Part 1 of this thesis addresses the control of myelination in the central nervous system (CNS). We have a sound knowledge of myelin structure, particularly the molecules and cells involved in its make-up. However, our understanding of the control of myelin formation is scanty. Myelination of CNS tracts during development follows a strictly ordered schedule suggesting local control by axons. Here I present evidence that CNS axons need to form synaptic connections before they can be myelinated. I have used myelin renewal during regeneration of the fish optic nerve as a model system: the axons withstand target deprivation and their behaviour in these circumstances is well characterised from earlier studies of synaptic plasticity. Depriving the regenerating optic nerve of its primary target, the contralateral optic tectum, delays myelination until the regenerating axons find the intact ipsilateral tectum and form synapses there. Facilitating this process hastens the onset of myelination and denying the axons of any opportunity to form synapses abolishes it. I investigated possible mechanisms by which the synaptogenesis-timed signal is mediated and also compared myelination during regeneration and development. Part 2 describes the phenotypic characterisation of a novel zebrafish mutant: akineto (aknu45). Aknu45 mutant embryos cannot contract their skeletal muscles. Here I show that this is caused by incomplete sarcomere assembly. Myofibrils of mutants lack properly assembled thick filaments. Our understanding of the process of myofibrillogenesis, particularly relating to thick filament assembly, is mostly speculative at present. Although the aknu45 gene is unidentified so far, I am currently engaged in efforts to identify it. Subsequently the aknu45 mutation may become a useful tool to further unravel the process of myofibrillogenesis.

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Environmental oestrogens have adverse effects on fish development and reproduction. The availability of models to study the differential effects of endogenous and exogenous oestrogens and to test for endocrine disrupting chemicals (EDCs) would be advantageous. Vitellogenins (VTGs) are precursors of egg yolk proteins and phospho-lipo-glycoproteins, and belong to a large lipid transfer protein superfamily. VTGs are synthesised differentially in the liver of females under the oestrogenic regulation of the oestrogen receptor (ER) and are distributed to oocytes via the circulatory system. During the last decade, in vitro systems such as fish cell lines have become very important in aquatic ecotoxicology. Thus, a zebrafish liver cell line (ZFL, CRL-2643) was developed for the investigation of VTGs and ERs induction potential oestrogens (E2/EE2). Surprisingly the levels of transcribed ERa and VTGs are very low in ZFL cells and cannot be induced by oestrogens. The weak ERa mRNA was proposed to be the crucial reason of negative VTG induction in responses of oestrogens. Therefore, a recombinant ERa (human Esrl) plasmid 'was subsequently constructed and transfected into ZFL cells. Due to the low efficiency of transfection, Esr 1 stable transfected ZFL cells were only obtained after a long period of selection. However, an unexplained protein band was detected in Esrl transfected cells by western blot. Conversely, the expression of Esrl protein was detected in Esrl transient transfected EcoPack cells (HEK-293) with higher transfection efficiency in parallel assays. Polycyclic aromatic hydrocarbons (PAHs) are suspected to have inhibitory effects on the expression of VTG and ER genes in fish liver through aryl hydrocarbon receptor (AhR) which is a ligand activated transcription factor stimulating the expression of the genes encoding xenobiotic metabolizing enzymes such as cytochrome P450-1A (CYP-IA). ~- naphthoflavone (bNF) is a potent agonist of AhR and also an inducer of such detoxification enzymes as CYPs. As expected, the prominent induction of CYP-IA was detected in ZFL cells by bNF exposure. This result indicated the characterization of hepatic mechanism in ZFL cells and initiated the investigation of the mechanism behind the cross-talk between ERs and AhRs under the combination exposure (bNF and EE2). The results of quantitative real-time, reverse-transcriptase polymerase chain reaction (qPCR) assays revealed that transcript levels of CYP-IA and AhR2 were increased by bNF exposure in a time- and concentration-dependent manner, but no significant effect in AhRls or ERs, whereas ER~2 seemed to be repressed by bNF. There are multiple VTG genes and an in silico analysis of their distribution in the zebrafish genome has identified eight genes (VTG-l to -8) and a pseudogene (VTG-9). A cluster of eight VTGs is located in a single 271 kbp region of chromosome 22, whereas a single gene (VTG-3) is mapped on chromosome 11. Sequence analyses of nucleotide and protein show extensive identity between VTG-l, -4 to -7 and -9 which share a domain structure (LvH- Pv-LvL), and VTG-2 is similar to -8 in terms of length and the presence of a von Willebrand Factor domain (LvH-Pv-LvL-vWFD) whereas VTG-3 is dissimilar in sequence and structural features since it lacks the phosvitin domain (LvH-LvL). qPCR assays were developed to measure transcripts of all VTG and ER genes in adult livers. Transcript levels of VTGs and ERs in vitellogenic female were ordered: VTG-l»VTG-2::::VTG-5>VTG- 4>VTG-6>VTG-7>VTG-3»VTG-8>VTG-9 and ERa>ER~2»ER~I, respectively. In male liver, transcript levels ofVTGs, ERa and ER~1 were undetectable or very low while ER~2 transcript was present in appreciable quantity. According to the genomic organization and phylogenetic analysis, it was speculated that the origin and evolution of multiple VTG genes in zebrafish was based on a number of whole genome duplications (WGDs), gene duplications and conversion events. Furthermore, tests for selection suggested that VTGs in zebrafish had undergone purifying selection in combination with mixed process of concerted and birth-and-death evolution.

