“The scientist, by the very nature of his commitment, creates more and more questions, never fewer. Indeed the measure of our intellectual maturity, one philosopher suggests, is our capacity to feel less and less satisfied with our answers to better problems.” ~G.W. Allport, Becoming, 1955 It will be interesting to look back at this thesis in a few decades and reflect on how the questions and interpretation of data in the field of developmental biology have changed. Indeed, a biologist currently in their twilight years might reflect on their youth, before the discovery of hereditary material, and compare that bookend with the range of genome sequences and related knowledge currently available. How long will it take before this thesis reads like a debate about whether the male or female contributed the ‘homunculus,’ a miniature preformed human to the embryo that grows into an adult? In this thesis I asked three related questions: whether the role of Oct4 during embryogenesis provides insight into its contribution to pluripotency; how surfaceome changes contribute to functional maturation of neural stem cells and to what extent the murine genome is imprinted. Our data indicate that Oct4 is required for posterior expansion. We propose that the function of the protein is conserved, but that its expression has been coopted to yield different cell types based on its combination with different factors. We show that fundamental aspects of cell biology are altered during the maturation from pluripotent populations to neural stem cells, and identify mediators of proliferation, survival and adhesion that distinguish neural stem cell regulation from their precursors. Finally, we validated discovery of a dozen novel imprinted transcripts using a genomic approach. These discoveries will contribute to a holistic view of the causes and consequences of imprinting, but do not support a paradigm shift in the scale and consequences of imprinting.

“The scientist, by the very nature of his commitment, creates more and more questions, never fewer. Indeed the measure of our intellectual maturity, one philosopher suggests, is our capacity to feel less and less satisfied with our answers to better problems.” ~G.W. Allport, Becoming, 1955 It will be interesting to look back at this thesis in a few decades and reflect on how the questions and interpretation of data in the field of developmental biology have changed. Indeed, a biologist currently in their twilight years might reflect on their youth, before the discovery of hereditary material, and compare that bookend with the range of genome sequences and related knowledge currently available. How long will it take before this thesis reads like a debate about whether the male or female contributed the ‘homunculus,’ a miniature preformed human to the embryo that grows into an adult? In this thesis I asked three related questions: whether the role of Oct4 during embryogenesis provides insight into its contribution to pluripotency; how surfaceome changes contribute to functional maturation of neural stem cells and to what extent the murine genome is imprinted. Our data indicate that Oct4 is required for posterior expansion. We propose that the function of the protein is conserved, but that its expression has been coopted to yield different cell types based on its combination with different factors. We show that fundamental aspects of cell biology are altered during the maturation from pluripotent populations to neural stem cells, and identify mediators of proliferation, survival and adhesion that distinguish neural stem cell regulation from their precursors. Finally, we validated discovery of a dozen novel imprinted transcripts using a genomic approach. These discoveries will contribute to a holistic view of the causes and consequences of imprinting, but do not support a paradigm shift in the scale and consequences of imprinting.

The ERK-MAPK pathway is a dynamic signaling module, conserved across Eukarya, and capable of processing a myriad of environmental and cellular signals. It has been implicated in controlling important cell fate decisions in many cell types and species. In mES cells, growth factor activation of the ERK-MAPK pathway is involved in the earliest stages of lineage segregation, however very little is currently known about the mechanism by which this is accomplished. Taking a loss-of-function gene targeting approach I have reexamined the relative contribution of ERK2 activity to FGF-ERK signaling. Although ERK2 depletion results in an attenuation of the combined ERK1/2 activity, this is compensated for by the hyperactivation of the remaining ERK1 isozyme. Normal ERK1/2 function can be restored to ERK2 deficient cells by transgenic expression of either ERK1 or ERK2, indicating a degree of functional redundancy between both isoforms. When subjected to the appropriate cues, lineage commitment proceeded normally in ERK2 deficient cells, however increased self-renewal was observed under standard culture conditions. Several attempts were made to further probe ERK1/2 function by siRNA depletion, and dominant negative inhibition of ERK1 in Erk2 knockout cells, however both approaches failed to provide further insight. Furthermore, taking a candidate approach, the role of Srf, a canonical target of ERK1/2 signaling, was examined. Initial experiments indicated a role for SRK in neural differentiation, however due to issues of culture adaptation and instability in several cell lines it was not possible to conclude this line of research within the time frame of this thesis. IP-MS/MS analysis identified several proteins known to interact with ERK2 and indicated an involvement in nuclear pore function through TPR as well as transcriptional and translational regulation through RSK proteins. Moreover, this study identified DUSP6 and DUSP9 as the primary induced dual specificity phosphatases that regulate ERK2 activity in mES cells. To further probe the functional significance of the ERK:p90RSK interaction I examined a mES cell line genetically depleted for PDK1, a crucial regulator of p90RSK function. This cell line exhibits no detectable p90RSK activity, however in contrast to studies in other cell lines, p90RSK activity is dispensable for mitogen-induced cFos expression in mES cells. Subsequent experiments demonstrated a requirement for PDK1 activity in either the specification or maintenance of mES cell derived neurons. Further analysis indicated that p90RSK may be involved in a negative feedback loop regulating ERK1/2 activity, and if so may represent a point whereby ERK1/2 activity can be manipulated. To examine this I determined the effect pharmacological inhibition of p90RSK has on ERK1/2 activity and self-renewal using a novel p90Rsk inhibitor, BI-D1870. Although treatment with BI-D1870 correlated with enhanced ERK1/2 phosphorylation, the offtarget effects this molecule exhibits made it impossible to draw any firm conclusions from these experiments. Overall this study has demonstrated a degree of redundancy between ERK1/2 isozymes in mES cells. It has highlighted the complex nature of ERK1/2 regulation as well as the robustness of this pathway to perturbations in ERK dose. Furthermore, it has underscored some of the common pitfalls encountered when studying differentiation phenotypes in mES cells. Although this study failed to highlight anything more than a coincidental relationship between ERK1/2 activity and self-renewal capacity of mES cells, it has helped to highlight some important behavioral characteristics of the FGF-MAPK pathway in mES cells and provide a platform for further study.

