Replacement of the prototype promoter recognition factor (PRF) TFIID by alternative PRFs or changes in its subunit composition have recently been implicated in differentiation processes during development. TFIID is composed of TBP (TATA-binding protein) and 13 TAFs (TBP-associated factors). I comprehensively studied the roles of Tafs during vertebrate development using zebrafish model. Taf knock down (kd) phenotypes generated in an antisense morpholino oligonucleotide (MO) screen suggest differential functions of Tafs during zebrafish development. The kd phenotypes also propose a special requirement of DNA-binding Tafs during zebrafish development. In conjunction with zygotic mutant phenotype analysis of zebrafish taf8-mutants compared to taf6-mutants I investigated a potential coactivator function of Taf8 for Pparγ, which has been suggested by in vitro data. Oil Red O (ORO) stainings of 5 dpf (days post fertilisation) taf8- and taf6-mutant larvae revealed a specific lipogenesis defect in liver and intestine of taf8-mutant larvae. The results from treatments with a PPARγ inhibitor suggest that this lipogenic process is Pparγ-dependent. To convincingly establish a coactivator function of Taf8 for Pparγ during early zebrafish development, future work has to link the 5 dpf lipogenesis phenotype of taf8-mutants to defects in Pparγ-dependent transcription.

I: Ambystoma mexicanum is a useful model for the study of heart development as it has a naturally occurring mutant, the cardiac (c/c) lethal, in which the embryos lack organised myofibrils and have low levels of tropomyosin. Antisense oligonucleotides specific to axolotl tropomyosin disrupted myofibril formation in normal heart, whereas sense oligonucleotides encouraged myofibrillogenesis in the mutant hearts, demonstrating the importance of tropomyosin in cardiac muscle development and the effectiveness of cationic liposome transfection system. A novel isoform of the human tropomyosin, TPM1κ, was discovered and found to be cardiac specific in humans. Ectopic expression of GFP.TPM1κ fusion protein promoted myofibrillogenesis in the cardiac mutant axolotl heart. Tropomyosin N- and C-termini modification did not affect its function. II: The neotenous form had vagal preganglionic neurons (VPN) in the dorsal vagal nucleus (DVN) only. In the metamorphosed, VPN were found in the DVN but 18% were relocated in a ventro-lateral position. Neotenous ventilation rate followed heart rate. In the metamorphosed, ventilation caused heart rate variability. This may have been induced by the ventral-lateral VPN, which may be equivalent to a primitive nucleus ambiguus, an area that generates rhythmic sinus arrhythmia in mammals. Thus, ventrolateral VPN may be involved in cardiorespiratory control.

No description available.

Activation of Ras/mitogen-activated protein kinase (ERK MAPK) signalling controls the differentiation of mouse embryonic stem (ES) cells. An established modulator of the ERK MAPK pathway is the IRS-1 (Insulin Receptor Substrate 1) family adaptor protein Gab1 (Grb2-associated binder 1). Gab1 is ubiquitously expressed and is activated by a wide range of cell surface receptors, mediating growth factor, cell-cell and cell-substratum interactions. The N-terminal region of Gab1 contains a pleckstrin homology (PH) domain required for membrane binding and a nuclear localisation sequence (NLS) that facilitates nuclear translocation. Undifferentiated mouse ES cells preferentially express high levels of a novel form of Gab1 (Gab1β) lacking the N-terminal region. Based on its novel structure and abundance, Gab1β may act in a dominant negative manner by binding and mislocalising downstream effectors. Alternatively, it may have a deregulated function unrestrained by the PH or NLS domains. Data presented here shows that Gab1β is tyrosine phosphorylated in response to the self-renewal factor Leukemia Inhibitory Factor (LIF) and/or Foetal Bovine Serum (FBS) stimulation. This then leads to the formation of complexes with Shp2 and the p85 subunit of PI3K. Experiments comparing the responses of wild-type and Gab1β knock-out ES cells indicate that Gab1β enhances ERK and potentially AKT phosphorylation in response to LIF. In contrast, Gab1β has a negative effect on ERK and AKT phosphorylation in response to IGF-1 (Insulin Growth Factor 1). These results suggest that the contribution of Gab1β to signalling activity is receptor specific and may imply that the response of ES cells to ERK activation is context specific. By reintroducing fluorescently tagged Gab1 proteins into Gab1β knockout ES cells, I investigated the localisation of Gab1β in ES cells. Gab1β localised at the cell membrane as well as in a perinuclear body. I next investigated the potential role of Gab1β in the differentiation of ES cells into neural precursors. A monolayer differentiation protocol was used to differentiate Gab1β wild-type and knock-out cells into neural precursors. Furthermore, the effect of insulin on the emergence of neural precursors from Gab1β-targeted cells was also explored.

