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No description available.

This thesis establishes methods to investigate early spleen development in the mouse embryo. An expression analysis of early <i>Nkx2.5 </i>expression is reported, along with the finding that <i>Nkx2.5 </i>may be the earliest marker of splenic precursors in the mouse. Expression of <i>Nkx2.5</i> is also shown for the first time to overlap considerably with that of <i>Nkx3.2 </i>– a major gut development gene upstream of <i>Nkx2.5</i>. An <i>in silico </i>analysis of the evolutionary conserved regions upstream of <i>Nkx2.5 </i>is presented along with the establishment and analysis of stable transgenic reporter lines expressing <i>LacZ </i>under the control of an <i>Nkx2.5</i> gut regulatory sequence (<i>NGRS</i>). <i>NGRS </i>confers spleen, posterior stomach, and pyloric sphincter expression, with very little of the cardiac expression associated with endogenous <i>Nkx2.5. </i>This enhancer is thus ideal for gut studies, providing a tool for directing gut-specific expression and genetic manipulations. <i>NGRS </i>was also found not to require <i>Nkx3.2 </i>for its activity. Finally, <i>NGRS </i>is demonstrated to have the potential to mark abnormal spleen development, in a previously unreported splenic mutant: the <i>Rwhs </i>mutant. Potential uses for <i>NGRS </i>are explored. An approach was taken to mark and follow spleen development, using <i>NGRS-LacZ </i>in an organ culture system. Data generated from these experiments shed some light on how the E11.5 spleen develops, providing evidence for migration of splenic precursors along the stomach, and suggesting that an inhibitory “anchor” effect is normally exerted by the posterior spleno-pancreatic mesenchyme, disruption of which permits precocious spleen development. Finally, an analysis of the role of Wnt signalling in development of the spleno-pancreatic region is presented. Wnt signalling is active in the developing spleen at E11.5, E12.5 and E14.5. A number of <i>Wnt </i>and <i>Frz </i>genes are expressed in the E14.5 spleen.

Patterning mechanisms are controlled by regulatory networks of genes of different cells interacting together to act like a cellular computer. Traditional approaches worked on a case by case basis, analyzing gene networks responsible for specific pattern formation phenomena and exploring how they function by reverse genetics. More recently systems biology approaches have been used to explore how these gene networks function, extracting features that are only apparent on a system scale. These approaches require the computational modelling of the underlying gene regulatory networks controlling the individual patterning phenomena in a spatial context. They have been successful in identifying widespread phenomena such as the robustness of network function to mutation and noise. Due to the advent of more powerful computers, we now believe systems biology can go much further. Instead of limiting ourselves to the gene networks we know are responsible for pattern formation in specific systems, we can model all possible gene networks up to a particular level of complexity (number of genes and interactions). By modelling all possible regulatory networks in a spatial context we are exploring how network structure (genotype) relates to the possible gene expression patterns (phenotype). Fundamental design and evolutionary principles can be extracted by mapping out the space of possibilities in this fashion, which is not possible by analyzing real gene networks on a case by case basis. As a by product, by using a realistic model of gene regulation, new non-intuitive patterning mechanisms can also be discovered and suggested to account for observed patterning phenomena in real biological systems.

Genetic and developmental studies have shown that upon the presence or absence of the Y-linked sex-determining gene SRY, the bipotential gonad will develop either as a testis or ovary. Since the discovery of SRY, several other genes (such as WT1, SF1, DAX1 and SOX9) have been isolated which play an important role in gonadal development and sex determination. Despite these advances our understanding of the mammalian sex determination process remains incomplete. To identify new genes involved in gonadal development, a differential screen using Affymetrix GeneChip arrays was performed. Wild-type male and female gonads were dissected from mouse embryos at El2.5 (during the differentiation process) and subjected to microarray analysis. From this extensive analysis, several novel transcripts were identified, which show a sex-specific expression pattern. The validity of this approach was verified by analysing the expression of these novel transcripts by in-situ hybridisation in male and female gonads at E12.5. Transcripts with a confirmed sex-specific expression pattern where then selected for detailed insitu hybridisation of male and female gonads at El 1.5, E13.5 and E14.5. Moreover, real-time PCR of these candidates was carried out to establish a sex-specific gene expression profile during gonadal development, ranging from 17 to 36 tail somites. This thesis also describes the novel approach of applying small interfering RNAs (siRNAs) to a gonadal organ culture system. Several known sex determination genes were successfully targeted and knocked-down by applying this technique. Importantly, siRNA against Sry effectively blocked male gonad differentiation resulting in a lack of expression of male specific markers. Taken together these results suggest that the siRNA approach in gonad cultures can be used to efficiently analyse the function of candidate genes isolated in our sex determination screen.

