The aim of this project is to investigate genetic mechanisms of early development of vertebrate embryos using model fish species. Zebrafish (Danio rerio) and medaka (Orizias latipes) have been used extensively for molecular genetics and developmental biology studies because these fish produce many eggs, which can be manipulated from the 1 cell stage and are ideally suited for analysing gene expression, function, and embryonic phenotypes. These species have already been extensively used to generate many mutants which show clear phenotypes during early embryonic development. The development of other model species for mutant screening and analyses is likely to provide scope to analyse gene function from uncharacterised/under-characterised genes. Therefore we have developed and tested a small number of early developmental mutants from the mangrove killifish (Kryptolebias marmoratus). To achieve my aim, embryos from zebrafish, medaka and the mangrove killifish have been used as models to study gene function and understand the molecular mechanisms for early patterning genes. We focused in particular on development of neural ectoderm and non-neural ectoderm (epidermis) and anterior-posterior patterning (head, trunk and tail development). As different model animals have different advantages, we used these model animals for different purposes. Zebrafish and medaka were used with chemical treatment (specific inhibitors of target genes) and morpholino analyses because they give many synchronized eggs every morning allowing highly replicated analyses. On the other hand, the mangrove killifish were used for developing and testing novel mutants and associated loss (or gain) of gene function. Firstly, zebrafish was used to study maternal fibroblast growth factor (FGF) signaling at pre-maternal zygotic transition (Pre-MZT) and consequent neural induction at the gastrula stage (Chapter 3). This study found the important role of acquiring maternal FGF signaling in stem cells to achieve neural induction during the zygotic gene expression stage. An FGF signaling inhibitor SU5402 was tested using RNA–seq, ATAC-seq, in.situ hybridization and immunohistochemistry methods. Through these techniques, we found that the maternal FGF signaling provides competence to the ectodermal stem cells for neural induction possibly via epigenetic modification of histone trimethylation. To examine the role of a specific FGF molecule (FGF2), gene knockdown was conducted to study fgf2 gene function during early development in zebrafish (Chapter 4). In situ hybridization and immunostaining with tissue-specific markers at the gastrula stage were used to discover a novel role for fgf2 in development of the epidermis. The final stage of my project involved characterization of mutations underlying two mutant phenotypes (short tail/stl and ball tail/stl), that exhibit defects in tail development using the self-fertilizing mangrove killifish (Chapter 5). Using a small scale RNA-seq, the mutated genes responsible for the stl and btl mutations were instantly identified as noto and msgn1 respectively. The mutant phenotype was phenocopied by morpholino injections in medaka. This study revealed crucial roles of the two genes in tail bud development. Defects of these genes affected the motility of progenitor cells in the tail bud by suppressing cell translocation to the axial mesoderm in the noto mutation and to the paraxial mesoderm in the msgn1 mutation. The study demonstrated similarity of gene function and redundancy in the mangrove killifish and medaka that is different from the function of these genes in zebrafish, revealing the importance of research on different model animals to fully characterise the gene function. From these data, it can be considered that mangrove killifish is very powerful model for mutation screening, suggesting that this animal model can be applied in various genetic studies alongside or in addition to other vertebrate models.

This dissertation reports on the discovery of a new method of patterning bacteria (Pseudomonas aeruginosa PAO1) on a surface using a drying sessile drop. This work identifies bacterial suspension age and the length of time mica is exposed to the laboratory atmosphere as the key parameters which impact the behavior of the sessile drop and the resulting residue. Possible origins of mica aging and bacterial suspension aging are discussed in light of the literature and the experimental conditions. The residue area and the fraction of the residue area on which substantial bacteria and salt deposits remained after the drying of the drop (fill-in fraction) were measured via analysis of optical micrographs. In general, smaller residues are more filled in. For fresh bacterial suspensions, and short mica exposure times, the residue covers the largest area and is characterized by rings formed during discrete depinning events as the solvent evaporates. As the exposure time increases and the mica surface slowly picks up contaminants from the atmosphere, the drop residue shrinks in size and bacteria are deposited in a regular cellular film in the interior of the drop residue. The fraction of the interior area covered by the cellular film is well correlated with the mica exposure time. For sufficiently aged bacterial suspensions, residues are small and more filled-in than residues formed from fresh suspensions on similarly aged mica. In addition, the interior deposition pattern transitions from a cellular film characteristic of fresh suspensions to a cracked carpet pattern for aged suspensions. Suspension aging related changes in the residues are attributed to accumulation of organic materials such as DNA, RNA, proteins, and other bacterial components in the suspension. The suspension aging process is also observed to be at least partially dependent on ventilation of the suspension during aging.

