2 pp. / Plant growth regulators are commonly applied to wheat and barley grown under high-yielding conditions to reduce the risk of lodging. Plant growth regulators reduce lodging by reducing plant height and strengthening the stem.

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Embryonic stem cells (ESCs) represent a promising source of cells for cell replacement therapy in the context of muscle diseases; however, before ESC-based cell therapy can be translated to the clinic, we must learn to modulate cell-fate decisions in order to maximize the yield of myocytes from this systems. In order to gain a better understanding of the myogenic cell fate, we sought to define the molecular mechanisms underlying the specification and differentiation of ESCs into cardiac and skeletal muscle. More specifically, the central hypothesis of the thesis is that myogenic signalling cascades modulate cell fate via regulation of transcription factors. Retinoic acid (RA) is known to promote skeletal myogenesis, however the molecular basis for this remains unknown. We showed that RA expands the premyogenic progenitor population in mouse stem cells by directly activating pro-myogenic transcription factors such as Pax3 and Meox1. RA also acts indirectly by activating the pro-myogenic Wnt signalling cascade while simultaneously inhibiting the anti-myogenic influence of BMP4. This ultimately resulted in a significant enhancement of skeletal myogenesis. Furthermore, we showed that this effect was conserved in human embryonic stem cells, with implications for directed differentiation and cell therapy. The regulation of cardiomyogenesis by the Wnt pathway was also investigated. We identified a novel interaction between the cardiomyogenic transcription factor Nkx2.5 and the myosin phosphatase (MP) enzyme complex. Interaction with MP resulted in exclusion of Nkx2.5 from the nucleus and inhibition of its transcriptional activity. Finally, we showed that this interaction was modulated by phosphorylation of the Mypt1 subunit of MP by ROCK, downstream of Wnt3a. Treatment of differentiating mouse ESCs with Wnt3a resulted in exclusion of Nkx2.5 from the nucleus and a subsequent failure to undergo terminal differentiation into cardiomyocytes. This likely represents part of the molecular basis for Wnt-mediated inhibition of terminal differentiation of cardiomyocytes. Taken together, our results provide novel insight into the relationship between myogenic signalling cascades and downstream transcription factors and into how they function together to orchestrate the myogenic cell fate in stem cells.

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Embryonic stem (ES) cells are derived from the inner cell mass of the blastocyst stage embryo. They exhibit unlimited proliferation in culture and have the ability to differentiate into all three germ layers of the developing organism, a property defined as pluripotency. Previously it was reported that growth factor-bound protein 2 (Grb2) is required for differentiation of the epiblast, the embryonic tissue that harbours the pluripotent founder cells of the foetus. GRB2 is an adapter protein involved in the activation of the mitogen-activated protein kinase (MAPK) pathway in response to extracellular signals. It has also been implicated in the activation of the phosphoinositol-3-kinase (PI3K) pathway in response to fibroblast growth factor (FGF) signaling. The work presented in this thesis examines the role of Grb2 in ES cells and describes previously unreported contributions of this adaptor protein in regulating ES cell growth and differentiation. It has been previously been shown by others that Grb2 deficient (Grb2-/-) cells grow relatively normally in ES growth medium containing serum. However, in serum free conditions (N2B27 medium) in this project, proliferation of Grb2-/- cells is reduced compared with wild type and “restored” Grb2-/- cells stably expressing a Grb2 cDNA mini gene. Under serum free conditions, Grb2-/- cells grow in tight, refractive colonies. Nanog expression was uniformly upregulated, in contrast to the heterogeneous pattern reported in serum-based medium. Colony expansion on the substratum appears to be compromised, although there is no apparent defect in the initial attachment of Grb2-/- cells. Cell cycle analysis indicates that the slower growth of Grb2-/- cells in serum free medium could be due to lengthening of the G1 phase of the ES cell cycle. In an attempt to identify the signalling deficiency responsible for the growth defect of Grb2-/- cells, MAPK activation was restored by two methods, PMA a ligand that bypasses the requirement for Grb2, and Raf-ER, a conditionally regulated component of the MAPK pathway that acts downstream of Grb2 in the MAPK pathway. Although both approaches increased MAPK signalling they were unable to rescue the growth defect. This suggests that MAPK is not required or alone is not sufficient. Inhibition of Glycogen synthase kinase 3 β (GSK3 β ) is known to augment growth of ES cells under MAPK inhibition. Surprisingly, GSK3 β inhibition did not enhance Grb2-/- cell growth. Under GSK3 β inhibition, Grb2-/- ES cells fail to thrive. It is hypothesised that under these conditions cells undergo hyper-self-renewal at the cost of growth. Grb2-/- ES cells are reported to exhibit limited differentiation potential. To examine the potency of Grb2-/- cells, these cells were subjected to embryoid body (EB) and monolayer differentiation. Analysis of EBs showed a loss of Gata4, Gata6 and endoderm marker gene expression. However, markers of ectoderm (Sox1, Pax6, MAP2), the late epiblast/nascent mesoderm (Brachyury) and markers associated with gastrulation (Twist and Snail) were expressed. Outgrowths of morphologically and immunohistochemically identifiable neuronal cells confirmed differentiation of ectodermal cell types, indicating Grb2 is not required for neuronal differentiation. However, beating cardiomyocytes could not be identified in Grb2-/- EBs, though readily found in restored Grb2-/- cells expressing the Grb2 cDNA. This suggests that there is an essential role for Grb2 in the mesoderm/cardiomyocyte differentiation pathway. This may be due to a defect in GATA factor expression since these factors are essential for cardiogenesis. In serum-free monolayer differentiation, Grb2-/- cells formed neuronal cells. Additional inhibition of the MAPK pathway using a small chemical inhibitor failed to prevent this differentiation. However, biochemical analysis of the cells indicates that this occurs when ERK activation is very low, indicating differentiation was not MAPK-independent. Grb2 mediates FGF-MAPK induced exit from the naïve ground state. These data suggest a Grb2-independent pathway can also facilitate this transition. Grb2 is dispensable for differentiation in to some lineages. However as differentiation of Grb2-/- ES cells is restricted, this indicates Grb2 is required for true pluripotency.