No description available.

This thesis is in two major sections: (1) Segmental sensory innervation: During the segmental, sensory innervation of the periphery, neurones situated in the dorsal root ganglia grow out axons at different rates. These vary with segmental level of the ganglia. The most rapid rates of axonal advancement are seen from those ganglia which innervate limb as opposed to non-limb ganglia. How are these differential axonal growth rates controlled? This problem has been addressed using cell culture techniques. No evidence for a segmentally regulated, intrinsic growth mechanism has been found. Further some common constituents of the peripheral fiels of these neurones were also, on the whole, ineffective in regulating neurite growth rates in culture. I conclude from this that some, as yet, unidentified factor in the periphery is reponsible for the enhanced growth rate of axons into limb tissue. A number of differences in the neurite growth patterns of neurones isolated from embryos at different stages of development were noted. This suggests that neurite growth is regulated developmentally if not segmentally. Neurite lengths from cultured neurones were found to benon-normally distributed. They showed a positively skewed, multimodal form. This was not explained by differences in neurite length, branch pattern or the stage of development of the neurones. In the light of this, a quantal hypothesis of neurite growth is proposed, and modelled using Poisson statistics as a theoretical basis. (2) Neuronal toxicity of cytosine-B-D-Arabinofuranoside: Cytosine-B-D-Arabinofuranoside (AraC) is a commonly used anti-mitotic agent in neural cultures. In this study it is also shown to be toxic to cultured neurones and to inhibit neurite growth. This toxicity is dose dependent and inhibited in the presence of 2'DeoxyCytidine (a metabolic precursor of AraC). A relationship between high metabolic demand (neurite growth) and AraC toxicity is discussed in the light of other experimental work.

Coordinated cell movements during zebrafish gastrulation shape the embryo, forming three germ layers and establishing the embryonic body plan. Through convergent extension (CE) movements, mesodermal and neurectodermal cells intercalate at the dorsal midline, extending the anteroposterior axis. CE has been shown to be regulated by non-canonical Wnt signalling, homologous to planar cell polarity (PCP) signalling in Drosophila. In this thesis, I undertook several different approaches to understand the molecular mechanisms by which non-canonical Wnt/PCP signalling regulates CE. Firstly, I characterised the novel PCP gene pklb, one of three zebrafish homologues of the PCP gene prickle (pk). pk1a was shown to regulate CE. Like pk1a, pklb is expressed during gastrulation. Abrogation of pklb function enhances CE defects in the non-canonical Wnt/PCP pathway mutants silberblicklwntl 1 (sib) and trilobiteistbm (tri). Secondly, I investigated the hypothesis that non-canonical Wnt signalling interacts with the Drosophila axon-pathfinding ligand Slit and its transmembrane receptor Robo in regulating CE. The zebrafish homologues slitla, slit2, slit3, robol, robo2 and robo3 are expressed during gastrulation, while gain of function of slit2 in zebrafish was shown to severely disturb CE movements. Abrogation of slitla, slit2, robo2 and robo3 function via morpholino knockdown and a dominant-negative approach yielded CE phenotypes in wildtype embryos and enhanced CE defects on non-canonical Wnt/PCP mutant/morphant backgrounds, suggesting that non- canonical Wnt/PCP and Slit/Robo signalling pathways cooperate in the context of CE. Finally, I characterised a mutant identified in a screen for dominant enhancers of sib that we have r provisionally named airbag (abg). Homozygous abg embryos exhibit a slightly shorter body axis and thickened yolk extension. The pk1a, pklb and stbm morphant phenotypes are enhanced on the abg background. I explored the abg phenotype in contexts separate to CE prior to the mapping of this mutation.

