Search

The extraembryonic endoderm of mammals is essential for nutritive support of the foetus and patterning of the early embryo. Visceral and parietal endoderm are major subtypes of this lineage with the former exhibiting most, if not all, of the embryonic patterning properties. Extraembryonic endoderm (XEN) cell lines derived from the primitive endoderm of mouse blastocysts represent a cell culture model of this lineage, but are biased towards parietal endoderm in culture and in chimaeras. Here, I further characterise XEN cells and show that these cell lines exhibit high levels of heterogeneity. In an effort for XEN cells to adopt visceral endoderm character different aspects of the in vivo environment were mimicked. I found that BMP4 and laminin promote a mesenchymal-to-epithelial transition of XEN cells with upregulation of epithelial markers and downregulation of mesenchymal markers. Gene expression analysis showed the differentiated XEN cells most resembled extraembryonic visceral endoderm. Correspondingly, inhibition of Erk and BMP signalling drives XEN cells toward parietal endoderm fate. Finally, I show that BMP4 treatment of freshly isolated parietal endoderm from Reichert’s membrane promotes its visceral endoderm differentiation. This suggests that parietal endoderm is still developmentally plastic and can be transdifferentiated to a visceral endoderm in response to BMP. Generation of visceral endoderm from XEN cells uncovers the true potential of these blastocyst-derived cells and is a significant step towards modelling early developmental events ex vivo.

Histone proteins provide a means of packaging DNA over 10,000-fold in order to allow the accommodation of genetic material as chromatin in the nucleus of the cell. However, the chemical manipulation of histones underpins an array of additional biological functions of chromatin. The unstructured N-terminal tails of histones are covalently modified in a variety of fashions, with many of these modifications implicated in the regulation of gene expression. Lysine specific demethylase 1 (LSD1), which demethylates mono- and di-methylated histone H3 lysine 4 (H3K4) as part of a complex including CoREST and histone deacetylases (HDACs), is essential for embryonic development in the mouse beyond embryonic day (E)6.5. The aim was to determine the role of LSD1 during this early period of embryogenesis through generation and analysis of conditional knockout mouse embryonic stem (ES) cells, which are the in vitro counterpart of the epiblast. Prior analysis of post-implantation loss-of-function genetrap embryos revealed that LSD1 expression, and therefore function, is restricted to the epiblast. Conditional deletion of LSD1 in mouse ES cells revealed a reduction in CoREST protein and associated HDAC activity, resulting in a global increase in histone H3K9 and H3K56 acetylation, but only minor increases in global H3K4 methylation. Despite this biochemical perturbation, LSD1 deleted ES cells proliferate normally and retain stem cell characteristics. However, differentiation of these ES cells is associated with significant cell death. Loss of LSD1 causes the aberrant expression of 588 genes, including transcription factors with roles in anterior/posterior patterning and tissue specification. Brachyury, a key-regulator of mesodermal differentiation, is a direct target gene of LSD1 and is over-expressed in E6.5 Lsd1 genetrap embryos. Thus, LSD1 regulates the expression and appropriate timing of a key developmental regulator, as part of the LSD1/CoREST/HDAC complex, during early embryonic development. Notably, rescue experiments show that the catalytic activity of LSD1 is not required for gene repression and it is proposed that the regulatory role of LSD1/CoREST/HDAC complex is governed by the ability to target HDACs to genomic regions or to prevent promoter access of gene-activating complexes with H3K4 tri-methylation catalytic activity.

The related genes lin28a and lin28b code for conserved RNA-binding proteins, which contain two key RNA-binding motifs that determine their functions. Previously, our laboratory identified lin28a as a putative downstream target of FGF signalling in the early Xenopus embryo. This was found to occur at gastrulation stage, during which key signalling pathways such as the FGF pathway are active in specifying germ layer development. lin28 is a heterochronic gene in C. elegans and controls the timing of developmental events. In vertebrates, the lin28a gene shows pluripotent-specific expression, and has come to particular interest as one of four factors used to successfully re-program differentiated somatic cells into induced pluripotent stem cells. Both lin28a and lin28b have a high prevalence of ectopic expression in cancer. A well characterised target of both lin28a and lin28b is the microRNA let-7, inhibiting biogenesis to the mature microRNA form. The lin28 proteins have also been shown to potentiate translation of numerous mRNA targets. The aim of this project was to identify if lin28 has an important developmental role in vertebrates. Work in the Xenopus tropicalis embryo found that both lin28a and lin28b were targets of FGF signalling at gastrulation, and were required for correct germ layer patterning at this stage. In order to explore conservation in humans, mesenchymal stem cells were used to model the effects of FGF signalling upon the mesodermal germ layer, and whether lin28 played a role in this. Additional pluripotent cell models were investigated for their suitability in which to study lin28 function. Analysis of lin28 targets in Xenopus revealed that the miR-17-92 cluster family of miRNA may be positively regulated by the lin28 proteins, with a direct interaction possible with a member of these: pre-mir-363. These miRNAs may have further important developmental roles in response to this regulation by the lin28 proteins.

The processes which drive morphogenesis during Drosophila development are well studied. However, the mechanisms employed to preserve the structures formed as a result of these events are poorly understood. The work of this thesis examines the role of localised cell signalling in effecting the morphogenesis of epithelial tissues. The Drosophila embryonic hindgut is a curved epithelial tube which undergoes elongation and rotation to form a shepherd's crook shaped organ which breaks with the symmetry of the embryo. Work within this thesis describes how asymmetric JAK/STAT signalling, on the inside of the curve, positively regulates protein levels of the homophilic adhesion molecule Fasciclin III (FasIII). Increased levels of FasIII lead it to become asymmetrically distributed throughout the lateral cell membrane. Loss of either JAK/STAT signalling or FasIII leads to a reduction in the magnitude of the hindgut curve. Furthermore, preliminary data indicates that the spatial regulation of JAK/STAT signalling, and lateral FasIII, is also required for correct directional control of hindgut rotation. Secondly, this thesis describes a role for localised JAK/STAT signalling and lateralised FasIII in maintaining the shape of folds within the prospective hinge of the 3rd instar larval wing disc. Further investigation of these folds indicates that their development is required for correct wing posture within the adult. This thesis confirms FasIII as a mediator of cell-cell adhesion. It is therefore hypothesised that the lateralised FasIII domain increases tissue stability within the hindgut curve and the wing disc folds. Loss of this intrinsic support results in these structures being unable to maintain their form during development.

