Lowe syndrome is a rare X linked disorder, characterized by renal, ocular and cerebral defects, caused by mutation in the protein OCRL1. OCRL1 has been implicated in a plethora of cellular functions, and loss of its catalytic conversion of PtdIns(4,5)P2 into PtdIns(4)P is proposed to underly many of the cellular phenotypes associated with lack of OCRL1. The interaction with other proteins such as IPIP27A are also required for the correct function of OCRL1. Renal tubular dysfunction similar to that seen in Lowe syndrome patients is seen in zebrafish models of ocrl1 and ipip27a mutation. Using zebrafish as a model of Lowe syndrome, a reduction of increased PtdIns(4,5)P2 levels in ocrl1-/- embryos is shown to alleviate the renal tubular dysfunction. This demonstrates that targeting PtdIns(4,5)P2 is a viable option for therapeutic treatment of Lowe syndrome. Novel transgenic zebrafish lines are also described, that provide megalin specific, fluorescent and luminescent readouts of proximal tubular endocytic function. These will be an important tool to perform high throughput screens for compounds that alleviate the symptoms of Lowe syndrome. The importance of the binding of IPIP27A to its interaction partners OCRL1 and SH3 containing proteins such as PACSIN2 is demonstrated by rescue of the ipip27a-/- mutant with ipip27a with mutated binding sites. The phenotype of ipip27a-/- mutant embryos is further characterised to demonstrate there is no long term growth defect or defect in tubular polarity, however tubular dilation is seen, suggesting possible mild ciliary defects. In the zebrafish proximal tubule in fish with no functional IPIP27A or OCRL1, a more severe defect in 10 kDa dextran endocytosis is seen, as well as hydrocephaly and curved body axis, cilia impairment related phenotypes. This indicates that IPIP27A and OCRL1 are acting in the same pathway, and therefore depletion of both exacerbates phenotypes. Finally, transgenic lines expressing ubiquitous or pronephric tubule specific fluorescently tagged Rab proteins as markers of membrane compartments in zebrafish are described.

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Within the ventral midbrain of the developing vertebrate embryo there is a transient area of conserved gene patterning called the midbrain arcs, this patterning influences the formation and position of nearby nuclei, as well as the position of the MLF (medial longitudinal fascicle) axon tract in the early axon scaffold. A hypothetical regulatory loop has been identified between the genes Nkx1.2 and Emx2 during midbrain arc patterning and this project aimed to identify if a highly conserved non-coding sequence named Nkx1.2.1 acted as an enhancer for the Nkx1.2 gene, and bound directly with the Emx2 protein. Electroporation of a reporter construct containing the Nkx1.2.1 sequence in the chick embryo identified the element was active specifically in the ventral midbrain during the time of midbrain arc patterning. The Nkx1.2.1 sequence was then analysed for binding affinity with the Emx2 protein using EMSA. The full Emx2 sequence could not be produced in a soluble form, but two artificial sub-forms of the protein were produced. The closest binding affinity identified was a value of 0.2 K_dμM, compared to the control experiment using a non-specific sequence of DNA of 2.6 K_dμM.

Gangliosides have been measured in three parts of the brain (forebrain, cerebellum and brainstem) in three species namely man, the pig and the rat at different stages of normal development. Sub fractionation of gangliosides into the four major types was carried out in each brain part by thin-layer chromatography. DNA content was studied as a measure of cellularity. Samples of the three brain parts from malnourished children were studied. Brain parts from pigs undernourished prenatally (runts) and pigs malnourished postnatally (protein deficient or calorie deficient) and also from animals rehabilitated for different periods of time were included in this study. The brain of rats undernourished postnatally and of the offspring of rats reared from weaning on a moderately low protein diet and also those of the offspring of rats which were on a low protein diet during gestation and lactation were used in this investigation. The studies of the ganglioside content of the three parts of the brain of normal pigs and rats showed that there are differences in the pattern of development in each part. In the development curve for the forebrain, there were two peaks in both species. However, there was an overall similarity in the pattern for each of the brain parts in these two species. The large difference was in the timing of the first peak in the curve for the forebrain. In all three species, the principal ganglioside in the forebrain was a disialoganglioside, G[3], and that in the cerebellum was the trisialoganglioside, G[1]. The ganglioside content was found to be low for the chronological age in all three parts of the brain of the malnourished children and animals; the value was particularly low for weight in the forebrain of malnourished children, the cerebellum of the undernourished pigs and the brainstem of the severely malnourished rats. Incorporation of [14]C-glucosamine into the brain gangliosides of the severely malnourished rats, in vivo, resulted in a low total activity compared to the controls. The G3 ganglioside, which normally accounts for the largest proportion of the major gangliosides in the forebrain, was proportionally reduced considerably in the forebrain of malnourished children. Rehabilitation for different periods of time did not particularly correct the ganglioside content of the cerebellum in the undernourished pigs.

