly eliminating an unwanted protein from the cell, and is therefore a potentially effective tool for regulating cellular behaviour. Ubiquitin-mediated proteolysis is involved in such diverse physiological functions as growth control, cell signalling, differentiation and the immune response. The aim of this research has been to investigate its role in <i>Drosophila </i>embryogenesis. Protein ubiquitination is a stepwise process carried out by three classes of enzyme known as E1s, E2s and E3s. The E1 (ubiquitin-activating enzyme), generates a thiolester linkage with a ubiquitin cysteine residue. The activated ubiquitin is then transferred to an E2 (ubiquitin-conjugating enzyme) which, with the help of an E3 (ubiquitin-protein ligase), recruits the substrate protein which is to be degraded. I examined the embryonic expression patterns of several known and novel genes encoding each type of ubiquitinating enzyme. The E2 <i>UbcD4</i> is transcribed during early to mid-embryogenesis in a variety of tissues, with specific germcell expression in stage 10 embryos. This suggested a possible role for UbcD4 in germ cell migration towards the somatic gonadal precursors. <i>UbcD4 </i>mRNA was also abundant in git and nervous system during germband retraction and dorsal closure. I screened for UbcD4 - interacting proteins using the yeast two-hybrid system, and identified several putative substrates for, as well as ancillary factors involved in, ubiquitination by UbcD4. These included a novel E3 of the Hect-domain family. In an attempt to examine the function of UbcD4 directly, I used RNA interference to disrupt <i>UbcD4</i> function. The results suggest a post-germband retraction requirement for UbcD4.

Retrospective single cell marking experiments indicate a stem cell progenitor for the myotome and the spinal cord in the primitive streak and its descendant, the tail bud. However, the identity and exact location of the progenitors is unclear from these analyses. First, I performed a detailed gene expression analysis using genes known to play a role in axis elongation <i>(T (Brachyury), Fgf8 </i>and <i>Wnt3a) </i>and the homologues of genes expressed in the <i>Xenopus </i>tail <i>(Evx1 (Xhox3), Foxa2(Pintallavis) </i>and <i>Cdx2 (Xcad3). </i>Comparison of the gene expression patterns showed that either these genes were expressed in domains of the primitive streak that corresponded to specific tail bud regions or showed a consistent set of expression domains that was continuous throughout axis elongation. This gives further support to the idea that tail bud formation in the mouse, as shown in other vertebrates, is a continuation of gastrulation. However, in the mouse, we have not detected a new wave of gene expression coinciding with tail elongation, as seen in <i>Xenopus.</i> Secondly, from experiments started during my MRes and finished during my PhD, we have shown that ingression of ectodermal cells to the mesoderm layer continues even after posterior neuropore closure around the 35­somite stage, as previously observed in chick. Furthermore, grafting of one of the regions of the tail bud, named the chordoneural hinge (CNH) region by analogy to an equivalent structure in Xenopus and chick, showed it had properties expected of a stem cell population. When transplanted to earlier (8.5dpc) embryos, which were then cultured in vitro during a period of extensive axis elongation, the CNH could incorporate to all dorsal host axial tissues and still retain progenitor cells in the tail bud. Indeed, these cells could be serially passaged through 3 successive generations of embryos without apparent loss of their ability to differentiate and retain progenitors in the tail bud. Therefore, the CNH has the characteristics of a population of axial stem cells. A third set of experiments was then performed to localise the putative axial stem cells at earlier stages. Using isotopic grafts to 8.5 dpc embryos, I tested the fate of the node region itself, the region just posterior to it that represents the border between the node and streak (named border) and the anterior primitive streak (APS) region, and found that only cells from the border region contribute to all the dorsal axial tissues and to the CNH, while its neighbours showed a much restricted set of fates. Taken together, these results point to the existence of axial stem cells with equivalent gene expression that reside in the border region at primitive streak stages and later in the CNH in the tail bud.

In order to examine the function of the whole <i>Myosin VI</i> molecule in epithelial tissues, the whole <i>Myosin VI</i> molecule or its separate domains (the head + neck domains or the tail domain) tagged to Green Fluorescent Protein (GFP) were expressed in somatic cells under the control of Ga14 protein and their localisation in the cells was observed. The expression of the tagged proteins under the control of a <i>Myosin VI-Ga14</i> line allowed us to observe all the epithelial cells that express <i>Myosin VI</i> during the whole life cycle. In many epithelial cells the head domain seems to pull the whole molecule towards the cell nucleus, where the minus end of the actin filaments is localised, however it was found that the tail is the domain that anchors the whole molecule to specific areas of the cells away from the nucleus, probably by the binding to cargo molecules. The role of <i>Myosin VI</i> in dorsal closure was found when four <i>Myosin VI</i> mutants that were produced in our laboratory failed to close the dorsal hole during embryogenesis. The genotype and phenotype of two of the mutants, <i>jar^R39</i> and <i>jar^R235</i> are described. Deletion of <i>Myosin VI</i> caused detachment of cells in the leading edge and the amnioserosa and folding-in of the tissue. A similar phenotype was observed when a <i>Myosin VI</i> dominant negative was expressed. In regions of ruptured tissue DE-cadherin and Armadillo was mislocalised, therefore <i>Myosin VI</i> seems to interact with these proteins during cell adhesion. Actin filaments were found to be disorganised at the leading edge and in the lateral epidermis at regions of ruptured tissue, suggesting that <i>Myosin VI</i> is also necessary for actin dynamics during dorsal closure.