Understanding how the germ layers are formed is one of the key questions of developmental biology. Abundant studies in the anuran amphibian Xenopus laevis have described that maternal and vegetally localised mRNAs for VegT and Vg1 contribute greatly to the formation of mesoderm and endoderm in the developing embryo. Within Xenopus mesendoderm gene-regulatory network (GRN), Wnt/β-catenin as well as Nodal and Mix family members have been shown to play important roles. The involvement of several members of the Nodal and Mix gene families with redundant functions makes the mesendoderm GRN surprisingly complex and difficult to study in Xenopus laevis. By contrast, mouse and humans have only single copies of Nodal and Mix. Since urodeles have an embryology that is basal to amphibians and that has most likely also been conserved during the evolution of amniotes, including mammals, we have investigated the Mix and Nodal genes in the urodele Axolotl in the hope that their gene families contained fewer members. We cloned one Mix and two Nodal orthologs from the axolotl and showed by Southern blot analysis that there are likely no further copies in the axolotl genome. Morpholino and rescue experiments furthermore showed that AxNodal-1, Mix and Brachyury play essential roles in mesoderm specification in axolotl embryos, suggesting that the urodele Axolotl has a more simplified mesendoderm GRN. In this context, we demonstrate that Mix acts to induce Brachyury expression during mesoderm induction. Mixl1 shRNA knowdown in mouse embryonic stem cells (mESCs) shows that Mixl1 is involved in the production of mesoderm in mESCs too. Analysis of the localisation of the VegT and Vg1 mRNAs in oocytes revealed that they are neither vegetally localised in the Axolotl, nor in the basal fish species lungfish and sturgeon. Furthermore, gain and loss of function assays examining the roles of maternal VegT and β-catenin demonstrated that VegT is not required for mesoderm induction, whereas β-catenin is necessary and sufficient for mesoderm induction by activating AxNodal-1 expression in the axolotl. As these results reveal additional similarities to the GRN in mammals they further support our hypothesis that the regulatory network in the axolotl is more closely related to that in amniotes rather than anuran amphibians.

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.

Nitric oxide (NO) is a highly diffusible signalling molecule that serves a wide range of physiological functions. During development, biosynthetic enzymes for NO are often expressed in nascent neurons, although little is known of how this molecule regulates in vivo nervous system development. This thesis aims to address this problem by examining the functional roles of NO signalling in the zebrafish embryo, focusing specifically on nitrergic regulation of spinal locomotor network assembly. The first aim of this study is to characterise the spatiotemporal distribution of NO synthase 1 (NOS1), the enzyme responsible for NO biosynthesis in the zebrafish nervous system. NOS1 transcript and protein was observed in discrete regions of the brain as well as a distinct class of spinal interneuron from early stages of embryonic development. The second aim was to examine functional roles of NO signalling during in vivo spinal cord development. Using molecular antisense and pharmacological approaches, NO levels were disrupted during early life and the consequences to spinal circuit maturation assessed. NO was found to specifically regulate the growth of spinal motoneurons that innervate axial trunk muscles. Abrogation of NO signalling dramatically increased the number of motor axon branches formed within the muscle across the first three days of life whilst exogenous elevation of NO levels had the opposite effect. The third aim was to determine downstream signalling pathways underpinning NOs effects. Pharmacological studies revealed that NO regulates motoneuron branching through the cyclic guanosine monophosphate pathway and subsequent analysis revealed that this pathway can modify neuromuscular synapse density, with high NO levels suppressing synaptogenesis and retarding locomotor maturation and low NO levels having the converse effect. In summary, the work presented in this thesis identifies a novel and important role for NO signalling, demonstrating that it functions to sculpt neuromuscular synapse assembly and modify locomotor maturation.

A conserved feature of early vertebrate embryos is the formation of simple epithelial layer of cells which surrounds the embryo and protects it from the external environment. This epithelium is called the trophectoderm in mammals, the superficial layer in Xenopus and the enveloping layer in zebrafish. This project investigates what promotes differentiation of this cell type. In Xenopus embryos aPKC and Notch signalling were found to be unable to promote differentiation of the superficial layer. In contrast, BMP signalling can promote expression of a number of transcriptional regulators, including members of the Grhl and Msx families and differentiation of the superficial layer. This pathway is initiated in the underlying deep cells, but not all target genes are activated so differentiation does not occur in these cells. The role of BMP signalling in mouse development was investigated by using mouse embryonic stem cells (mESCs) as the model. BMP4 is sufficient to induce mESCs to form a polarised epithelial cell type and that these epithelial cells appear trophoblast in fate. BMP signalling activates Grhl and Msx genes in mESCs, as it does in Xenopus embryos. This suggests that similar target genes are activated by BMP signalling in the first epithelium of Xenopus and mouse. Based on this data it is tempting to propose that BMP signalling acts in a conserved manor to promote differentiation of the first epithelium in diverse vertebrates.

