In mammals, the timing of sex determination and a small number of the genes involved in gonadal development have been established. However, other so far unidentified genes are clearly involved in gonadal development, in avian and other species. Chick gonadal development was investigated by studying the morphology of the developing gonads and by attempting to gain a better understanding of the genes involved in the initial stages of this developmental process. The first approach was a histological analysis of chick gonadal development over the period from the indifferent gonad stage to the initial stages of ovary and testis differentiation. Secondly, we analyzed the expression of chick homologues to genes involved in mammalian gonadal development, in an attempt to compare gene regulation and timing of gonadal development between mammals and avians. Finally, we used the technique of differential display to both ascertain the feasibility of this approach in the identification of transcripts in a complex developmental system and to isolate novel genes involved in chick gonadal development. As a result of this study we were able to predict that the sex determination event in chicks occurred earlier in embryogenesis than previously documented. We also established that genes involved in mammalian gonadal development were expressed with similar expression profiles in the developing chick gonads. Finally, twelve candidate clones were isolated by differential display, five of which represented novel sequences. Expression profiles, during chick gonadal development and differentiation, were analyzed by Northern analysis and whole mount <I>in situ</I> hybridization to confirm differential expression.

Using microarray technology it has been possible to identify genes both induced by LIF and differentially expressed upon Oct4 knockout in the ZHBTc4.1 cell system. Analysis of the biological function of two genes from the LIF pathway (<i>Egr1 and Gbx2</i>) revealed altered morphology, transcriptome and putative ability to self-renew upon over-expression. The analysis of genes enriched in ES cells compared with trophoblast and neural stem cells, which are subsequently down-regulated upon Oct4 deletion revealed 42 genes including; <i>Nr0b1 </i>and <i>8430410A17Rik. </i>Enrichment analysis combined with examination of the convergence between the LIF/Stat3 and Oct4 pathways revealed <i>Klf2, Klf4 </i>and <i>Klf5 </i>as genes which are tightly associated with pluripotency. <i>Klf2 </i>has the ability to engage long term LIF-independent self-renewal in <i>Lifr^-/- </i>ES cells. Long term culture of mES cells is associated with karyotypic changes which seemingly enhance self-renewal at the expense of pluripotent contribution. Understanding selection may identify genes and loci with novel functions in self-renewal. Using array based comparative genome hybridisation (aCGH) it was possible to identify a recurrently aberrant region on chromosome 14, which contains <i>Rb1, Spry2 </i>and <i>Klf5. </i>Trisomy chr. 11, amplification of a 37Mb region containing the <i>Nanog </i>locus on chromosome 6 and an 11Mb deletion on chromosome 17 were also discerned. The regions on chromosomes 6 and 11 are syntenic with recurrently aberrant regions in human ES cells, implying conservation of either the mechanism of aberration, or a selective pressure that is retained across different species of ES cell. Utilisation of array-based technologies has facilitated the identification of number genes which are enriched in mES cells and are rapidly down-regulated upon Oct4 deletion, some of which are also implicated in the LIF/Stat3 pathway. One gene in particular; <i>Klf2 </i>a member of the Krüppel-like family is capable of maintaining cytokine independent self-renewal upon over-expression. <i>Klf4 </i>and <i>Klf5 </i>also seem to be entwined with Oct4 and LIF/Stat3 function. Karyotypic aberrations in mES cells were also identified, which represent putative selective loci which may shift the balance between self-renewal and survival over differentiation and death.

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In <I>Drosophila </I>the three <I>yolk protein (yp) </I>genes are expressed in the adult female fat body and ovary. The yolk proteins (YPs) are stored in the oocyte for utilisation during embryogenesis. Within the intergenic region of <I>yp2 </I>and <I>yp2</I>, a fat body enhancer (FBE) and two ovarian enhancers have been found to direct YP1 and YP2 tissue- and sex-specific expression. The male and female forms of Doublesex protein (DSX<SUP>M</SUP> and DSX<SUP>F</SUP> respectively) have been found to bind to the FBE and are thought to confer the sex-specificity of <I>yp</I> expression to the fat body of females only. The <I>dsx</I> gene is at the end of the sex determination pathway, other genes that operate from this branch of the pathway include <I>hermaphrodite (her) </I>and <I>intersex (ix)</I>, and it is possible that HER and IX proteins could also regulate <I>yp </I>gene expression. Upstream of <I>yp3</I> are sequences that direct female-, fat body-specific <I>yp3</I> expression (FBE3) and ovarian-specific <I>yp3</I> expression (OE3). This project involved analysis of FBE3 and OE3 to gain further understanding of the regulation of <I>yp3</I> expression. The role of DSX in the regulation of <I>yp3</I> has been investigated; the FBE3 was divided into smaller subfragments and it was determined that one of these fragments can bind DSX <I>in vitro. </I>These subfragments were cloned into reporter gene constructs to test their capabilities in directing correct <I>lacZ</I> expression pattern <I>in vivo</I>, using P-element mediated transformation. This revealed that the fragment capable of binding DSX <I>in vitro,</I> could direct <I>lacZ</I> expression in fat body cells, but this expression was not sex-specific, both male and female fat bodies showed reporter gene expression. C/EBP was reported to bind to the FBE <I>in vitro</I> and considered to be a potential activator of <I>yp</I> gene expression. From C/EBP mutant analysis, there was no observed difference from wild type in the level of YP expression <I>in vivo</I>; thus it is unlikely that C/EBP has a regulatory role in <I>yp</I> gene expression in the fat body.