This thesis presents the study of organic layers covalently assembled onto carbon surfaces. As a result of their attachment, the properties of carbon surfaces were controllably adjusted so that these surfaces could be used for desired applications. In order that a wide range of properties were imparted onto the carbon surface, many different modifiers were attached and thoroughly characterised. Three applications that the modified carbon surfaces were used for were the subsequent coupling of molecular species, adsorption of protein and assembly of aldehyde/sulfate-functionalised polystyrene (PS) and citrate-capped gold nanoparticles (NPs). Finally, patterning of different organic layers at pre-determined spatially defined locations on the one carbon surface was also investigated. The carbon surfaces used in this work were glassy carbon (GC) and pyrolysed photoresist film (PPF) surfaces. For PPF, methods for the reproducible fabrication of electrochemically suitable surfaces were investigated. The properties of GC and PPF surfaces are very similar apart from the surface roughness. PPF has near atomic smoothness and has RMS roughness values that are approximately four times smaller than GC. The first series of modifier layers attached to the carbon surfaces was via the oxidation of seven different primary amines. The different layers allowed the modulation of the wettability of the surface. Both n-tridecylamine (TDA, monoamine) and 1,12-diaminododecane (DAD, diamine) are able to form multilayers. The stability of TDA and DAD layers were tested by scanning, soaking and sonicating the layers in different media. Changes in the layer were monitored by the probe response of ferrocene monocarboxylic acid (FCA). However, atomic force microscope (AFM) depth profiling experiments showed that changes in the probe response did not indicate cleavage of the covalently attached layer and mechanisms are proposed to account for the changes in the response of the probe. Surface concentrations of the amine modifiers were estimated by the coupling of an electrochemically active species, FCA and nitrobenzoyl chloride (NBC). The electrochemical reduction of the 4-nitrophenylethylamine (NPEA) layer in acid caused the layer to 'shrink'. Surface concentration estimates of NPEA from acid reduction of layers with different thicknesses suggested that only a limited fraction of the p-nitrophenyl groups were reduced in acid. However, in ACN (acetonitrile)/0.1 M [Bu4N]BF4 (tetrabutyl ammonium fluoroborate) the relationship between the concentration of electroactive surface groups and layer thickness was linear. The other series of modifiers that was attached to alter the surface properties was performed by the reduction of aryl diazonium salts. Subsequent coupling reactions of tetraethylene glycol diamine (TGD) to para methylene carboxylic acid phenyl (MCA) and NBC to electrochemically reduced para nitro phenyl (NPh) layers were carried out. Surface concentrations of NPh as estimated from reduction scans was higher when reduction was performed in ethanol/water compared to acid. Four peaks at N1s binding energies were observed in x-ray photoelectron spectroscopy (XPS) spectra for both acid and ethanol/water reduced layers. The ability of attached amine and aryl layers to modulate the adsorption of protein was investigated using fluorescently labelled protein, bovine serum albumin-fluorescein isothiocynate (BSA-FITC) and fluorescence microscopy. TGD, para methyl phenyl (MP), para hexyl phenyl (HP) and para polyethylene glycol phenyl (PEG)-modified GC surfaces promoted protein adsorption relative to as-prepared GC, whereas n-hexylamine (HA) and polyethylene glycol diamine (PGD) layers reduced protein adsorption. The assembly of two types of NPs, aldehyde/sulfate-functionalised PS and citrate-capped gold NPs, onto amine-containing modifiers layers was examined. Citrate-capped gold NPs were synthesised and characterised. The surface coverage of the gold NPs was controlled by using different modifiers of different chemical compositions, tuning the modification conditions and adjusting the immersion time, concentration and pH of gold NP solution. Approaches to creating patterns of modifiers in pre-determined spatially defined locations on GC and PPF surfaces using poly(dimethyl)siloxane (PDMS), poly(vinyl)alcohol (PVA) and thin metal films were investigated. With the "fill-in" approach using PDMS, the smallest pattern of modifiers was the parallel lines with a line width of 20 µm and straight edges and was created by performing electrochemistry in PDMS microchannels which has not been previously investigated. Visualisation techniques, based on optical and scanning electron microscopy, were demonstrated for the molecular patterns.