The past two decades have highlighted the utility in using the fruit fly Drosophila as a model organism for unravelling the molecular and functional complexities of two important fields of research: the systems that guide retinal determination (RD) and circadian rhythms (the daily body clock or oscillator). RD and clock factors are of great interest as they are: (1) highly conserved in vertebrates; (2) also essential for other physiological systems; (3) implicated in several congenital disorders and other diseases. The RD factors operate within a network in which several of their interactions have been described. Two such factors, eyes absent (eya) and sine oculis (so), are known to function as a unit to direct transcriptional regulation during photoreceptor (PR) differentiation. The regulation of eya and so by a transcriptional repressor at the heart of the clock mechanism, vrille (vri), is here investigated. Two distinct observations advocated exploration of a link between vri and eya/ so is of interest. (1) vri is a core component of the clock and interacts with RD but the RD function is unknown. (2) Recent evidence suggests that an RD factor directly upstream of eya and so, twin-of-eyeless (toy), interacts with the oscillator mechanism through direct and indirect pathways. It is possible that the indirect influences of toy on the oscillator are mediated via eya and/ or so. Interactions between eya, so and vri during RD and within oscillator cells are investigated here.Eye development function was studied using immunohistochemistry and transgenic manipulation. VRI is not expressed within the developed PRs; rather, expression of VRI is down-regulated prior to differentiation. In addition, conversely to the hypothesised role, VRI is co-expressed with EYA in some regions. Together with data from transgenic manipulation of VRI regional expression, I propose that VRI is predominantly part of a developmental pathway but can attenuate eya and so expression.The VRI binding site has been described previously and several sites were identified within eya/ so loci, some of which were tested in an in vitro binding assay. Two such sites were located adjacent to a known enhancer of so. I generated two transgenic fly lines containing: 1) an extension of the original enhancer to contain the VRI sites; and 2) a similar construct with the VRI sites ablated. Comparison of the original enhancer to those from the current study confirmed that the VRI sites attenuate expression and that intervening regions must contain binding sites for other transcription factors.In adult brains over a circadian light-dark cycle, EYA protein was expressed in three of the central brain clock neurones. Furthermore, expression of eya and so transcripts in adult heads, PRs and the brain, changed over the light/ dark cycle independently of the clock - indicating that their expression is modulated over the light-dark cycle but not by the oscillator mechanism. These data suggest interactions between eye development factors eya/ so and oscillator components, or, the light/ dark cycle exist. These interactions may be important for tissue-specific circadian physiology as well as the overall oscillator mechanism and offer an intriguing route for future investigation.

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.

Cilia are cellular organelles involved in processing components of the hedgehog (hh) signalling pathway and determining left-right (L-R) axis formation in the embryo. An embryonic lethal mouse mutant, called gasping 6 (gpg6), was identified that demonstrated morphological and molecular defects associated with L-R development and hh signalling. gpg6 mutant embryos also demonstrate abnormally short cilia, which was hypothesised to be the primary morphological defect in gpg6 mutants. The underlying genetic lesion in gpg6 is a mutation in the DNA repair gene Atmin. The base pair change results in an amino acid substitution in a critical residue in the third zinc finger of Atmin. The consequence of this change is the failure to activate transcriptional targets of Atmin. This raised the possibility that previously unidentified Atmin target genes are important for ciliogenesis. Consistent with this hypothesis, Dynein light chain-LC8 (Dynll1) is downregulated in gpg6 mutants. LC8 (a homolog of mouse Dynll1) is required, in the single cell eukaryotic organism Chlamydomonas, for retrograde intraflagellar transport (IFT), a process crucial for ciliogenesis. These data led to the following hypothesis: Atmin activates expression of Dynll1, which functions in retrograde IFT to enable normal ciliogenesis. Knockdown of Atmin in a ciliated kidney cell line resulted in abnormally short cilia. Thus, Atmin functions in ciliogenesis. Investigation of gpg6 has therefore identified a novel role for Atmin in ciliogenesis and has added to the growing knowledge of genes that control cilia formation and embryonic development.

Calcium ions (Ca2+) are fundamental to the regulation of many cellular processes; however, the coordination of these signals during embryogenesis is not well understood. Ryanodine receptors (RyR) are a family of important intracellular ion channels that are responsible for the release of Ca2+ and they regulate the cytosolic Ca2+ concentration. Humans have three differentially expressed ryr genes (ryr1, ryr2 and ryr3) and mutations can cause both skeletal and cardiac diseases. Although the primary function of RyR is to mediate excitation-contraction coupling in muscle, they may also regulate Ca2+ signalling during developmental processes. The project has addressed the role of RyR during embryonic development, using the zebrafish as an in vivo vertebrate model. Five zebrafish RyR genes (ryr1a, ryr1b, ryr2a, ryr2b and ryr3) were characterised and a comprehensive overview of their spatial and temporal expression in the embryo was determined. At 24 hours post-fertilisation (hpf), ryr1a, ryr1b and ryr3 are expressed in the skeletal muscle, ryr2a in specific neuronal populations and ryr2b in the cardiac muscle. Semi-quantitative PCR data and wholemount in situ hybridisation revealed strong maternal expression of ryr3 during the cleavage and blastula periods and into adulthood. The early expression of the ryr3 gene suggests that this receptor functions during the initial stages of development; a role that has not been described previously. The functional significance of RyR3 during early embryogenesis was investigated in a loss-of- 3 function model using antisense morpholino oligonucleotides. The ryr3 specific knockdown experiments appeared to affect the establishment of embryonic axis prior to the segmentation periods (before 10 hpf). In addition, by 19 to 20 hpf ryr3 morphants failed to exhibit spontaneous muscle contractions and displayed a defect in neuromuscular development. In conclusion, this study has characterised the ryr genes and provided an overview on their temporal and spatial expression. The work provides evidence that ryr3 expression provides the Ca2+ vital for myofibrils organisation and that is required for the spontaneous movements during zebrafish embryonic development. The knowledge of RyR tissue distribution in zebrafish has provided a strong foundation for loss-of-function studies aimed at addressing their role in development. In the long term, the work will also facilitate more focused studies on disease.

Understanding the gene regulatory networks driving the differentiation of the distinct cell types in embryos is a key component of understanding development. Amongst the first types of cell to differentiate during vertebrate development are blood cells, and haematopoiesis is tightly regulated by proteins known as haematopoietic transcription factors (HTFs). Although the mRNA expression patterns of many HTFs are now well characterised, little is known about their protein expression patterns or how their transcription regulation activity is controlled during early development. Knowledge of their spatial and temporal expression patterns and their post-translational regulation would greatly enhance our understanding of the genetic regulatory networks that produce blood. Antibodies have been successfully raised and characterised that recognise Xenopus Hex, fli1 and Tal1. Western blot experiments have shown that Hex and fli1 are expressed in oocytes and expression continues throughout development. This maternal expression is unexpected but is reminiscent of another embryonic HTF, Gata2. At stage VI of oogenesis the majority of fli1 and all Hex protein was localised within the cytoplasm, suggesting an alternative second function for each of these proteins. An intensively sampled developmental time-course carried out for fli1 between stages 24 and 31, revealed striking post-transcriptional control over fli1 expression. Neither Hex or fli1 proteins have been shown to be absent from the regions containing sites of haematopoiesis and both proteins were eluted in the same fractions following size exclusion chromatography using Xenopus extract, suggesting the possibility that they may be in complex together. Finally, either Tal1 protein is undetectable during early Xenopus embryogenesis, or is heavily modified. Since, at least for fli1, knockdown of the zygotic protein specifically affects blood development a number of questions are raised. What is different about the maternal and zygotic proteins, which means that the zygotic form is needed for haematopoiesis? What is the function of the maternal proteins – are they even capable of transcription regulation?

