TLE proteins are a closely related family of vertebrate corepressors. They have no intrinsic DNA binding ability, but are recruited as transcriptional repressors by other sequence specific proteins. TLE proteins and their homologues in other species have been implicated in many developmental processes including neurogenesis, haematopoiesis and the formation of major organs. They have also been implicated in early lineage specification in vertebrates but a direct role in this has not been found in mammals. The aim of my PhD is therefore to analyse the function of TLE proteins in early lineage specification and cell fate decisions using mouse embryonic stem cells (ESCs) as a model. The investigation of this has previously been complicated, firstly by the large array of transcription factors that TLEs interact with and secondly by redundancy between similar TLE proteins hindering loss of function approaches. To circumvent these problems, I have used two complementary experimental strategies. The first was identification of point mutations in TLE1 that affect specific classes of DNA binding. Two of these mutations L743F and R534A were of particular interest and were reversibly overexpressed in ES cells to correlate phenotypes to biochemical activity. The second strategy was the mutation of the two primary TLC genes in ES cells and early mouse embryos, TLE3 and TLE4. Complementary evidence from these approaches revealed a role for TLEs in the promotion of ES cell differentiation by repression of pluripotency/self-renewal associated genes. Additionally, neural specification was increased by TLE1 expression especially by the TLE1 point mutations, highlighting opposing roles for negative effects on mesendodermal differentiation. Early mesoderm/primitive streak was increased by loss of TLE, probably through Wnt antagonism. Anterior endoderm was increased by reduced TLE, but a critical level of TLE was still necessary and TLE1 overexpression also upregulated some anterior endoderm markers suggesting both negative and positive roles for TLE proteins in this process.

Embryonic cells of many species polarise and the cell polarity is often important for the normal developmental progression. In the mouse embryo, the prototype of epithelial cell polarity, namely apico-basal polarisation, become established at the 2.5 days' post-fertilisation, when the embryos are at the 8-cell stage. The formation of apical domain is necessary and sufficient for the first segregation of extra-embryonic and embryonic cell lineages, as well as the following up morphogenetic transitions, such as the blastocyst formation. This study aims to explore the molecular pathways triggering the first cell polarity establishment in the mouse embryo, and to reveal the mechanism that programmes the timing of this event in the mouse embryo. The results showed that cell polarity establishment during the 8-cell stage development can be divided into two major phases: in the first phase actomyosin complex became polarised to the cell-contact free surface; and in the second phase apical proteins recruited to the actomyosin enriched cell-contact free cortex, they further became centralised in the cell-contact free surface, excluding the local actomyosin meshwork, resulting in the formation of actomyosin ring. The activation and assembly of actomyosin meshwork during the first phase, but not its contractility, was essential for apical protein recruitment. Factors responsible for actin cytoskeleton reorganisation included Phospholipase C (PLC) - Protein Kinase C (PKC) pathway components, they directly activated actomyosin in the first phase through the Rho proteins such as RhoA. Further results showed that the apical protein centralisation step required a proximate transcriptional input that was induced by two transcription factors, Tfap2c and Tead4. RNAi and Genetic depletion of these two factors prevented apical protein centralisation and the final apical domain assembly. The protein expression profile indicated that Tfap2c and Tead4 expression, and therefore their activity, were induced by zygotic genome activation. Significantly, overexpression of Tfap2c, Tead4, together with constitutively activated Rho proteins were sufficient to advance the timing of apical domain formation, indicating that the timer of cell polarity establishment at the 8-cell stage is set by the Rho proteins activation, and the zygotic transcriptional accumulation of Tfap2c and Tead4. Together, these results characterised the molecular events during the cell polarity establishment at the 8-cell stage mouse embryo, and identified the timing regulation of this event.