The brain is our most complex organ, with an estimated 1011 neurons. With the spinal cord, it forms the central nervous system which controls our movements and our senses, holds our memories and creates our thoughts. Because of this, neurodegenerative disorders can be extremely distressing and a thorough understanding of how the nervous system develops is essential if progress is to be made in finding ways to treat them. Critically, this includes understanding how the nervous system forms, i.e., the nature of the signals that promote neural identity (neural induction) and determine correct positional information (patterning). The zebrafish (Danio rerio) has become established as a model for embryological studies due to ease of experimental manipulation. Taking advantage of this, the aims of this PhD were to contribute to unravelling the molecular mechanisms of neural induction and patterning, using a variety of embryological and molecular methods. In the first project, functional analyses of the eve1 gene identified a key factor for posterior neural development. Eve1 was found to be a critical posteriorising factor, with an additional role in posterior neural induction. An outstanding question in neural induction is the relative contribution to this process of two key developmentally important signalling pathways, Bmp and Fgf. In the second project, differential analyses of maternal versus zygotic Bmp and Fgf signalling revealed crucial maternal roles for these two pathways in neural development as neural and epidermal capacitators. The results further suggested that Fgf signalling may be the critical neural inducer. Finally, as a third project, a zebrafish ectodermal explant assay was developed using the organiser-deficient ichabod mutant. The aim was to develop a system to analyse how key molecules directly affect ectoderm and neural development, free of mesoderm and endoderm influences, as signalling from these layers can directly or indirectly influence neural development.

Induction of vertebrate heart development requires inhibition of canonical/<i>β</i>-catenin dependent Wnt signalling, activation of non-canonical/<i>β</i>-catenin independent Wnt signalling and transcription factor activation. Wnt/<i>β</i>-catenin signalling may also have a later regulatory role in cardiogenesis. The recent discovery of Wnt6 expression next to and within the developing heart during the relevant stages of cardiomyogenesis, combined with knockdown and over-expression data suggests that Wnt6 may have a role in the regulation of this process. Inhibition of canonical signalling leads to increased expression of cardiac associated transcription factors such as members of the Nkx2 and GATA family. These families are expressed in overlapping regions which specify the early heart field prior to the expression of the later cardiomyocyte-specific genes. This study demonstrates the ability of <i>β</i>-catenin to inhibit cardiogenesis during later developmental stages, before the cardiac mesoderm begins to differentiate into myocardium (heart muscle) and that the newly discovered Wnt6 exerts inhibition of cardiogenesis in a <i>β-</i>catenin<i> </i>dependent manner. This inhibition of cardiogenesis by <i>β-</i>catenin can occur in a cell-autonomous manner and is a result of direct inhibition of cardiac transcription factors of the GATA family. Over-expression of these pro-cardiogenic transcription factors GATA4 and GATA6 can restore the cardiomyogenic differentiation programme in embryos where it has previously been inhibited by <i>β</i>-catenin. In conclusion GATA factors are the relevant targets of Wnt/<i>β-</i>catenin signalling in the inhibition of normal cardiac development. The subsequent loss of cardiac gene expression observed is therefore a result of insufficient GATA expression and function.

No description available.