CAMSAP 1 is a spectrin associated, Calmodulin regulated protein that is a member of a large ubiquitous family of cytoskeletal proteins in the animal kingdom, defined by a novel C-terminal domain, the CKK domain. The role of the CAMSAP proteins is unknown but studies using rat PC12 cells have shown that CAMSAP1 plays a key role in neurite outgrowth. It has been shown to colocalise with microtubules in cultured cells and binds to microtubules in vitro via the CKK domain. The CAMSAP family member in the fruit fly Drosophila melanogastor is encoded by the gene short spindles4 (ssp4). Little is known about this gene but a role in microtubule dynamics has been shown in cultured cells. I have interrogated bioinformatics databases and compared Ssp4 with Human CAMSAP proteins and found many similarities, and some differences, between the proteins. Using in situ hybridisation I show that ssp4 transcripts are expressed in the gut, head and central nervous system during embryogenesis and an antibody that recognises the Ssp4 C-terminus reveals expression throughout the development of the gut and nervous system, and in a discrete population of cells in the head. I have investigated the effects of two independent P-element induced mutant alleles of ssp4 and show that mutant flies die in late embryonic or early larval stages. Disruptions to the locus do not seem to affect the nervous system but mutants were found to have aberrant head involution. I present preliminary evidence that suggests this defect may be the result of reduced apoptosis in the embryo Head involution is a complex process, dependent upon co-ordinated changes to cell shape and the movement of groups of cells from different origins. As Ssp4 is a multidomain cytoskeletal protein that is required for embryonic development, it may play a role in processes that are common to these morphogenetic events.

The core promoter is the ultimate target of all transcriptional regulatory processes. The recently discovered diversity of core promoters and basal transcription factors suggests a regulatory role in differential gene expression. However, the direct contribution of the core promoter remains poorly understood. I investigated core promoters and their putative role in differential gene regulation using the zebrafish embryo as an in vivo model system. To analyse the functional requirement for the general transcription factor TATA-box binding protein (TBP), a diverse set of promoters was tested for their TBP dependence. This analysis revealed a differential requirement of TBP for promoter activity. To further explore the roles of core promoters the ability of various core promoters to interact with tissue-specific enhancers was investigated. A high-throughput pipeline combining automated imaging with custom-designed software for registration of spatial reporter gene activity in thousands of zebrafish embryos was developed. The technology was applied in a large-scale screen analysing the tissue specific activities of 202 enhancer - core promoter combinations. A variety of interaction specificities observed suggests an important role of the core promoter in combinatorial gene regulation. Overall, these findings indicate that the core promoter significantly contributes to differential transcriptional regulation in the vertebrate embryo.

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.

Here I show that the so-called core components of NMD are not all essential genes, since flies that lack the function of Upf3 are viable, in contrast to flies that lack the function of Upf1 and Upf2. By removing the upf1 and upf2 maternal contribution during oogenesis, I demonstrate that Upf1 and Upf2 are required during embryogenesis, as was the case in mice and zebrafish, exemplifying the fly as a valuable NMD model system for vertebrates. In addition, upf1 and upf2­ mutant eggs exhibit a specific dorsal appendage phenotype, possibly the result of developmental perturbation. I have observed that upf1 and upf2, but not upf3, mutant cell clones are unable to grow, suggesting that upf1 and upf2 mutant cells do not proliferate and, in addition, undergo apoptosis. This supported the hypothesis that Upf1 and Upf2 have Upf3-independent functions that are essential for cell and organism viability, and that NMD is not required for the development of an organism. NMD target gene expression levels were analysed using qPCR. My results suggested that potential NMD targets can be separated into distinct groups depending on which NMD factors are most important for mediating their degradation. In adult flies, genes with known PTCs required Upf2 and Upf3, but not Smg1 for their full degradation by NMD, whereas targets with as yet unidentified NMD-inducing features were fully stabilised only in upf2 mutants. These results suggest that Smg1 is a weak inducer of the pathway, rather than a core component, and that Upf3 is only essential for NMD of specific target mRNAs. In embryos, Upf1 and Upf2 were essential for NMD whereas Smg1 and Upf3 had only moderate effects.

Interferon (IFN)τ is the luteotrophic signal in ruminants and is secreted by bovine blastocysts both in vivo and in vitro. IFNτ secretion is highly variable and its control is only partly understood. Most studies on the effects of environmental factors on IFNτ production have evaluated IFNτ production during the time of embryo elongation and attachment. There is less knowledge of how IFNτ production at the blastocyst stage is modulated. Therefore, the hypothesis of this thesis was that the amounts of IFNτ expressed and/or secreted by bovine blastocysts produced in vitro were modulated by environmental factors. In the first set of experiments, bovine embryos were incubated with a cytokine (granulocyte macrophage colony stimulating factor, GM-CSF). GM-CSF had been shown previously to promote embryo viability in a range of species and to modulate IFNτ secretion by ovine blastocysts and thus was classified as a beneficial environmental factor. Three experiments were conducted to test whether GM-CSF stimulated bovine blastocyst development and IFNτ secretion. Embryos were incubated with a range of different concentrations of GM-CSF (2, 5, 10 and 50 ng mL-1) and at different stages of development (1 to 3 and 1 to 9 days post-insemination). Bovine embryos were unresponsive to GM-CSF in terms of IFNτ secretion, pyruvate oxidation, rate of development, blastocyst yield, morphological quality and apoptotic index, irrespective of timing of exposure and/or concentration of GM-CSF. In the second part of the thesis, bovine blastocysts were exposed to a mild heat treatment (42°C for four h) to determine whether heat stress affected IFNτ expression by bovine blastocysts. A novel multiplex reverse-transcription polymerase chain reaction methodology was validated to detect IFNτ and heat shock protein (HSP)70 mRNA in individual bovine embryos relative to an endogenous gene (YWHAZ) and an exogenous mRNA (α-globin) and results were expressed both in absolute terms and in relation to the endogenous control. Heat treatment upregulated IFNτ mRNA expression, suggesting that detrimental environmental factors may influence IFNτ expression. Heat treatment also caused an increase in HSP70 mRNA expression but did not affect blastocyst morphology, suggesting that the level of stress caused by the heat treatment was great enough to activate the cellular stress response, but mild enough not to cause a change in morphology. In addition, the positive correlation between HSP70 and IFNτ transcript levels and the higher IFNτ expression by embryos which showed signs of degeneration and collapse compared to those which progressed in development suggested that IFNτ expression may be indicative of stress. The relationship between IFNτ expression and secretion in vitro with morphology, pyruvate metabolism, apoptotic index and cell number was inconsistent, suggesting that IFNτ production did not correlate with ‘quality’ (defined as an index of viability). Blastocyst yield, day of blastulation and change in morphology index did account for at least part of the variation in IFNτ production, suggesting that some intrinsic factors may regulate IFNτ secretion. These intrinsic factors, however, did not explain all the variation in IFNτ secretion between blastocysts. Therefore, the amount of IFNτ secreted by bovine blastocysts is modulated by both intrinsic and environmental factors. A model was proposed where different levels of stress affect survivability to different extents, and the ability to respond to mild levels of stress may be indicative of improved survivability.