Neural stem cell (NSC) transplantation is a promising therapy for the treatment of brain damage. Although the “proof of principle” for NSC transplantation therapy has been demonstrated in a variety of animal models of brain injury (stroke, traumatic brain injury, ageing) and in a clinical setting (Parkinson’s disease), the mechanisms by which grafted stem cells survive, migrate and differentiate in host brain are yet to be elucidated. Initial studies have demonstrated that, after transplantation of the MHP36 neural stem cell line in a focal ischaemia model, the lipid transport protein apolipoprotein E (apoE) is upregulated and co-localised to differentiated cells in parallel with functional recovery. ApoE has been shown to have a critical role in the response to brain injury and repair processes. Furthermore, in humans, three different forms of apoE exist (E2, E3, E4 encoded by the alleles e2, e3, e4) and each of these has a different ability to promote repair, with the E4 form associated with an impaired capacity. This thesis tests the hypothesis that apoE is critical in stem cell integration and investigates whether this effect is APOE genotype dependent, in a mouse model of global cerebral ischaemia. This model was chosen as it produces diffuse selective neuronal damage in the striatum and hippocampus, which also occurs in other conditions such as ageing and Alzheimer’s disease. The studies described in this thesis were designed to test the hypothesis and are outlined as follows: I. Characterisation of neural stem cell grafts in a mouse model of global ischaemia In order to investigate the potential influence of apoE on stem cell grafts, it was first essential to characterise stem cells grafts in mouse brain. Thus, the initial aim of the thesis was to characterise MHP36 grafts in a mouse model of ischaemic neuronal injury. The effect of cyclosporin A (CsA) immunosuppression was also investigated. C57Bl/6J mice underwent an episode of transient global ischaemia induced by bilateral common carotid artery occlusion. Three days following ischaemia, mice received a unilateral striatal graft of fluorescently labelled MHP36 neural stem cells or vehicle; the mice also received CsA or saline. The mice were terminated at either XVII 1 or 4 weeks post-transplantation. This study determined that MHP36 grafts survived and migrated robustly in host ischaemic brain at both 1 week and 4 weeks post-transplantation. Grafted MHP36 cells differentiated into neurons and were able to reduce the extent of ischaemic neuronal damage. An acute host inflammatory response was evoked following MHP36 grafting, but this decreased dramatically by 4 weeks post-transplantation. CsA immunosuppression did not affect MHP36 survival and migration or reduce the host inflammatory response. The successful transplantation and characterisation of MHP36 grafts in mouse brain allowed for future investigation into the genetic factors underlying stem cell graft integration via the use of apoE transgenic mice. II. Influence of apoE on neural stem cell grafts in a mouse model of global ischaemia The aim of this study was to investigate whether endogenous apoE influenced MHP36 survival, migration and differentiation and then to determine potential signalling pathways that may be involved. ApoE deficient mice on a C57Bl/6J background (APOE-KO) and control wildtype C57Bl/6J (WT) mice were subjected to an episode of transient global ischaemia, as in Experiment 1. Two weeks following ischaemia, all mice received unilateral striatal and hippocampal grafts of MHP36 cells. All mice received CsA immunosuppression. Mice were terminated 4 weeks post-transplantation. MHP36 survival and migration was significantly increased in WT as compared to APOE-KO mice. In addition, neuronal differentiation was significantly increased in WT as compared to APOE-KO mice. Increased astrocytic differentiation was observed in the hippocampus, but not striatum of WT as compared to APOE-KO mice. Measurement of the levels of signalling proteins associated with cell survival, extracellular signal-regulated kinase (ERKs) and c-Jun amino-terminal kinase (JNKs) and their phosphorylated forms (pERK and pJNK), indicated selective alterations in JNK with no change in ERK in APOE-KO as compared to WT mice, suggesting that JNK may underlie the apoE effects in stem cell integration. This study demonstrated that apoE strongly influences the survival, migration and differentiation of grafted MHP36 cells and provides initial evidence for the signalling pathways involved. XVIII III. Influence of APOE genotype on neural stem cell grafts in a mouse model of global ischaemia Following the demonstration that endogenous mouse apoE has a critical role in MHP36 graft survival, migration and differentiation, this study sought to investigate whether these effects are influenced by human APOE genotype. Transgenic mice expressing human APOE-e3 or e4, (on an APOE-KO background) and a control group of APOE-KO mice underwent transient global ischaemia and two weeks later MHP36 cells were transplanted unilaterally into the striatum and hippocampus. 1 week after grafting the mice were started on a series of tests for motor balance and coordination using the rotarod, and taken for histology 4 weeks post-transplantation. MHP36 graft survival was significantly improved in APOE-e3 mice compared to APOE-KO and APOE-e4 mice. However, the migration and differentiation of MHP36 cells and motor performance of grafted mice were similar in all three APOE groups, indicating a comparable fate and functional activity within a 4 week survival time. Thus the data indicate that APOE genotype may influence cell survival with minimal effect on stem cell migration and differentiation. The data presented in this thesis demonstrate that endogenous apoE strongly influences MHP36 graft survival, migration and differentiation. Although there was minimal evidence that human APOE genotype influences cell migration and differentiation, stem cell survival was markedly improved in a human APOE-e3 allelic environment, which may affect the effectiveness of stem cells in APOE-e4 individuals.