The Notch cell-cell signalling pathway plays a key role both in development and in adult life, controlling cell fate decisions in a wide variety of animal tissues. At its core lie the Notch receptors and their ligands, the proteins of the Delta and Jagged/Serrate family. Both ligand and receptor are transmembrane proteins that interact via their extracellular domains, yet the intracellular domains of the Notch ligands are also important for the activation of Notch receptors on neighbouring cells. In all vertebrates we find a conserved subset of Deltas ending in the C-terminal motif ATEV*, a potential PDZ-binding sequence. This motif mediates the interaction between Delta and the members of the MAGI family of scaffolding proteins. Surprisingly, blocking this interaction has no effect on Delta-mediated processes, indicating that it is not essential for normal Delta function. However, in embryos in which the interaction between Magi and DeltaD is blocked, we find that primary motor neurons are misplaced, a phenotype not seen in the DeltaD loss of function mutant after eight. These data suggest that MAGI proteins may somehow function to restrict Delta function. Recent updates to the zebrafish genome database have revealed a novel ATEV-Delta that is orthologous to mammalian Delta-like 4 (D114), a Notch ligand essential for normal vascular development. Zebrafish dll4, like dll4 in the mouse, is expressed in arterial endothelial cells. Embryos lacking D114 initially develop normally, but at later stages exhibit an overproduction of arterial endothelial cells and excessive angiogenic sprouting. A similar phenotype is seen in embryos lacking the receptor Notch lb and in embryos in which all Notch signalling is blocked with the gamma-secretase inhibitor DAPT. Over-activation of the Notch pathway produces an opposite effect. The excessive angiogenesis observed in embryos lacking D114 is blocked by inhibition of the vascular endothelial growth factor (VEGF) signalling pathway. Thus, D114-Notch signalling acts as an angiogenic "off switch, making endothelial cells deaf to VEGF.

The posterior lateral line primordium migrates along the horizontal myoseptum to the tip of the tail. The migration of the primordium utilises the CXC-type chemokine receptor cxcr4b which is expressed in a graded fashion within the primordium with strongest expression at the leading edge. The ligand for CXCR4b is stromal-derived factor 1a (SDF1a). sdfla is expressed along the length of the horizontal myoseptum. Loss of cxcr4b, or sdfla, results in defects in migration. However, various observations suggest additional systems guiding the primordium. We have performed an expression screen to identify genes with differential expression in the migrating primordium and as a result have identified the chemokine receptor cxcr7b, which is expressed in a complimentary fashion to cxcr4b. Functional analyses reveal that cxcr7b is essential for initiation and guidance of primordium migration. This work reveals that the posterior lateral line primordium is formed by fusion of several distinct groups of placodal cells and the fusion of these groups correlates with the initiation of directional migration. Inhibition of cxcr7b function leads to retarded primordium migration and failure to complete coalescence. Ectopic expression of cxcr7b on the path results in stalled migration of the leading cells suggesting a role for cxcr7b in altering the response of the primordium to sdfla, possibly by inhibiting its effect. It is likely that expression of cxcr7b in the trailing part of the primordium acts to render it incapable of responding to the underlying sdfla stripe, thus producing directional guidance of migration. Preliminary results from this work also demonstrate the presence of FGF signalling in parallel to the cxcr4b/cxcr7blsdf1a pathways which may have roles in the initiation and guidance of the migration of the posterior lateral line primordium. In the absence of cxcr4b/cxcr7blsdfla pathways the primordium often migrates aberrantly towards the pectoral fin bud due to the presence of FGF signalling.