In undifferentiated ES cells, many Polycomb Repressive Complex 2 (PRC2) target genes carry not only repressive H3K27me3 but are also enriched for conventional indicators of active chromatin including methylated H3K4. This so-called bivalent domain structure is thought to silence key developmental regulators while keeping them poised for future activation (or repression). Consistent with this hypothesis, bivalent genes assemble RNAP II preferentially phosphorylated on Serine 5 residues (poised RNAP II) and are transcribed at low levels. Productive expression is, however, prevented by the action of PRC1. Here, I have focused on the pre-implantation stage of mouse development to evaluate whether bivalent or poised chromatin signatures are indeed specific attributes of emerging pluripotent cells and investigate how the fate of key developmental genes is specified while the first lineage decision event (extra-embryonic lineage formation) occurs. Using blastocyst-derived stem cells and chromatin immunoprecipitation (ChIP), I have shown that lineage-inappropriate genes retain bivalent histone marking in extra-embryonic trophoblast stem (TS). However, and in contrast to ES cells, PRC1 (Ring1B) and poised RNAP II are not recruited to these loci in TS cells, indicating that gene priming is a unique hallmark of pluripotent cells in the early embryo. To investigate the intricate relationship between lineage identity and dynamic chromatin changes, I exploited the potential to convert ES cells into trophoblast-like stem (TSL) cells using a previously established artificial system dependent on doxycycline (Dox) induced repression of an Oct4 transgene. I demonstrated that Suv39h1-mediated H3K9me3 alongside DNA methylation is targeted to PRC2-bound bivalent, lineage-inappropriate genes upon trophectoderm lineage commitment. A change in chromatin conformation was observed upon differentiation of ES cells to TSL cells comparable to that seen in TS cells derived in the traditional manner from the trophectoderm (TE) of blastocyst stage embryos. Most importantly, I have begun to explore when epigenetic differences are specified, at the locus level, from 8-cell stage embryos onwards using newly designed Carrier ChIP technology. This data validated the occurrence of bivalent chromatin domains in vivo and further support the view that alternative strategies operate in the TE to silence key developmental regulators upon blastocyst lineage segregation.

Cranial neural crest cells (cNCC) emigrate from the neuroepithelium to form the cartilaginous structures of the face and much of the peripheral nervous system of the head. Several genetic disorders and drug compounds can disrupt the patterning of cNCC-derived tissues during development. I have studied cNCC patterning in the chick embryo, focussing on one genetic disorder (X-linked Opitz syndrome, caused by mutations in the MIDLINE1 (MID1) gene) and one drug compound (retinoic acid). I demonstrate the expression pattern of the chick orthologue of MID1 and show that the Mid1 protein, via its effects on Protein Phosphatase 2A, controls the migration of cNCCs and regulates the timing of cranial gangliogenesis, likely by altering cNCC protease activities. Retinoic Acid (RA) is a diffusible morphogen and well-known teratogen that causes patterning defects in cNCC migration. I provide evidence that the cranial mesenchyme adjacent to rhombomere 3 of the hindbrain is a potent responder to the presence of RA, resulting in cNCC mis-migration. Furthermore, I demonstrate that the 9-cis RA isoform is more potent than the all-trans RA isoform at causing cNCC mis-migration.

Polycomb proteins are a group of repressor proteins that form multiprotein complexes, Polycomb repressor complexes (PRC) that mediate gene silencing. Two major complexes that have been widely studied are PRC1 and PRC2.Both complexes have enzymatic activities that modify specific histone residues, monoubiquitination of H2AK119 (H2AK119u1) and methylation of H3K27 (H3K27me3) respectively, and it is thought that this is the basis for their silencing activity. The basis for recruitment of PcG proteins in mammalian systems remains to be fully elucidated. A key concept is the hierarchical model explaining co-occupancy of PRC1 and PRC2 at target loci. Here it is proposed that a chromodomain in the protein polycomb (Pc), a core PRC1 subunit, recognises the H3K27me3 histone modification catalysed by PRC2. Although some observations appear inconsistent with this idea it is nevertheless widely accepted. In this study the Ink4b-Arf-Ink4a locus was used as a model to study PcG recruitment both by examining PcG localization and by setting up a reporter system that could be used to screen for novel regulators of PcG recruitment. The former analysis revealed that Exon E2, which is shared between Ink4a and Arf is strongly bound by PcG proteins, suggesting it as a good candidate for a recruitment centre. However, deletion of exon E2 does not inhibit the recruitment of PcG to other regulatory regions within the locus. In the latter study P1-derived artificial chromosome (PAC) recombineering was used to produce a modified PAC in which fluorescent marker proteins replaced endogenous Ink4a and Arf open reading frames. This will provide a useful tool for screening for factors that contribute to Ink4a-Arf activation including PcG recruitment factors. Preliminary data from the lab indicated that global levels of PRC1 mediated H2AK119u1 are largely unaffected in cells lacking H3K27me3, an unexpected outcome based on the hierarchical recruitment model. Chromatin immunoprecipitation (ChIP) was carried out on Eed mutant (H3K27me3 depleted) ESCs and consistent with the hierarchical model, occupancy of the core PRC1 proteins RING1B and MEL18 was strongly reduced. However, H2AK119u1 was clearly detectable at target loci, suggesting PRC1 recruitment independent of H3K27me3. Genome wide analysis of RING1B occupancy substantiated this conclusion, demonstrating significant RING1B levels at polycomb target loci in Eed-/- ESCs. From this study it can be concluded that PRC1 and PRC2 are recruited independently to sites that they co-occupy. Additionally evidence is provided that the primary function of H3K27me3 is to increase the residency of PRC1 at target loci and thereby to contribute to the stability of PRC1 mediated silencing.