The notochord is a vital and defining organ in vertebrates. Mutagenesis screening in zebrafish identified seven 'dwarf mutants that lack notochord development grumpy, sleepy, bashful, dopey, happy, sneezy and doc. This thesis is concerned with the identification and positional cloning of the doc locus as well as the confirmation and characterisation of the dopey and happy loci. Previous positional cloning efforts identified the grumpy, sleepy and bashful genes, demonstrating a requirement for the laminin chains pi, yl and al in formation of the notochord basement membrane (Parsons et al., 2002b Pollard, 2002) and the mutant sneezy, which has been shown to encode the COPI subunit a (Coutinho et al., 2004). This thesis establishes that the doc locus lies within a 0.5Mb region on linkage group 18, containing several genes, including a novel gene encoding a predicted protein with 14 WD40 domains. Antisense morpholino (MO) knock-down of doc results in a phenocopy of doc and insitu expression demonstrates that this gene is expressed specifically within the notochord during development. Expression analysis of echidna hedgehog (ehh) demonstrated that MO knock-down of this gene results in a lack of notochord differentiation. I therefore expect this novel gene is doc. Analysis of the mutants dopey and happy has demonstrated that they encode the coatomer subunits COPp' and COPp respectively. Expression of these and other COPI subunits demonstrate that the majority of COPI subunits are up-regulated within the notochord during development and maintained abnormally in COPI deficient embryos. I have investigated the mechanism of coatomer gene regulation and found that loss of coatomer function leads both to up-regulation of coatomer mRNA and activation of the unfolded protein response (UPR). Suggesting that the UPR is the regulator of mRNA expression, functioning to maintain the secretory network during development, though work to provide definitive proof remains.

Surveys of UK rivers have shown a high incidence of sexual disruption in populations of wild roach (Rutilus rutilus) living downstream from sewage treatment works (STW). Moreover, the degree of intersex (gonads containing both male and female structural characteristics) has been correlated with the concentration of effluent in those rivers.

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Though the zebrafish is an important laboratory model organism, little is known about its natural ecology, and few studies have been conducted on wild populations. It is a small, shoaling cyprinid, native to the floodplains of the Indian subcontinent. A survey conducted in Bangladesh showed that it is most abundant in shallow lakes, ponds and ditches, often associated with rice cultivation. Length-frequency analysis indicates that the zebrafish is an annual species, recruitment being linked to the monsoon season. Its diet, based on gut content analysis, consists primarily of zooplankton and insects. Zebrafish are group spawners and egg scatterers, though females are choosy with respect to sites for oviposition and males defend territories around such sites. The reproductive success of both females and males is affected by density; females produce smaller clutches at higher densities; at low densities territorial males achieve greater reproductive success than non-territorial males, but at higher densities they are no more successful than non-territorials. Female mating preferences, when assessed in the absence of male-male competition, do not correspond to male dominance. The opportunity for selection appears to be weak in zebrafish. Zebrafish show innate and learned preferences for visual cues, both in social and foraging contexts. They shoal preferentially with their own colour pattern, but when individuals are cross-reared with groups of an alternative colour pattern, they prefer to shoal with fish of the appearance with which they were reared; there appears to be a strong learned component to species recognition. In a foraging context, zebrafish display both innate and learned colour preferences. When fish were reared on diets consisting of different colours, each group respond most strongly to red, although there was also an effect of conditioning. This project provides a basis for further studies linking adaptive behaviour with gene expression in zebrafish.