Growing evidence from animal and clinical studies suggests that cardiac cell therapy can restore perfusion and improve function in the ischemic/infarcted myocardium. However, cell therapy is hindered by insufficient cell numbers, inefficient cell homing and engraftment, and inadequate cellular interactions. Furthermore, the biological mechanisms and local effects of transplanted cells have not been well-elucidated. The research presented herein attempts to address some of these issues. In manuscript #1, a new subpopulation of circulating progenitor cells (CPCs), termed derived CD133+ cells, was generated from the CD133- fraction of human peripheral blood. The derived CD133+ progenitors appeared to have superior vasculogenic potential in vitro, which may prove to be beneficial in inducing vasculogenesis in ischemic tissues. Positron emission tomography (PET) with direct cell labeling and reporter gene techniques were employed to assess the fate of transplanted human CPCs in vivo at different subjects of investigation, and different stages of cell transplantation. In manuscript #2, PET imaging with 2-[18F]fluoro-2-deoxy-D-glucose (18F-FDG) direct cell labeling was used to demonstrate that collagen-based matrices improve the early homing and retention of delivered CPCs in a rat ischemic hindlimb model. This mechanism conferred by the matrix may have implications on cell therapy at the early stages after transplantation. In manuscript #3, a more efficient, stable and accurate labeling method, hexadecyl-4-[18F]fluorobenzoate (18F-HFB) direct cell labeling, was developed to quantify cell distribution of transplanted CPCs in a rat myocardial infarction model. PET imaging of 18F-HFB-CPCs revealed significant cell washout from the myocardium immediately after intramyocardial injection, with only a small proportion of transplanted CPCs remaining in the target area in the first 4 hours after delivery. In manuscript #4, human CPCs transduced with lentiviral vectors showed stable expression of PET reporter genes. This reporter gene based-cell labeling technique can be developed for noninvasive tracking cells within a bioengineered matrix by PET, while preserving cell phenotype, viability and function. These studies contribute important insights into the biology and physiology of transplanted stem cells and the ability of delivery matrices to improve transplanted cell engraftment, survival, and function. I believe with further refinement, cell expansion, tissue engineering and PET imaging could facilitate the clinical applications of cell therapies in years to come.

Growing evidence from animal and clinical studies suggests that cardiac cell therapy can restore perfusion and improve function in the ischemic/infarcted myocardium. However, cell therapy is hindered by insufficient cell numbers, inefficient cell homing and engraftment, and inadequate cellular interactions. Furthermore, the biological mechanisms and local effects of transplanted cells have not been well-elucidated. The research presented herein attempts to address some of these issues. In manuscript #1, a new subpopulation of circulating progenitor cells (CPCs), termed derived CD133+ cells, was generated from the CD133- fraction of human peripheral blood. The derived CD133+ progenitors appeared to have superior vasculogenic potential in vitro, which may prove to be beneficial in inducing vasculogenesis in ischemic tissues. Positron emission tomography (PET) with direct cell labeling and reporter gene techniques were employed to assess the fate of transplanted human CPCs in vivo at different subjects of investigation, and different stages of cell transplantation. In manuscript #2, PET imaging with 2-[18F]fluoro-2-deoxy-D-glucose (18F-FDG) direct cell labeling was used to demonstrate that collagen-based matrices improve the early homing and retention of delivered CPCs in a rat ischemic hindlimb model. This mechanism conferred by the matrix may have implications on cell therapy at the early stages after transplantation. In manuscript #3, a more efficient, stable and accurate labeling method, hexadecyl-4-[18F]fluorobenzoate (18F-HFB) direct cell labeling, was developed to quantify cell distribution of transplanted CPCs in a rat myocardial infarction model. PET imaging of 18F-HFB-CPCs revealed significant cell washout from the myocardium immediately after intramyocardial injection, with only a small proportion of transplanted CPCs remaining in the target area in the first 4 hours after delivery. In manuscript #4, human CPCs transduced with lentiviral vectors showed stable expression of PET reporter genes. This reporter gene based-cell labeling technique can be developed for noninvasive tracking cells within a bioengineered matrix by PET, while preserving cell phenotype, viability and function. These studies contribute important insights into the biology and physiology of transplanted stem cells and the ability of delivery matrices to improve transplanted cell engraftment, survival, and function. I believe with further refinement, cell expansion, tissue engineering and PET imaging could facilitate the clinical applications of cell therapies in years to come.

Stem and progenitor cells have the ability to regulate fate decisions through asymmetric cells divisions. The coordinated choice of cell division symmetry in space and time contributes to the physiological development of tissues and organs. Conversely, deregulation of these decisions can lead to the uncontrolled proliferation of cells as observed in cancer. Understanding the mechanisms of cell fate choices is necessary for the design of biomimetic culture systems and the production of therapeutic cell populations in the context of regenerative medicine. Environmental signals can guide the fate decision process at the single level but the exact nature of these signals remains to be discovered. Gradients of factors are important during development and several methods have been developed to recreate gradients and/or pulses of factors in vitro. In the context of asymmetric cell division, the effect of the soluble factor environment on the polarization of cell surface receptors and intracellular proteins has not been properly investigated. We developed a finite-element model of a single cell in culture in which epidermal growth factor (EGF) was delivered through a micropipette onto a single cell surface. A two-dimensional approach initially allowed for the development of a set of metrics to evaluate the polarization potential with respect to different delivery strategies. We further analyzed a three-dimensional model in which conditions consistent with single cell polarization were identified. The benefits of finite-element modeling were illustrated through the demonstration of complex geometry effects resulting from the culture chamber and neighboring cells. Finally, physiological effects of in vitro polarization were analyzed at the single cell level in HeLa and primary cells. The potential of soluble factor signaling in the context of directed fate control was demonstrated. Long term phenotypical effects were studied using live-cell imaging which demonstrated the degree of heterogeneity of in vitro culture systems and future challenges for the production of therapeutic cell populations.