The complex organisation of the vertebrate brain starts off as a simple arrangement of axon tracts in the early embryo termed the early axon scaffold. These initial axon tracts were important for the correct guidance of later, follower axons. Yet, little is known about the temporal and spatial control of neuronal differentiation, or the control of axon guidance for the early axon scaffold. The aim of this study was to provide a detailed anatomical description of the early axon scaffold as a basis for functional experiments, to identify candidate genes involved in the differentiation of the early neurones, and to gain insight into the axon guidance for a particular early tract. The anatomical formation of the early axon scaffold in the chick embryonic brain has been analysed in detail using immunohistochemistry and axon tracing. The early tracts in the chick were directly compared with cat shark, Xenopus, zebra finch and mouse. These results highlight the conservation of early axon scaffold development. The medial longitudinal fascicle (MLF) was shown to be the most conserved tract forming first in all vertebrates, apart from mouse where it forms later. Since the genes involved in specification of neurones to an MLF fate are unknown, microarray analysis was used to identify candidate genes with a possible role in MLF neurone specification. CRABPI was shown by in situ hybridisation to be specifically expressed by the MLF neurones. Another highly conserved early tract is the tract of the posterior commissure (TPC). Its neurones were shown to be located in the ventral diencephalon of the chick embryonic brain. While the TPC neurones were intermingled with the MLF neurones, their axons project along very separate paths, suggesting that their outgrowth is directed by different guidance cues present. Netrin1 and Netrin2 were identified as candidate genes for repelling the TPC axons along their correct path. Gain-of-function experiments led to reduction or loss of the TPC, suggesting that Netrins act as repellents on the TPC axons in the chick embryonic brain.

Using Xenopus laevis embryos, the aim of this project is to establish a new experimental model to help understand the mechanism that regulates cardiac cell diversification and heart morphogenesis. In order to achieve these goals we use two assays. The cardiogenesis assay involves the use of animal cap explants excised from the animal pole of blastua embryos. Here it is shown that GATA-4 reliably induces the expression of ventricular and proepicardial markers, providing an assay to study the mechanisms of cardiac cell fate diversification. However, cardiomyocytes generated in animal pole explants do not undergo significant morphogenesis and physiological maturation. In order to study these later aspects of heart development we required a different assay to generate a structure similar to the heart. Using GATA-4 injected AC explants transplanted into host embryos we obtained secondary beating hearts in which regionally restricted cardiac gene expression was observed in addition to growth and a limited degree of morphogenesis. We demonstrated that the host plays an essential role as it provides a wide range of permissive regions which allow the development of the SH. Moreover, we also showed that the competence to generate a secondary heart is lost in reaggregates transplanted at stage 28. The host cells however do not contribute to the SH indicating that the role of the host is providing signals which allow the development of the SH.

The connections between cell cycle exit and terminal differentiation remain poorly understood. Cyclin dependent Kinase Inhibitors (CKIs) provide a possible link between entry into the quiescent state and differentiation. The initial aim of this project was to further investigate if the CKI p27Xic1 could promote differentiation in addition to, and independently of, its well characterised cell cycle exit function. p27Xic1 has been shown to be involved in cell fate determination during gliogenesis, neurogenesis, myogenesis and cardiogenesis and many mammalian Cip/ Kip CKI homologues of p27Xic1 have been described as important regulators of cellular processes beyond control of cell division. We aimed to investigate these roles during development of the embryonic kidney, the pronephros. We discovered that p27Xic1 does not affect differentiation during pronephrogenesis, but instead controls pronephric organ size through its cell cycle exit function. In addition we identified a previously unrecognised role for the cell cycle exit function of p27Xic1 in allocation of the somites during paraxial mesoderm segmentation. Preliminary results had suggested p27Xic1 expression in the pronephros was under the control of the Notch signalling pathway. Over-expressing a constitutively active form of Notch, Notch-ICD, and a dominant negative form of the Delta ligand, DeltaSTU, showed that both mis-activation and suppression of Notch signalling inhibited p27Xic1 expression. However, when investigating the effects these overexpressions had on pronephros development, we identified novel results indicating the Notch signalling pathway, which has previously been implicated in pronephros development, is essential for the separation of the proximal lateral and medial pronephric mesoderms. This process we propose is mediated by the Notch signalling pathway through the establishment of a boundary between these two distinct populations of cells, permitting both compartments to develop in isolation. The results in this thesis suggest novel mechanisms by which cell division controls X. laevis segmentation and organ size and how the Notch signalling pathway is able to pattern the pronephros anlagen such that the different compartments of the mature pronephros are able to develop, and thus function.

During development, polarity is a common feature of many cell types. One example is the polarisation of whole fields of epithelial cells within the plane of the epithelium, a phenomenon called planar cell polarity (PCP). It is widespread in nature and plays important roles in development and physiology. Prominent examples include the epithelial cells of external structures of insects like the fruit fly Drosophila melanogaster, polarised tissue morphogenesis in vertebrates and sensory hair cells in the vertebrate ear. In this work we focus on the wing and the abdomen of Drosophila, where PCP becomes obvious in the alignment of hairs and bristles. The underlying dynamics are not fully understood yet, but two distinct protein networks centred around the transmembrane proteins Frizzled and Dachsous, respectively, have been shown to play essential roles. We will present and analyse five models for different aspects of the process of planar cell polarisation. The first two models assess the nature of PCP in a generic setting, ensuring that the results are valid for whole classes of PCP models. Models three and four are existing more complex models that include detailed assumptions about the underlying protein interactions of the Frizzled system in the Drosophila wing. Model five considers the Dachsous system in the Drosophila abdomen. We describe the features of the different types of mechanisms and determine the conditions under which they can yield polarity. All five models can establish wild-type polarity for a wide range of parameter values. We find, however, that for model one, three and four an inhomogeneous pattern exists for the same parameter values as the polarised state. Therefore, in these cases either specific initial conditions, which are unlikely in nature, or a global bias are necessary to ensure correct polarisation. Furthermore, we present the effects of clonal clusters of cells on the polarity of the surrounding cells in our models and relate them to the phenotypes observed in experiments. Model one and five show the largest discrepance between the numerical and the experimental results. We discuss the biological relevance of these findings and indicate outstanding questions.