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This study investigates the role of present day crustacean bioerosion and bioturbation in the development and taphonomy of a muddy coral reef environment (Phuket, South Thailand). At the main locality, a 200 m wide, 400 m long, intertidal fringing reef flat drops 3 m on to a muddy sea bed where fine surface sediments comprise 80% terrigenous clays. Adjacent intertidal sand waves and tidal pools are composed of gravel - to sand-size sediments. <I>In situ</I> experimentation involved transect and quadrat surveys, resin casting and airlift excavation of burrows, coring, measurement of sediment turnover rates and direct observation of sediment mixing using tracer sediment. Grain size analysis, carbonate content determination and microscopy studies of sediments, borers and coral growth patterns were carried out in the laboratory. Crustacean bioerosion, especially 'passive' boring by hapalocarcinid crabs, pyrgomatid barnacles and alpheid shrimps, alters coral colony morphology which in turn affects reef development. Reef progradation relies on the continued growth of coral boulders that have fractured along large upogebiid and alpheid boreholes and then toppled from the reef front on to the fore-reef slope. Bioerosion also results in the production of calcareous sediment. Active erosion by upogebiid and alpheid shrimps directly produces grains in the gravel to silt size range. Coral colonies weakened by boring are prone to physical disintegration into boulder- to silt-size fragments. Crustacean bioturbation, mainly by callianassid and alpheid shrimps and <I>Dotilla</I> crabs, is abundant in both intertidal and subtidal reef sediments. The subtidal sediment slope shows an offshore zonation of crustacean burrowers. Simple, sloping burrows of near-reef alpheids give way to deep, spiral, off-reef alpheid burrows and then to complex, Thalassinoides-type burrow networks of callianassids offshore. Burrow distribution seems to be related to the grain size and sediment thickness of the areas they inhabit. Tiered burrows, in densities of up to 350/m<SUP>2</SUP>, were measured subtidally. Depths of turnover of up to 25cm (subtidal callianassid and alpheid shrimps) and turnover rates of up to 0.4 m<SUP>3</SUP>/m<SUP>2</SUP>/year (550kg/m<SUP>2</SUP>/year (550kg/m<SUP>2</SUP>/year; <I>Dotilla</I> crabs) were calculated. Subtidal bioturbation rates are three times the rate of sediment accumulation on the fore-reef slope. Biogenic sorting of sediment occurs, with subtidal callianassids burying coarse grains in shelly pockets at depths of >25cm, maintaining a relatively gravel-free surface layer. In contrast, intertidal and near-reef alpheids sort grains to produce a heterogeneous distribution of gravel patches exposed at the sediment surface.

<i>Sox1</i> is upregulated during ES cell differentiation into neural precursors, and its misexpression causes an EC cell line to differentiate into neurons. <i>Sox1 </i>expression during nervous system development is associated with proliferating cells of the CNS, expression being lost as cells exist mitosis and terminally differentiate. Its expression pattern is more restricted than that of most other markers for early neural cells such as nestin, making it a good marker gene for the study of neural development both <i>in vivo</i> and <i>in vitro</i> from ES cells. Here I have used gene targeting to generate a reporter ES cell line (46C) in which the <i>Sox1</i> open reading frame is replaced by the gene encoding enhanced green fluorescent protein linked to a selectable marker. The use of EGFP enables the observation of <i>Sox1</i> expression in live cells. The expression of the reporter faithfully recapitulates the normal expression of <i>Sox1</i> <i>in vivo </i>in mice generated from the 46C cells. This cell line has been used to analyse the differentiation of ES cells to neural fates, in particular to characterise a newly discovered, monoculture differentiation system. <i>Sox1-</i>EGFP expression is monitored by flow cytometry, which enables quantification of the differentiation process. The effect of proteins and inhibitors implicated in neural determination has been monitored quantitatively and over time using this system. Acquisition of neural fate occurs rapidly in the absence of any inducers or serum, and without formation of multicellular aggregates. BMP-4 completely blocks this neural fate specification, similarly to the situation in the frog and the chick. Collectively, the results indicate that restriction of ES cells pluripotency to neural fates occurs in a manner resembling default neural induction in amphibians.