No description available.

To understand how the notochord controls morphogenesis in the mesencephalon, I have undertaken a study to fully characterize the microsurgical manipulation that perturbs development. I have discovered that the same phenotype can be achieved by merely separating, rather than removing, the notochord from the overlying neural tube and that this effect is stage- and regional-specific. It is known that the development of the mesencephalon is regulated by an organizing centre situated at the metencephalic boundary, referred to here as the MHB. I have addressed the contribution of the MHB, neutral tube and notochord towards the phenotype by analyzing the expression patterns of a variety of developmental control genes after the manipulation. In the MHB, <I>Wnt1, En1 </I>expression are initially reduced after the manipulation, and <I>Wnt1</I> recovers to control levels by 48 hours; no change is seen in <I>Fgf8</I> expression. The presence of notochord and floor plate was also assessed by the expression of Sonic hedgehog (SHH). Interestingly, while Stage 11 embryos maintained expression of <I>Shh</I> in both structures, Stage 12 embryos lost notochord expression after 24 hours and showed a reduced floor plate expression after 48 hours. This correlates with the stage specificity seen for the manipulation. These results provide an <I>in vivo</I> model to study the consequences of a conditional loss of notochord signalling after axial patterning. To examine the response of later embryos, a histological analysis was performed and revealed a reduction in neutral tissue and ventricular space.

No description available.

The paternally expressed gene 1 (Peg1), also known as mesodermally expressed specific transcript (Mest), is one of approximately 60 genes so far discovered, the expression of which is dependent on the parental origin of each gene copy. Human Peg1 and its highly conserved mouse counterpart are expressed primarily from the paternal genome. Initial investigations into the distribution of <i>Peg1</i> expression have concluded that its mRNA is found exclusively in tissues of mesodermal origin and that expression levels decline substantially after birth. A lack of <i>Peg1</i> expression in mice has been associated with a modest growth retardation at late gestation and abnormal maternal behaviour. Aside from this however, very little is known about the biochemical function or biological role of Peg1 in mammalian development. The data presented here show that from E7.5 onwards, <i>Peg1</i> expression was strong in tissues of mesodermal origin, but there was clear evidence of additional expression in ectodermal tissues at the anterior end of the embryo, deriving from the neural plate. Mice lacking <i>Peg1</i> expression were 5 – 15% growth retarded in embryo, placenta and kidney from embryonic day 14 onwards. They also exhibited specific irregularities in organogenesis, consisting of a 22% reduction in the total volume of maternal blood spaces in the labyrinthine placenta, and a 26% reduction in glomerular volume fraction in the kidney, attributed to fewer glomeruli in the mutant mouse. Significantly, <i>Peg1</i> expression was absent in the trophoblast tissue that forms the maternal spaces in the labyrinthine placenta, being confined instead to the adjacent endothelial layer surrounding the fetal blood villi. In the kidney, <i>Pe1l</i> expression was strong in undifferentiated metanephric mesenchyme and also in glomeruli. Expression of human Peg1 <i>in vitro</i> caused substantial morphological change and cell death in one human fibroblast and two epithelial cell lines. Cell death induced by exogenous expression of Peg1 exhibited features common to both necrotic and apoptotic pathways.

The renal or Malpighian tubules (MpTs) are the major excretory organs of insects. <i>Drosophila </i>has 4 MpTs, an anterior and posterior pair. During embryogenesis these MpTs undergo a morphogenetic programme such that cell rearrangement by convergent extension causes the MpTs to elongate so the short fat tubules become longer thin tubules. In addition directed migration or pathfinding occurs that the MpTs take up a specific location within the body cavity. In this study an analysis of wild type development demonstrates that several aspects of normal MpT morphogenesis are invariant whilst other aspects are less defined. The shape of an organ is a consequence of both extrinsic factors and intrinsic properties. I demonstrate that external tissues and other regulators are controlling morphogenesis of the MpTs through alterations in the cytoskeleton. I find that when the visceral mesoderm is disrupted genetically the MpTs have an aberrant morphology; the visceral mesoderm acts as an extrinsic cue for normal MpT pathfinding. The visceral mesoderm secretes Decapentaplegic (Dpp) and my analysis suggests that Dpp is an attractant controlling the normal MpT migratory behaviour. Downstream activators of the <i>dpp</i> signalling pathway are present in the MpTs and ectopic expression of Dpp in tissues near the extending MpTs affects their morphogenesis. The convergent extension process itself may be intrinsic to the MpTs and can be disrupted when signalling via the Rho family of small GTPases is perturbed. In order to identify novel genes involved in MpT morphogenesis I have analysed lines from a mutagenesis screen previously performed to select for MpT defects. One locus was mapped and characterised as an allele of <i>D-Cbl</i>, previously shown to be in inhibitor of the EGF pathway. I present evidence to show that when the regulation of EGF signalling is disrupted subtle defects on MpT morphology are observed, thus revealing a requirement for the activity of this pathway during MpT morphogenesis.

No description available.