The neural crest is a transient population of cells that forms a diverse range of derivatives in vertebrate embryos. Neural crest cells also migrate extensively throughout the embryo. The specification of a number of neural crest derivatives, including pigment cells and neurons and glia of the peripheral nervous system, is dependent on the transcription factor Sox10. In sox10 mutant zebrafish embryos, these neural crest derivatives fail to specify and subsequently the cell differentiation and migration fails leading to apoptosis. Sox10 mutant embryos also display an ear defect although the precise role of Sox10 in the ear is less well defined. Thus Sox10 controls an extensive gene regulatory network that drives the development of an important subset of neural crest derivatives and also functions during ear development. This gene regulatory network is currently poorly defined. The aim of this project was to identify genes that are both direct and indirect targets of Sox10 to further elucidate this gene regulatory network. To achieve this, a microarray approach was adopted. Initially, fluorescence activated cell sorting was employed to enrich for sox10 expressing cells from 24 hours post fertilization sox10:GFP transgenic embryos. The transcriptomes of WT and sox10 mutant cells were compared by microarray analysis to identify differentially regulated genes. A large number of target genes were identified by this method and by an unbiased in situ hybridization screen, 28 genes were validated. Of these, 23 genes were expressed in cells of the neural crest and down-regulated in sox10 mutant embryos. The majority of these genes were expressed in cells of the melanocyte and xanthophore lineages. 5 genes were expressed in the ear (otic vesicle) of which three otic vesicle genes were down-regulated while two otic vesicle genes were up-regulated in sox10 mutant embryos. Unfortunately due to time constraints, a study into the function of one of these target genes could not be completed. The series of validated genes identified during this project has opened new opportunities for research and has identified a number of highly expressed marker genes that will be useful in future studies. In addition, the microarray data presented will be a useful resource to aid the identification of further targets of Sox10.

Thisbe (Ths) and Pyramus (Pyr) are the ligands for the Fibroblast-Growth-Factor (FGF)receptor Heartless (Htl), which is expressed in all mesodermal cells during gastrulation. To understand how these two FGFs orchestrate mesoderm spreading in gastrulation and mesoderm differentiation during organogenesis, loss and gain of function studies were performed. In an approach of functional analysis, a single mutant allele of ths was generated, ths759, for comparison of the single mutant conditions of ths and the null mesodermal cells to migrate and differentiate in a precise pattern.

The idea of morphogen gradients encoding positional information for a developing organism has long been discussed in the field of developmental biology, but only recently have quantitative models been proposed that relate measured transcription factor concentrations to enhancer activity. However, successful models are typically computationally time-consuming, thus limiting full exploration and interpretation of the data. This thesis addresses these problems using standard statistical techniques applied to a comprehensive data set with the even skipped (eve) locus as a test case. The first part of the thesis introduces the data set. This is the precellular Virtual Embryo from the Berkeley Drosophila Transcription Network project. It comprises expression measurements of almost 100 genes in more than 6,000 individual nuclei at six time points. Different modelling approaches are evaluated in the context of this data set leading to a justification of logistic regression and the methods used to prepare the data set for further analysis. The second part applies logistic regression to describe the response of the eve enhancers to known regulating transcription factors such as Hunchback. Predictions of behaviour under regulator perturbation are consistent with experimental results and the functional form is shown not to be arbitrarily flexible, both in terms of the regulators and regions of the embryo included. The third part uses the framework developed above to find minimal explanatory models in the context of statistical model selection. It is found that the best scoring models depend on well-known regulators. The model selection techniques are then extended by directing the process using previous biological observations to analyse the eve 2 and eve 3+7 enhancers. The results are consistent with published research, but suggest specific additional hypotheses for the enhancers' regulation. Finally, the thesis concludes by proposing a general model of positional information and discussing the biological implications of the results. Overall, the results show how transcriptional control can be allocated to discrete enhancers and that characterising their activity in relatively simple terms is sufficient to explain their precise spatially-defined response to transcription factor concentrations.