Though development of the cerebral cortex is of singular importance to human cognition, it remains very poorly understood. Microcephaly, or "small head," is a neurodevelopmental disorder causing significantly reduced cerebral cortex size, and the disease has proved to be a useful model system for elucidating the steps essential for proper cortical development and cognitive function. Many known microcephaly gene products localize to centrosomes, regulating cell fate and proliferation, however, the elucidation of different microcephaly genes with different functions may shed light on previously unidentified key steps of brain development. We identify and characterize a nuclear zinc finger protein, ZNF335/NIF-1, as a causative gene for severe microcephaly, small somatic size, and neonatal death. Znf335-null mice are embryonically lethal and conditional knockout leads to severely reduced cortical size. RNA-interference and postmortem human studies show that Znf335 is essential for neural progenitor self-renewal, neurogenesis, and neuronal differentiation. ZNF335 is a component of a vertebrate-specific, trithorax H3K4-methylation complex, directly regulating REST/NRSF, a master regulator of neural gene expression and cell fate, as well as other essential neural-specific genes. Our results reveal ZNF335 as an essential link between H3K4 complexes and REST/NRSF, and provide the first direct genetic evidence that this pathway regulates human neurogenesis and neuronal differentiation.

The stochastic nature of intracellular chemistry guarantees that even genetically identical cells sharing an environment will differ in composition. The question of whether this chemical diversity translates into significant phenotypic individuality is tied to the relative timescales of the processes involved. In order for cells in a population to have distinct functional identities, they must maintain their states for an appreciable period of time. Quantification of these timescales requires accurate time-lapse measurements covering tens or even hundreds of generations, a technical hurdle that has left these questions largely underexplored. In this thesis I present three pieces of work that aim to provide a foundation for the study of fluctuation timescales in bacteria. In the first part, I describe modifications to a recently developed microfluidic platform for continuous culture of cells under constant conditions. These revised devices enable the high-throughput, long-term measurement of gene expression dynamics while eliminating several confounding experimental factors that interfere with timescale measurements. In the second part, I employ one of these devices to survey fluctuation timescales in ~50 reporters for Eshcerichia coli gene expression. Under rich conditions, all reporters exhibited nearly identical, rapid fluctuation dynamics that were captured by a simple model of gene expression. In contrast, under poor nutritional conditions gene expression states became correlated over several cell divisions. However, accounting for instantaneous growth rate fluctuations eliminated these slow timescales, revealing an exceedingly simple behavior. In the third part, I describe our work to dissect the stochastic transition between the solitary motile state and sessile multicellular state in exponentially growing Bacillus subtilis</italic.. By enforcing static environmental conditions, we uncover the cell's internal strategies for state switching. The transition to the multicellular state occurs without regard to the cell's state history, whereas commitment to the multicellular state is tightly timed. By manipulating the genetic circuit responsible for the switch, we also expose surprising functional modularity in the commitment. I believe that the striking range of gene expression timescales we observe--from the fast fluctuations in E. coli gene expression to the feedback-amplified noise in B. subtilis--will serve as a useful starting point for future studies.

Studies on epidermal cell fate determination have been important for gaining insight into the genetic and molecular mechanisms leading to the differentiation and patterning of cells. In Arabidopsis, the organization and development of many epidermal characters including trichomes, root hairs and the seed coat have been found to be controlled by a single combinatorial transcription factor complex consisting of a WD-repeat containing protein, Transparent Testa Glabra 1 (TTG1), and various MYB and bHLH proteins. The work here consists of identification of Glabra2 (GL2) and Transparent Testa Glabra2 (TTG2) as direct transcriptional targets of the TTG1 combinatorial complex, further characterization of GL2 function, and identification of transcriptional targets of GL2 and TTG2. Both GL2 and TTG2 are important in the regulation of trichomes, root hairs and seed coat development. vii GL2 has been identified as an important regulator of epidermal cell fate for over fifteen years yet there is little known about its function and only three transcriptional targets are identified, all involved in root hair patterning. Through the examination of its function a nuclear localization signal was verified and shown that GL2 homodimerizes. Through analysis of available expression databases and differential sequence analysis using SOLiD sequencing technology, several direct targets of GL2 and many more possible transcriptional targets of both GL2 and TTG2 were identified in trichomes. Some of these targets are members of the TTG1 complex, and they are all specialized in the maturation of trichomes, suggesting that GL2 switches the focus of the complex by activating the TTG1 complex members involved in maturation of the trichome through a feedback mechanism. Examination of gl2 mutants shows that they do not produce trichome accessory cells which usually surround the trichome. An additional target of GL2 is At5g65300 which when overexpressed results in the elongation and proliferation of trichome accessory cells into a tall pillar of cells. This suggests that GL2 is involved in the regulation of accessory cell development through At5g65300. The work presented here represents important advances of our knowledge of epidermal cell fate through characterization of the major downstream regulators of epidermal development. / text