The thymus develops from the ventral portion of 3^rd pharyngeal pouch endoderm. At E9.5 this region is marked by expression of the transcription factor <i>Rhox4, Rhox4 </i>may therefore have a role in the developing thymic primordia. <i>Rhox4 </i>is a member of the recently described <i>Rhox </i>family of homeobox-containing genes, which are present in 3 clusters on the mouse X chromosome and are primarily expressed in reproductive and extra-embryonic tissues. The expression of <i>Rhox4 </i>in the thymus, in addition to the testis and placenta makes it unique amongst family members. Here we show that there are seven copies of <i>Rhox4 </i>present in a tandem array within the <i>Rhox </i><i>α</i><i>  locus. All </i>alleles of <i>Rhox4 </i>are expressed, although preferential expression was observed and differed between tissues. In contrast to reproductive tissues, no evidence of co-linear expression of the <i>Rhox </i><i>α</i><i> </i>cluster during thymus development was found. Two other members of the <i>α</i><i> </i>cluster, <i>Rhox2 </i>and <i>Rhox3 </i>are also present in the duplicated unit, both having 8 copies. The only predicted orthologue of <i>Rhox4 </i>is present in a single copy in the rat; however, no <i>Rhox4 </i>expression was detected in the developing or postnatal rat thymus. Examination of different sub-species of mice showed that all contain multiple copies of <i>Rhox4, </i>suggesting that the duplication event arose at the time of divergence of the rat and mouse lineages. Finally, no changes in <i>Rhox4 </i>expression were detected in mice with defects in thymus organogenesis, placing it upstream or outside of established transcriptional pathways.

Rates of RNA synthesis, and in particular poly(A) +RNA synthesis, in oocytes of Xenopus laevis have been measured by analysing the kinetics of incorporation of exogenous radioactive nucleoside, or of microinjected radioactive nucleoside triphosphate, into TOA precipitable material coupled with determinations of precursor pool specific activity. Poly(A) RNA was isolated by oligo (dT)cellulose chromatography. Stable RNA, over 80% of which was rRNA, was synthesized in stage 6 oocytes and accumulated in the cytoplasm at a constant rate of 650 pg/oocyte/hour for at least 100 hours. By isolating germinal vesicles, it was shown that more than 70% of the RNA synthesized initially in stage 6 oocytes was unstable nuclear RNA with a half-life shorter than 4 hours, much of which sedimented heterogeneously on sucrose gradients. In stage 6 oocytes all the poly(A) RNA which was synthesized at an initial rate of 30 pg/oocyte/hour appeared to turn over with an average halflife of 10 hour and the steady state amount accumulated was 0.5% of the stored pool of poly(A) RNA. Similar kinetics were observed in enucleated oocytes but othidium bromide inhibited poly (4) RNA synthesis by about 70%. These observations suggest that much of the poly(A) RNA synthesis in stage 6 Xenopus laevis oocytes is mitochondrial. Stage 1 oocytes synthesized stable RNA at a constant rate of 17 pg/oocyte/hour for at east 80 hours and over 80% of the RNA accumulated was 4S and 53 RNA. Poly(A) RNA, with slightly different sedimentation properties and poly(A) size from that in stage 6 oocytes, was synthesized at an initial rate of 0.7 pg/oocyte/hour most of which was unstable, having a half-life of about 12 hours. The discussion relates these rate measurements to the known patterns of RNA accumulation during oogenesis and the mechanisms of transcription of maternal mRNA and other RNA classes.

No description available.

The generation of mature Schwann cells from the neural crest occurs by a transition through two intermediate cell types, namely the Schwann cell precursor and the immature Schwann cell. Immature Schwann cells mature into the myelinating and non-myelinaling Schwann cells present in the adult nerve. These cell types are well characterised and can be readily distinguished from one another by their distinct antigenic profile, survival responses, and morphological changes. In this study I investigated the effects of BMP in the Schwann cell lineage in vitro. I found that BMP2/4 acts to maintain the immature Schwann cell type by promoting its differentiation from the Schwann cell precursor and inhibiting the upregulation of myelin proteins. I also found that survival responses to BMP2/4 differ between embryonic and postnatal Schwann cell. I examined the role of STAT3 in Schwann cells both in vitro and in vivo using mice with a conditional mutation of STAT3 specifically in Schwann cells. I found that STAT3 is activated by, and supports survival following stimulation by autocrine factors secreted by Schwann cells both in vitro and following nerve injury in vivo. I also discovered that STAT3 promotes the expression of myelin proteins in vitro and that it is involved in the timing of demyelination following sciatic nerve injury. I also investigated the repression of the transcription factor c-Jun, by itself and by Krox-20, in immature Schwann cells and found that this occurs mainly at the protein, rather than the transcriptional level.