Studies on new arthropod models are shifting our knowledge of embryonic patterning and morphogenesis beyond the Drosophila paradigm. In contrast to Drosophila, most insect embryos exhibit the short or intermediate-germ type and become enveloped by extensive extraembryonic membranes. The genetic basis of these processes has been the focus of active research in several insects, especially Tribolium castaneum. The processes in question are very dynamic, however, and to study them in depth we require advanced tools for fluorescent labelling of live embryos. In my work, I have used a transient method for strong, homogeneous and persistent expression of fluorescent markers in Tribolium embryos, labelling the chromatin, membrane, cytoskeleton or combinations thereof. I have used several of these new live imaging tools to study the process of cellularisation in Tribolium, and I found that it is strikingly different to what is seen in Drosophila. I was also able to define the stage when cellularisation is complete, a key piece of information that has been unknown until now. Lastly, I carried out extensive live imaging of embryo condensation and extraembryonic tissue formation in both wildtype embryos, and embryos in which caudal gene function was disrupted by RNA interference. Using this approach, I was able to describe and compare cell and tissue dynamics in Tribolium embryos with wild-type and altered fate maps. As well as uncovering several of the cellular mechanisms underlying condensation, I have proposed testable hypotheses for other aspects of embryo formation. The work presented in this thesis will serve as a foundation for future studies on cellularisation and tissue morphogenesis in Tribolium. Furthermore, the live imaging method, the fluorescent labelling constructs, and the analysis I carried out should be easily adaptable to other non-model arthropod species.

The research presented here considers automatic diagnosis support for skin cancer. The role of computer-based diagnosis, and its value within a primary care situation are examined resulting in synthesis of aims, requirements and properties for an effective system -a system based on digital optical images captured and processed using low-cost commercial computer technology. The issues involved in acquisition of lesion boundaries are discussed. The value of accurate and robust boundaries, in terms of both directly obtainable diagnostic features and in enabling lesion property evaluation, is identified. Previous research has proposed the edge focusing process. This work has addressed the improvement, in terms of potential for future development, evaluation and reuse, of this process through porting it to a highly modular form in the Khoros environment. The role of colour analysis and its value in terms of provision of diagnostically useful features is investigated, and the central importance of segmentation is identified. The fundamental properties of effective segmentation of lesion image colours are identified as a need to reflect human perception of colour similarity and a basis on local regions. A new region-based segmentation technique using data transformed to a perception-uniform colour-space is presented and shown to yield promising results. Finally the use of texture information is discussed. The nature and properties of the large-scale texture of skin patterning and its disruption are investigated and an abstracted representation proposed. A new technique is presented and shown to be effective in extracting the qualities of the skin patterning. Methods for analysing this representation of the patterning to quantify the disruption attributable to the lesion are proposed and developed. The combination of these extraction, analysis and disruption evaluation techniques is shown to be effective in relation to both visual assessment of disruption and diagnostic performance.

Abstract The mammalian metanephric kidney develops through ureteric bud branching morphogenesis and tubule formation and involves secreted inductive signals and possibly their antagonists to regulate the process. Sprouty (spry) genes encode antagonists of FGFs and the EGF signalling pathways. To get an insight to potential developmental roles of the spry genes, the expression of spry1, 2 and 4 was analyzed in developing kidney. Spry1 is expressed in the ureteric bud, and spry2 and 4 in the ureteric bud, the kidney mesenchyme and the nephrons deriving from it suggesting developmental roles for the sprys in kidney development. Spry function was addressed in vivo in the kidney by targeting hspry2 expression to the ureteric bud with a Pax2 promoter. Hspry2 expression led to development of small, ectopic and cystic kidneys. Ureter branching was reduced and there was less glomeruli in a smaller kidney compared to the wild type controls. Spry2 may antagonize signalling of FGF2 and lead to changes in FGFR1 and FGFR3 expression. In organ culture ectopic FGFs restored ureteric branching of the hSpry2 transgenic kidneys suggesting that hSpry2 may antagonize FGF signalling in embryonic kidney. In addition to changes in FGFs, hspry2 expression also lead to downregulation of GDNF and BMP4. We conclude that the Sprouty-FGFs-FGFR signaling is important for kidney development. Wnt2b is a recently identified member of the Wnt family of secreted growth factors, but its function in organogenesis is unknown. In the kidney Wnt2b is localized to the perinephric mesenchymal cells at the initiation of organogenesis. Wnt2b signalling supported ureteric bud growth and branching in vitro. Ureteric bud that was co-cultured with Wnt2b expressive cells or incubated with a known Wnt pathway regulator lithium, and then recombined with isolated kidney mesenchyme led to recovery of the expression of some ureteric epithelial marker genes and reconstitution of early kidney development. Hence, Wnt2b signalling is critical for induction of ureteric branching in vitro. Type XVIII collagen is a matrix molecule and may be involved in Wnt signalling. Roles of type XVIII collagen in kidney and lung organogenesis was analysed. Type XVIII collagen expression correlated with the differences in epithelial branching in both of these organs and its expression in the epithelial tissue was mutually exclusive. In recombinants of ureteric bud and lung mesenchyme, type XVIII collagen expression pattern shifted from kidney to lung type and was accompanied by a shift in epithelial Sonic Hedgehog (Shh) expression and by ectopic lung Surfactant Protein C in the ureteric bud. Blocking of type XVIII collagen function prevented ureteric development with lung mesenchyme and associated with reduction in the expression of Wnt2. Taken together, the findings suggest critical roles for Sprouty2, Wnt2b and type XVIII collagen in controlling pattern formation and the mode of ureteric bud branching in the embryonic kidney.