Protein phosphorylation by tyrosine kinases evolved in multicellular organisms to regulate intracellular signalling pathways associated with proliferation, differentiation and migration. In most tissues, basal protein tyrosine phosphorylation is maintained at low levels, but in the brain, basal tyrosine kinase activity is high and regulates key processes in the developing and mature brain and is dysregulated in neurological disorders. N1-Src is a neuronal splice variant of the ubiquitous proto-oncogene C-Src tyrosine kinase, which differs by a six amino acid insert in its SH3 domain. Since the SH3 domain confers substrate specificity, it is anticipated that both C- and N1-Src will have different substrates and functions. Specifically, N1-Src is highly active in the developing brain and has been implicated in neuronal differentiation. Studies also suggest a role for N1-Src in ion channel regulation, however, the mode of action of N1-Src remains poorly understood. The primary aim of this study was to further clarify the role of N1-Src in both the developing and adult brain. To achieve this, a multidisciplinary approach was adopted, which sought to 1) identify novel N1-Src substrates 2) determine the function of N1-Src in developing neurons and 3) dissect the signalling pathways downstream of N1-Src. Recombinant, active Src kinases were generated to undertake in vitro kinase assays with putative N1-Src substrates. Src-dependent phosphorylation of HCN1, a pacemaker channel identified as an N1-Src interactor in a yeast 2-hybird screen, could not be detected. This result was not conclusive as surprisingly, the assay did not detect Src or PKC phosphorylation of NR2A, an NMDA receptor subunit, previously characterised as a robust Src and PKC substrate. However, a screen of several putative N1-Src SH3 binding peptides revealed some encouraging candidates to pursue as substrates. To address the function of N1-Src in neuronal development, N1-Src was overexpressed or knocked down in cultured hippocampal neurons. Both manipulations were detrimental to neurite outgrowth and neuronal polarization, suggesting that N1-Src activates cytoskeletal remodelling pathways and precise levels of N1-Src are required for normal cellular development in vitro. The molecular mechanism of this phenomenon was investigated in a fibroblast cell line, in which N1-Src overexpression induces neurite-like processes. Using this model, an investigation into the role of N1-Src in RhoA signalling implied that N1-Src does not drive process outgrowth via the inhibition of RhoA, however constitutive activation of RhoA, prevented N1-Src mediated process extension. Preliminary results suggested that N1-Src overexpression enhances RhoA activation, which could form part of a negative feedback loop. Taken together, I have implicated N1-Src in neurite outgrowth, which provides a starting point for understanding the mechanistic role of N1-Src in pathways that dictate neuronal morphology.

Neuroanatomical and functional asymmetries are a widespread, probably universal, feature of the vertebrate nervous system. Although brain asymmetry is a fundamental characteristic of the CNS, how it is established is not known. The epithalamus is a subdivision of the diencephalic region of the forebrain and structural asymmetries in this region are widespread among vertebrates. The epithalamus of the zebrafish shows differences between the left and right sides in terms of neuronal organization, connectivity and gene expression and has become a focus for the study of establishment and elaboration of brain asymmetry. Unilateral Nodal expression in the dorsal diencephalon of zebrafish embryos is required for correct lateralisation of the epithalamus. However, in embryos lacking Nodal signalling asymmetries still develop, but their sidedness is randomised among siblings. This suggests that Nodal is not required for asymmetric development per se and that other signals are responsible for generating asymmetry. In order to uncover signalling pathways required to break symmetry in the brain, we aimed to identify mutant lines that do not show elaboration of CNS asymmetries. In this thesis I describe the phenotype of the/g/8 mutant, acerebellar (ace), which develops a symmetric epithalamus. The parapineal does not migrate and remains at the midline and later epithalamic asymmetries do not develop. However, unilateral Nodal signalling in the brain and body axis are largely unaffected in ace mutants. Fgf8 is expressed bilaterally in the epithalamus in wild type embryos, as are some Fgf-responsive genes. Additionally, several Fgf-pathway genes are expressed specifically in the migrating parapineal. Using a modified Fgf8 micro-bead implantation technique, I am able to rescue the lateralised migration of the parapineal in ace mutants. In situations of unbiased Nodal signalling, exogenous Fgf8 can impose laterality on the epithalamus. In summary, these studies demonstrate that Fgf8 can break neuroanatomical symmetry in the epithalamus through the regulation of the bi-stable left- or right-sided migration of the parapineal. I present a mechanism whereby the combined action of Nodal and Fgf signals ensures the establishment of neuroanatomical asymmetries with consistent laterality.