The digestive tract and its accessory organs - liver, pancreas, and the inner lining of the swim bladder - are derived from the endodermal germ layer. In zebrafish the digestive system starts out as a rod at the embryonic midline, which subsequently becomes patterned and outgrowth of its associated organs occurs. Liver formation can be subdivided into three distinct phases; specification, budding, and differentiation and growth. Despite the importance of the liver for body homeostasis, relatively little is known about the mechanisms of liver development. The transgenic line Tg(gutGFP)s854, expressing GFP throughout the developing digestive tract, was used to identify the mutant lines s436 and 4C1 in a forward genetic screen for mutants displaying defects in endodermal organogenesis. Initial analysis of both lines revealed a hypoplastic liver at 48 hpf, and in addition distinct defects in pancreas formation. Positional cloning of the s436 line identified a novel allele of histone deacetylase 1 (hdac1), which plays distinct roles in endodermal organogenesis in zebrafish. Loss of Hdac1 causes defects in timely liver specification and subsequent differentiation, while mosaic analyses revealed a cell-autonomous role for Hdac1 in the endoderm in these processes. In addition, I have demonstrated that Hdac1 has specific functions in endocrine pancreas morphogenesis, as well as roles in exocrine pancreas specification and differentiation. Finally, loss of Hdac1 results in an expansion of the foregut endoderm in the domain from which the liver and pancreas originate, suggesting a scenario whereby Hdac1 may directly or indirectly restrict foregut fates while promoting hepatic and exocrine pancreatic specification and differentiation. Phenotypic characterisation of endodermal organogenesis in 4C1 mutant embryos revealed a requirement for 4C1 in specification of the correct number of hepatic progenitors and hepatic bud morphogenesis. This is accompanied by a subsequent requirement for 4C1 in hepatic growth and duct morphogenesis. Additionally, 4C1 mutants exhibit defects in exocrine pancreas anlage outgrowth, leading to ectopic exocrine bud formation, suggesting a role for 4C1 in the morphogenesis of this organ. Preliminary analysis suggests that 4C1 may be required within the neighbouring tissue, the lateral plate mesoderm, to promote endodermal organogenesis. Taken together, I have characterised two novel mutant lines, which despite initial phenotypic similarities, exhibit very distinct defects in liver as well as pancreas formation, highlighting important roles for these genes in the processes underlying endodermal organogenesis.

During embryonic development, organiser centres form at compartment boundaries and provide a source of graded signals that instruct cells to specific identities in a concentration-dependent manner. The mid-hindbrain boundary (Geling et al.) is an organiser that arises in the developing neural tube at the interface between midbrain and hindbrain, and is crucial for the formation of tectum and cerebellum. Local organisers at boundaries have been best studied in invertebrates (Ahmed et al.) where the Notch signalling pathway is important. Recent studies suggest that this signalling cascade may also control boundary and organiser formation in vertebrates. In this thesis I have tested the hypothesis that Notch signalling is important during MHB development. I have found that genes involved in the Notch signalling pathway are specifically expressed in domains within the chick neural tube that demarcate the MHB from early somite stages, implicating Notch signalling in the establishment of this organiser. I examined the effect on cells of experimentally altering Notch activity levels. Activation of the Notch pathway regulates cell affinity properties, as cells containing activated Notch are excluded from rhombomeres 1 and 2. I tested the hypothesis that LFng acts as a switch to activate Notch only on the midbrain side of the boundary. Perturbation of LFng expression leads to disruption of the boundary and cell lineage restriction is lost. Ectopic activation of the Notch signalling pathway perturbs expression of key MHB organiser genes, Fgf8 and Wnt1. In contrast, in the absence of Notch signalling, the MHB fails to form properly. I propose that Notch signalling through the ligand Serrate1 is sufficient for the generation of boundaries in this region of the CNS. Together, these data suggest a role for Notch signalling both in the formation of the MHB, and also in the regulation of cell affinity differences necessary to stabilise and maintain the organiser.

Assignment of antero-posterior (AP) positional identity along the neuraxis is regulated by the Hox gene family. In order to establish the precise expression domains of Hox genes, a number of transcription factors and signalling molecules are required. Previous studies have implicated the involvement of vertical and planar signalling in the assignment of positional identity. However, it remains unclear to what extent vertical and planar signalling contribute to correct AP patterning and whether any competence differences exist along the dorso-ventral axis of the neural tube that may bias Hox induction. This study examines how various signalling mechanisms are involved in patterning the chick neural tube along its AP axis, using Hoxb4 as an axial marker. This work shows that signals from somites (vertical signals) are required but not sufficient to establish the correct expression pattern o HoxM in the neural tube thus indicating the need for additional signals emanating from more posterior tissue. Experiments conducted in order to elucidate the exact nature of these signals imply that they are planar signals. In addition, this work shows that the dorsal side of the neural tube is more responsive to the somite signal than the ventral side. This responsiveness seems to be caused by the action of BMPs in the dorsal neural tube. Therefore, it appears that BMP and the somite signal may work in conjunction to upregulate Hoxb4 expression in the neural tube.