Growing evidence from animal and clinical studies suggests that cardiac cell therapy can restore perfusion and improve function in the ischemic/infarcted myocardium. However, cell therapy is hindered by insufficient cell numbers, inefficient cell homing and engraftment, and inadequate cellular interactions. Furthermore, the biological mechanisms and local effects of transplanted cells have not been well-elucidated. The research presented herein attempts to address some of these issues. In manuscript #1, a new subpopulation of circulating progenitor cells (CPCs), termed derived CD133+ cells, was generated from the CD133- fraction of human peripheral blood. The derived CD133+ progenitors appeared to have superior vasculogenic potential in vitro, which may prove to be beneficial in inducing vasculogenesis in ischemic tissues. Positron emission tomography (PET) with direct cell labeling and reporter gene techniques were employed to assess the fate of transplanted human CPCs in vivo at different subjects of investigation, and different stages of cell transplantation. In manuscript #2, PET imaging with 2-[18F]fluoro-2-deoxy-D-glucose (18F-FDG) direct cell labeling was used to demonstrate that collagen-based matrices improve the early homing and retention of delivered CPCs in a rat ischemic hindlimb model. This mechanism conferred by the matrix may have implications on cell therapy at the early stages after transplantation. In manuscript #3, a more efficient, stable and accurate labeling method, hexadecyl-4-[18F]fluorobenzoate (18F-HFB) direct cell labeling, was developed to quantify cell distribution of transplanted CPCs in a rat myocardial infarction model. PET imaging of 18F-HFB-CPCs revealed significant cell washout from the myocardium immediately after intramyocardial injection, with only a small proportion of transplanted CPCs remaining in the target area in the first 4 hours after delivery. In manuscript #4, human CPCs transduced with lentiviral vectors showed stable expression of PET reporter genes. This reporter gene based-cell labeling technique can be developed for noninvasive tracking cells within a bioengineered matrix by PET, while preserving cell phenotype, viability and function. These studies contribute important insights into the biology and physiology of transplanted stem cells and the ability of delivery matrices to improve transplanted cell engraftment, survival, and function. I believe with further refinement, cell expansion, tissue engineering and PET imaging could facilitate the clinical applications of cell therapies in years to come.

Stem and progenitor cells have the ability to regulate fate decisions through asymmetric cells divisions. The coordinated choice of cell division symmetry in space and time contributes to the physiological development of tissues and organs. Conversely, deregulation of these decisions can lead to the uncontrolled proliferation of cells as observed in cancer. Understanding the mechanisms of cell fate choices is necessary for the design of biomimetic culture systems and the production of therapeutic cell populations in the context of regenerative medicine. Environmental signals can guide the fate decision process at the single level but the exact nature of these signals remains to be discovered. Gradients of factors are important during development and several methods have been developed to recreate gradients and/or pulses of factors in vitro. In the context of asymmetric cell division, the effect of the soluble factor environment on the polarization of cell surface receptors and intracellular proteins has not been properly investigated. We developed a finite-element model of a single cell in culture in which epidermal growth factor (EGF) was delivered through a micropipette onto a single cell surface. A two-dimensional approach initially allowed for the development of a set of metrics to evaluate the polarization potential with respect to different delivery strategies. We further analyzed a three-dimensional model in which conditions consistent with single cell polarization were identified. The benefits of finite-element modeling were illustrated through the demonstration of complex geometry effects resulting from the culture chamber and neighboring cells. Finally, physiological effects of in vitro polarization were analyzed at the single cell level in HeLa and primary cells. The potential of soluble factor signaling in the context of directed fate control was demonstrated. Long term phenotypical effects were studied using live-cell imaging which demonstrated the degree of heterogeneity of in vitro culture systems and future challenges for the production of therapeutic cell populations.