The study of pluripotent cells in mouse has revealed a core transcription factor network. Pluripotent cells have not been identified in many non-mammalian organisms, but cells with pluripotent properties are found in axolotl (Ambystoma mexicanum) a urodele amphibian. Similarities, between the morphological processes in amniotes and those in urodeles led to the suggestion that amniotes may have arisen from a urodele-like ancestor. Thus, studying the pluripotency network in axolotl may be key to understanding the evolution of mechanisms governing pluripotency in amniotes. This study describes the investigation of two of the core pluripotency trancription factors, Axnanog and Axoct4. Coexpression of axnanog and axoct4 was detected in the undifferentiated tissues of blastula and gastrula stage embryos, suggesting a conserved role in pluripotency/multipotency. Antisense morpholino oligonucleotides were employed to investigate the function of these two molecules. Gastrulation was disrupted in Axnanog morphant embryos. Additionally, they maintained expression of genes associated with pluripotency and early lineage specification, but only expressed low levels of terminal differentiation markers. There are two explanations for this phenotype, a cell migration defect or a developmental block. Axoct4 morphant embryos had a similar phenotype suggesting that Axoct4 may function in a common pathway. Primordial germ cells (PGCs) are the only cells that retain the ability throughout development to derive all of the tissues of the embryo, upon fertilisation, and these cells express many pluripotency-associated factors. Little is known about PGC development in axolotl. In this study, the roles of Axoct4, Axnanog and Axblimp1 were investigated. Neither Axnanog nor Axoct4 were found to have a role in PGC development. Axblimp1 is unlikely to have a role in PGC specification, as in mouse, but a role in PGC maintenance was not ruled out.

Germband extension (GBE) in the early Drosophila embryo provides a simple system in which to study convergence and extension, a type of tissue remodelling common in development. During GBE the embryonic trunk (“germband”) extends in the anterior-posterior (AP) axis, and narrows in the dorso-ventral (DV) axis. I have quantitatively analysed cell and tissue behaviours during GBE. Our collaborators have developed novel algorithms to measure the continuous impact of cell intercalation and cell shape change on tissue deformation. Here I apply these algorithms to describe the behaviours contributing to GBE. I find that wild type embryos not only undergo cell intercalation, but also cell shape change which accounts for up to 50% of the extension rate during the fast phase of GBE. I find that this cell shape change is elevated in AP patterning mutants that have a defect in cell intercalation, and that this elevation accounts for the initial rapid pulse of extension in these mutants. Regional analysis of these cell behaviours in wild type and AP patterning mutants indicate that cell intercalation is regionally autonomous, but that cell shape change is controlled at a more global level. An external force may be present during early GBE, stretching cells in the AP axis if they fail to intercalate properly. Additionally, I have examined the impact that other morphogenetic movements of gastrulation have on GBE. Mutant analysis shows that the ventral furrow affects the rate but not overall extent of tissue deformation, while the posterior midgut invagination appears to be required only for correct displacement of the tissue as it deforms, and not for tissue deformation itself, at least during early GBE. Thus, the amount of tissue extension, the rate at which it occurs, and the tissue displacement by which it is accommodated are separable phenomena.

Id proteins (Inhibitors of differentiation) inhibit differentiation by preventing bHLH DNA binding activity through the formation of inactive Id-bHLH heterodimers. Xenopus Ids (xIds) are dynamically expressed in a wide variety of tissues, with high expression levels detected at early stages of development. In this work, I have identified a requirement for Xenopus xId2 and xId3 in primary neurogenesis and muscle differentiation. Overexpression of xId2 and xId3 disrupts the expression of late neuronal and muscle differentiation markers, but not the expression of the early markers xNGN and xMyoD, suggesting that xIds inhibit primary neuron and muscle differentiation without affecting neuronal or myogenic commitment. To elucidate the mechanism through which xIds inhibit primary neurogenesis, I investigated the ability of xIds to affect xNGN activity, the most upstream activator of neurogenesis that is both necessary and sufficient to initiate neuronal differentiation in vertebrates. I found that xId2 and xId3 negatively regulate xNGN’s activity at a post-transcriptional level, albeit through distinct mechanisms. While both xIds are able to inhibit xNGN’s ability to activate transcription in reporter assays, only xId3 is able to significantly reduce the DNA binding ability of xNGN/E12 heterodimer, through the sequestration of xE12. Importantly, I also demonstrate that by sequestering xE12, xId3 induces xNGN degradation. These findings not only provide the first direct evidence that xId3 modulates xNGN activity and stability, but also emphasize that xId2 and xId3 act through distinct pathways to ensure a stronger short-term control of xNGN activity and consequently prevent premature primary neuron differentiation. This non-redundant role for xId2 and xId3 is further supported by the finding that xId2 and xId3 expression and activity is differentially regulated. Finally, I also provide evidence for a role of Casein Kinase II (CKII) mediated phosphorylation on xNGN activity.