This thesis examines the ultimate and proximate determinants of niche width, with a focus on how cognition and biological information processing may drive the evolution of niche width. Using both field and laboratory experiments I investigate how learning can alter resource use in syrphids. Modelling biological information processing using artificial neural networks I consider how various ecological factors interact and can impact information processing to determine decision accuracy (a proposed factor in the evolution of niche width). Finally the ability of artificial neural networks to overcome evolutionary dead ends due to specialisation and functional loss is examined. I found that syrphids were able to use external, inter-specific cues to alter their resource use. Specialist artificial neural networks decision accuracy was altered by the introduction of the ecological variables they were subjected to and the loss of functionality can create an evolutionary dead end scenario only in very extreme cases or under specific ecological pressures. I studied the syrphid (Episyrphus balteatus) both in the field and under laboratory conditions. There is a huge amount of literature describing how bees use scent marks to aid decision making before landing on flowers but there is currently no work on the syrphids ability to detect and utilise these scent marks. The question I posed was ‘Can syrphids modify their pattern of resource utilisation by using this scent mark information?’ The field work was carried out using motion detection cameras positioned above flowers of knapweed (Centaurea nigra). The flowers had two different treatments: one was bagged overnight to prevent pollinator access and the other was left unbagged allowing foraging insects to deplete the nectar and pollen. Visits from both conditions were recorded and compared. I found that previously bagged flowers received more visits from both bumblebees (Bombus spp.) and syrphids suggesting that syrphids could also detect when a flower was depleted without landing. iii The laboratory tests were conducted in an arena using artificial flowers. The experiment was split into a learning phase and a testing phase. I tested the syrphids ability to recognise and learn an association to two different compounds, bee scent marks or 1-Hexanol. I found that syrphids could learn to associate both bee scent marks and 1-Hexanol with negative rewards and use this information to change their foraging behaviour. I used artificial neural networks to investigate differences between the decision accuracy of specialists and generalists when foraging under ecological pressures. Previous work has shown that specialists had higher decision accuracy when non-host selection carried a mild reward and I was interested to see how ecological variables would impact this advantage. The ecological conditions I considered were search costs, resource availability and starvation. To do this I trained neural networks to recognise different numbers of binary images (hosts) over a range of positive and negative non-host rewards or punishments. The fewer hosts a network had the more specialised it was. I found that both starvation and resource availability reduced the range of non-host values across which specialist networks had a fitness advantage over generalists. Interestingly I found that introducing search costs shifts the range of non-host values where specialist advantage occurs rather than narrowing them as in the previous conditions. Specialists suffering from search costs performed better when non-host selection carried a high to intermediate punishment. Finally, I used artificial neural networks to investigate the evolutionary dead end theory. This theory states that specialist organisms will lose genetic variation and will be unable to respond as effectively to ecological change. I first trained networks as specialists. These networks were then re-trained as generalists. While re-training networks had a percentage of their weights fixed to simulate the suggested reduction in evolutionary potential of specialists. Ecological conditions in these simulations were either non-host penalties, search costs or a combination of the two. I found that networks were relatively robust to loss of evolutionary iv potential. All of the networks performed well even at intermediate (50%) weight fixation. The application of search costs reduced overall network fitness but this effect was not as pronounced as when non-host penalties were introduced. Non-host penalties had the greatest effect on the fitness of networks. These results suggest that specialisation should only become an ‘evolutionary dead end’ under very specific and severe conditions.

The localization of L- and S-periaxin was studied in the developing axon-Schwann cell unit where both proteins were localized to myelin-forming cells. During initial axonal ensheathment L-periazin was detected at the Schwann cell plasma membrane and in uncompacted myelin whorls. In early postnatal nerve it was concentrated in the adaxonal (apposing the axon) and abaxonal (apposing the basal lamina) membranes, but as the myelin sheath matures, L-periaxin became predominantly localized to the abaxonal Schwann cell membrane demonstrating a dynamic change in localization during development and ensheathment. This shift in localization of the protein after completion of the spiralization phase of myelination suggests that it participates in stabilizing the mature myelin sheath. In contrast, S-periaxin was not associated with membranes but appeared to be present throughout the Schwann cell cytoplasm. Both proteins were excluded from compact myelin. During embryogenesis Schwann cell precursor cells develop from migrating neural crest cells. At around E14.5 in the mouse sciatic nerve the precursor cells differentiate to form embryonic Schwann cells which in turn become either myelin-forming or non-myelin-forming Schwann cells in the mature PNS. In contrast to the mRNA for the major myelin protein P0, which is expressed in neural crest cells, L-periaxin mRNA and protein were first detected in embryonic Schwann cells. S-periaxin was detectable somewhat later at post-natal day 1. L-periaxin protein was not expressed in the non-myelin-forming Schwann cells of the sympathetic trunk and is therefore the earliest known marker for myelin-forming Schwann cells, also indicating that as early as E14.5 Schwann cells are committed to either a myelin-forming or non-myelin-forming lineage. L-periaxin was initially detected in the nuclei of embryonic Schwann cells; however it was predominantly localized to the plasma membrane by E17.5. The regulation of the <I>periaxin</I> gene was investigated by transgenesis. It was shown that a region of 5.5 kb upstream from the transcription initiation site could direct expression to a subset of myelinating Schwann cells as well as cells in the central nervous system indicating the presence of a neuronal silencer in the <I>periaxin </I>gene.