Abstract How cell and tissue interactions lead to complex structures and differentiated cell types during organogenesis is still one of the most fundermental questions in modern molecular biology. Laminin appears to have a role in branching morphogenesis during organ development. Laminin5 (α3β3γ2) is an epithelium-specific isoform of laminin and previous report has shown that two alternative transcripts for the γ2 chain, the longer γ2 and the shorter γ2*, result from alternative use of the last exon in the human LAMC2 gene. But the transcription of murine laminin γ2 and γ2* and their biological significance have remained unclear. Type XVIII collagen is a newly identified member of the collagen family. It may be involved in the Wnt signaling pathway, since its longest N-terminal variant contains a frizzled domain, which is part of the Wnt receptor and could antagonize Wnt signaling when secreted. Wnt2b is a new member of the Wnt family. Also its function in organogenesis is unknown. In this study, we have investigated the expressions of laminin γ2 and γ2*, type XVIII collagen and Wnt2b during mouse organogenesis. The function of type XVIII collagen in developing lung, kidney and a recombination of ureteric bud and lung mesenchyme tissue and the function of the Wnt-2b gene during kidney organogenesis were studied by using the combined methods of traditional experimental embryology and modern molecular biology. Two alternative laminin γ2 transcripts were demonstrated in mouse. In the developing kidney, the shorter γ2* form was localized in the mesenchyme, whereas the longer γ2 form was only present in the epithelium of the Wolffian duct and in the ureteric bud, indicating different functions for the γ2 variants. Type XVIII collagen was expressed throughout the respective epithelial bud at the initiation of lung and kidney organogenesis. It becomed localized to the epithelial tips in the early-stage lung, while it was confined to the epithelial stalk region and was absent from the nearly formed ureteric tips in the kidney. In recombinants of ureteric bud and lung mesenchyme, the type XVIII collagen expression pattern in the ureteric bud shifted from the kidney to the lung type, accompanied by a shift in epithelial Sonic Hedgehog expression. The lung mesenchyme was also sufficient to induce ectopic lung Surfactant Protein C expression in the ureteric bud. A blocking antibody for the type XVIII collagen reduced the number of epithelial tips in the lung and completely blocked ureteric development with lung mesenchyme, which was associated with a notable reduction in the expression of Wnt2. The shift in type XVIII collagen expression in ureteric bud and lung mesenchyme tissue recombinant was also accompanied by the significant morphological changes in the branching pattern in ureteric bud development. Wnt2b was expressed in numerous developing organs in the mouse embryo, but it was typically localized in the perinephric mesenchymal cells in the region that partly overlaps the presumptive renal stroma at E11.5. Functional studies of the kidney demonstrated that 3T3 cells expressing Wnt2b were not capable of inducing tubule formation but rather stimulated ureteric development. Recombination of ureteric bud treated with cells expressing Wnt2b and isolated kidney mesenchyme resulted in recovery of the expression of epithelial marker genes and better reconstituted organogenesis. Lithium, a known activator of Wnt signaling, was also sufficient to promote ureteric branching in reconstituted kidney in a manner comparable to Wnt2b signaling. Our data suggest that different organ morphogenesis is regulated by an intraorgan patterning process that involves coordination between inductive signals and matrix molecules, such as type XVIII collagen. In the mouse kidney, Wnt2b may act as an early mesenchymal signal controlling morphogenesis of epithelial tissue, and the Wnt pathway may regulate ureteric branching directly.

Yes / The HOX genes are a group of homeodomain-containing transcription factors that play important regulatory roles in early development, including the establishment of cell and tissue identity. HOX expression is generally reduced in adult cells but is frequently re-established as an early event in tumour formation and supports an oncogenic phenotype. HOX transcription factors are also involved in cell cycle regulation and DNA repair, along with normal adult physiological process including stem cell renewal. There have been extensive studies on the mechanism by which HOX proteins regulate transcription, with particular emphasis on their interaction with cofactors such as Pre-B-cell Leukaemia Homeobox (PBX) and Myeloid Ecotropic Viral Integration Site 1 (MEIS). However, significantly less is known of how the activity of HOX proteins is regulated. There is growing evidence that phosphorylation may play an important role in this context, and in this review, we draw together a number of important studies published over the last 20 years, and discuss the relevance of phosphorylation in the regulation and function of HOX proteins in development, evolution, cell cycle regulation, and cancer.

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