Postembryonic neuroblasts (pNBs) in the <i>Drosophila</i> larval CNS are considered to be neural stem cells production most of the neurons of the adult <i>Drosophila</i> central nervous system (CNS). The pNBs are derived from the embryonic neuroblasts (NBs). After a period of quiescence at the end of embryogenesis they reactivate during larval life and proliferate extensively for a limited period. The pNBs are able to self-review in each division and also to produce a precursor cell that will generate two postmitotic cells that fully differentiate at metamorphosis. Therefore, the pNBs provide a good model to investigate the mechanisms involved in the regulation of neural stem cells. The aim of this research was to investigate whether Notch signalling and the transcription factor Grainyhead have roles in regulating pNBs processes. Before investigating the regulation of the pNBs behaviour it was first necessary to characterise in more detail the characteristics of these cells and their progeny. Several genes that are expressed in the embryonic CNS were selected to further study. Among these I identified some factors that are expressed only in specific pNB lineages, such as Gsb-p and others that are expressed at a specific stage in all lineages such as Prospero. Based on their expression pattern it appears that Gsb-p is likely to perform a similar function in the larval CNS as in the embryo. In contrast the distribution of Prospero suggest different roles in the pNB lineages. The result was first that the Notch pathway is active in the pNBs, indicated by the expression of Notch target gene <i>m</i><i>g. </i>However, the results obtained, using clonal analysis to manipulate Notch function in the pNB lineages, indicate that Notch does not have a role in maintaining the undifferentiated state of the pNBs or their proliferative state. In contrast to its function in vertebrate systems where it is able to regulate the uncommitted state of the neural progenitor cells. The analysis of <i>grh </i>mutant indicated that Grh has a role in regulating the proliferation and/or cell death of the pNBs.

The aim of my research was to understand how RPTPs are involved in retinal ganglion cell (RGC) axon outgrowth and guidance in the development of the <I>Xenopus </I>visual system. I first describe the cloning and expression patterns of a variety of RPTPs in the developing <I>Xenopus</I> embryo, focusing on the retinotectal system. All three members of Type IIa RPTPs; LAR, PTP-δ, and CRYP-α, are expressed in RGCs during periods of differentiation, axonogenesis, and axon guidance from the retina to the tectum. These three RPTPs, as well as PTP-p, are expressed in overlapping but distinct patterns in the developing <I>Xenopus</I> embryo. Expression patterns of putative ligands for CRYP-α and LAR were examined using receptor affinity probe in situ hybridisation in the developing retina and brain, and putative ligands for CRYP-α were found along the optic pathway and in the tectum. These results demonstrate that Type IIa RPTPs and their putative ligands are expressed in a spatial and temporal pattern consistent with their involvement in RGC axon guidance and outgrowth from the retina to the tectum. I also describe functional studies examining the role of RPTPs in the developing <I>Xenopus</I> visual system both in vitro and in vivo. In vitro analysis of RGCs expressing dominant negative RPTPs demonstrates that dominant negative PTP-δ inhibits RGC axon outgrowth, while dominant negative CRYP-α promotes outgrowth, in a substrate-dependent manner. In vivo analysis of RGC axon outgrowth and guidance demonstrates that although dominant negative PTP-δ and LAR reduce the rate of axon outgrowth, none of these RPTPs appear to be involved in axon guidance.

Id proteins (Inhibitors of differentiation) inhibit differentiation by preventing bHLH DNA binding activity through the formation of inactive Id-bHLH heterodimers. Xenopus Ids (xIds) are dynamically expressed in a wide variety of tissues, with high expression levels detected at early stages of development. In this work, I have identified a requirement for Xenopus xId2 and xId3 in primary neurogenesis and muscle differentiation. Overexpression of xId2 and xId3 disrupts the expression of late neuronal and muscle differentiation markers, but not the expression of the early markers xNGN and xMyoD, suggesting that xIds inhibit primary neuron and muscle differentiation without affecting neuronal or myogenic commitment. To elucidate the mechanism through which xIds inhibit primary neurogenesis, I investigated the ability of xIds to affect xNGN activity, the most upstream activator of neurogenesis that is both necessary and sufficient to initiate neuronal differentiation in vertebrates. I found that xId2 and xId3 negatively regulate xNGN’s activity at a post-transcriptional level, albeit through distinct mechanisms. While both xIds are able to inhibit xNGN’s ability to activate transcription in reporter assays, only xId3 is able to significantly reduce the DNA binding ability of xNGN/E12 heterodimer, through the sequestration of xE12. Importantly, I also demonstrate that by sequestering xE12, xId3 induces xNGN degradation. These findings not only provide the first direct evidence that xId3 modulates xNGN activity and stability, but also emphasize that xId2 and xId3 act through distinct pathways to ensure a stronger short-term control of xNGN activity and consequently prevent premature primary neuron differentiation. This non-redundant role for xId2 and xId3 is further supported by the finding that xId2 and xId3 expression and activity is differentially regulated. Finally, I also provide evidence for a role of Casein Kinase II (CKII) mediated phosphorylation on xNGN activity.

Germband extension (GBE) in the early <i>Drosophila </i>embryo provides a simple system in which to study convergence and extension, a type of tissue remodelling common in development. During GBE the embryonic trunk (“germband”) extends in the anterior-posterior (AP) axis, and narrows in the dorso-ventral (DV) axis. I have quantitatively analysed cell and tissue behaviours during GBE. Our collaborators have developed novel algorithms to measure the continuous impact of cell intercalation and cell shape change on tissue deformation. Here I apply these algorithms to describe the behaviours contributing to GBE. I find that wild type embryos not only undergo cell intercalation, but also cell shape change which accounts for up to 50% of the extension rate during the fast phase of GBE. I find that this cell shape change is elevated in AP patterning mutants that have a defect in cell intercalation, and that this elevation accounts for the initial rapid pulse of extension in these mutants. Regional analysis of these cell behaviours in wild type and AP patterning mutants indicate that cell intercalation is regionally autonomous, but that cell shape change is controlled at a more global level. An external force may be present during early GBE, stretching cells in the AP axis if they fail to intercalate properly. Additionally, I have examined the impact that other morphogenetic movements of gastrulation have on GBE. Mutant analysis shows that the ventral furrow affects the rate but not overall extent of tissue deformation, while the posterior midgut invagination appears to be required only for correct displacement of the tissue as it deforms, and not for tissue deformation itself, at least during early GBE. Thus, the amount of tissue extension, the rate at which it occurs, and the tissue displacement by which it is accommodated are separable phenomena.