Midline convergence of organ primordia is an important mechanism for shaping the vertebrate body-plan at various stages of development, such as the morphogenesis of the heart and endoderm. Down regulation of wnt or noncanonical wnt signalling components, such as dishelleved (Dvl) or RhoA GTPase (RhoA), impairs midline convergence of the heart primordia and endoderm in zebrafish. This suggests that wnt signaling plays an important role in regulating midline convergence. At the early patterning stage of the zebrafish kidney, the two pronephric-glomerular primodia (PGP), which derive from intermediate mesoderm, converge towards the midline and fuse to form a functional pronephros. In contrast, during development of the mammalian kidney, the pronephros degenerates as the mesonephros develops without midline convergence. The hypothesis is thus that there is/are mechanisms underlying midline convergence of PGP in zebrafish, which is/are in addition to the control of the non-canonical wnt/Dvl/RhoA pathway and specific to kidney morphogenesis. In this study, the aim was to identify genetic factors that are specifically involved in the mechanism of PGP midline convergence by establishing a cell lineage tracing transgenic system with Cre-loxP, followed by the analysis of selected mutant embryos using the cell lineage tracing system, whole-mount in situ hybridization (WISH) and immunostaining. The cell lineage tracing system was generated, and tested. Constructs, in which β-actin promoter drives the transcription of the reporter genes, were microinjected into zebrafish embryos at 1- to 4-cell stages. The mRNAs in microinjected embryos (5 dpf) were analyzed by RT-PCR. The results show that the constructs induced indiscriminate alternative splicing. RNA splicing mechanisms were not affected by transcription termination when the polyA signal was located in introns. To provide an alternative approach, three mutants were selected after screening of available ENU zebrafish mutants. These mutants were chosen not only because of their genetic importance in cell adhesion and motility but also because of their respective developmental defects in tissues surrounding the PGP, such as the notochord (no tail, ntl), somite (spadetail, spt), and endodermal tissues (zygotic oneeyed pinhead, Zoep). Spt and ntl are key targets of fibroblast growth factor (FGF) signalling in the trunk and tail respectively. EGP-CFC gene oep is a Nodal signalling cofactor. Firstly, a rapid genotyping technique was developed, which was applied in identifying the mutant alleles. Since the tools for tracing PGP using transgenes were unavailable, the three mutants were analyzed by WISH and immunostaining. Zygotic mutation of Zoep causes a PGP midline convergence phenotype of variable severity due to maternal Oep effects. In more than 90% of Zoep-/- embryos, PGP midline convergence was impaired. Even though the abnormality could be observed as early as 15 hpf, the differentiation of the PGP was not affected. Heart primordial phenotypes were also observed but they did not correlate with that of the PGP phenotypes. Embryos homozygous for mutations in T-box transcription factors, ntl or spt had normal heart midline convergence phenotypes. PGP midline convergence abnormality was observed in spt-/- but not in ntl-/- prior to 36 hpf. In addition the extracellular matrix (ECM) might play a key role in the mechanisms of PGP midline convergence. Furthermore, PGP midline convergence proceeds from 10 hpf (the specification of intermediate mesoderm) to 48 hpf (fused pronephric glomerulus) in wild type zebrafish embryos. The process was quantified by 2D image analysis of the PGP distance. Prior to 18 hpf, PGP midline convergence is closely correlated with the midline convergence of mesoderm but not at later stages in Zoep-/-. Spt is essential for PGP but not for cardiac primordium midline convergence. Data from this research suggests that there is not one universal mechanism, which controls all the midline convergence of organ primordia. Indeed, specific factors, which depend on tissues and development stages, are also required.

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 generation of mature Schwann cells from the neural crest occurs by a transition through two intermediate cell types, namely the Schwann cell precursor and the immature Schwann cell. Immature Schwann cells mature into the myelinating and non-myelinaling Schwann cells present in the adult nerve. These cell types are well characterised and can be readily distinguished from one another by their distinct antigenic profile, survival responses, and morphological changes. In this study I investigated the effects of BMP in the Schwann cell lineage in vitro. I found that BMP2/4 acts to maintain the immature Schwann cell type by promoting its differentiation from the Schwann cell precursor and inhibiting the upregulation of myelin proteins. I also found that survival responses to BMP2/4 differ between embryonic and postnatal Schwann cell. I examined the role of STAT3 in Schwann cells both in vitro and in vivo using mice with a conditional mutation of STAT3 specifically in Schwann cells. I found that STAT3 is activated by, and supports survival following stimulation by autocrine factors secreted by Schwann cells both in vitro and following nerve injury in vivo. I also discovered that STAT3 promotes the expression of myelin proteins in vitro and that it is involved in the timing of demyelination following sciatic nerve injury. I also investigated the repression of the transcription factor c-Jun, by itself and by Krox-20, in immature Schwann cells and found that this occurs mainly at the protein, rather than the transcriptional level.

The digestive tract and its accessory organs - liver, pancreas, and the inner lining of the swim bladder - are derived from the endodermal germ layer. In zebrafish the digestive system starts out as a rod at the embryonic midline, which subsequently becomes patterned and outgrowth of its associated organs occurs. Liver formation can be subdivided into three distinct phases; specification, budding, and differentiation and growth. Despite the importance of the liver for body homeostasis, relatively little is known about the mechanisms of liver development. The transgenic line Tg(gutGFP)s854, expressing GFP throughout the developing digestive tract, was used to identify the mutant lines s436 and 4C1 in a forward genetic screen for mutants displaying defects in endodermal organogenesis. Initial analysis of both lines revealed a hypoplastic liver at 48 hpf, and in addition distinct defects in pancreas formation. Positional cloning of the s436 line identified a novel allele of histone deacetylase 1 (hdac1), which plays distinct roles in endodermal organogenesis in zebrafish. Loss of Hdac1 causes defects in timely liver specification and subsequent differentiation, while mosaic analyses revealed a cell-autonomous role for Hdac1 in the endoderm in these processes. In addition, I have demonstrated that Hdac1 has specific functions in endocrine pancreas morphogenesis, as well as roles in exocrine pancreas specification and differentiation. Finally, loss of Hdac1 results in an expansion of the foregut endoderm in the domain from which the liver and pancreas originate, suggesting a scenario whereby Hdac1 may directly or indirectly restrict foregut fates while promoting hepatic and exocrine pancreatic specification and differentiation. Phenotypic characterisation of endodermal organogenesis in 4C1 mutant embryos revealed a requirement for 4C1 in specification of the correct number of hepatic progenitors and hepatic bud morphogenesis. This is accompanied by a subsequent requirement for 4C1 in hepatic growth and duct morphogenesis. Additionally, 4C1 mutants exhibit defects in exocrine pancreas anlage outgrowth, leading to ectopic exocrine bud formation, suggesting a role for 4C1 in the morphogenesis of this organ. Preliminary analysis suggests that 4C1 may be required within the neighbouring tissue, the lateral plate mesoderm, to promote endodermal organogenesis. Taken together, I have characterised two novel mutant lines, which despite initial phenotypic similarities, exhibit very distinct defects in liver as well as pancreas formation, highlighting important roles for these genes in the processes underlying endodermal organogenesis.