Pluripotent stem cells have the capacity to develop into different cell lineages and can be manipulated into certain cell types through the use of small molecule inducers. Retinoic acid (RA) signaling through retinoic acid receptors (RAR) and retinoid X receptors (RXR) has the ability to direct lineage determination but has yielded disappointing results in promoting skeletal myogenesis in embryonic stem (ES) cells. RXR is crucial in embryonic development although it is generally considered to act as a silent partner for other nuclear receptors such as RAR. Our findings demonstrate that rexinoid specific signaling enhances skeletal myogenesis and requires β-catenin but not RAR. Moreover, RXR signalling in mouse ES cells can efficiently enhance skeletal myogenesis and closely recapitulates sequential events observed in vivo. Since ES cells closely represent the properties of the developing embryo, efficiently generating skeletal muscle provides a means to further scrutinize signaling pathways in myogenic development in view of developing therapies for muscle related diseases.

Pluripotent stem cells have the capacity to develop into different cell lineages and can be manipulated into certain cell types through the use of small molecule inducers. Retinoic acid (RA) signaling through retinoic acid receptors (RAR) and retinoid X receptors (RXR) has the ability to direct lineage determination but has yielded disappointing results in promoting skeletal myogenesis in embryonic stem (ES) cells. RXR is crucial in embryonic development although it is generally considered to act as a silent partner for other nuclear receptors such as RAR. Our findings demonstrate that rexinoid specific signaling enhances skeletal myogenesis and requires β-catenin but not RAR. Moreover, RXR signalling in mouse ES cells can efficiently enhance skeletal myogenesis and closely recapitulates sequential events observed in vivo. Since ES cells closely represent the properties of the developing embryo, efficiently generating skeletal muscle provides a means to further scrutinize signaling pathways in myogenic development in view of developing therapies for muscle related diseases.

Pluripotent stem cells have the capacity to develop into different cell lineages and can be manipulated into certain cell types through the use of small molecule inducers. Retinoic acid (RA) signaling through retinoic acid receptors (RAR) and retinoid X receptors (RXR) has the ability to direct lineage determination but has yielded disappointing results in promoting skeletal myogenesis in embryonic stem (ES) cells. RXR is crucial in embryonic development although it is generally considered to act as a silent partner for other nuclear receptors such as RAR. Our findings demonstrate that rexinoid specific signaling enhances skeletal myogenesis and requires β-catenin but not RAR. Moreover, RXR signalling in mouse ES cells can efficiently enhance skeletal myogenesis and closely recapitulates sequential events observed in vivo. Since ES cells closely represent the properties of the developing embryo, efficiently generating skeletal muscle provides a means to further scrutinize signaling pathways in myogenic development in view of developing therapies for muscle related diseases.

Pluripotent stem cells have the capacity to develop into different cell lineages and can be manipulated into certain cell types through the use of small molecule inducers. Retinoic acid (RA) signaling through retinoic acid receptors (RAR) and retinoid X receptors (RXR) has the ability to direct lineage determination but has yielded disappointing results in promoting skeletal myogenesis in embryonic stem (ES) cells. RXR is crucial in embryonic development although it is generally considered to act as a silent partner for other nuclear receptors such as RAR. Our findings demonstrate that rexinoid specific signaling enhances skeletal myogenesis and requires β-catenin but not RAR. Moreover, RXR signalling in mouse ES cells can efficiently enhance skeletal myogenesis and closely recapitulates sequential events observed in vivo. Since ES cells closely represent the properties of the developing embryo, efficiently generating skeletal muscle provides a means to further scrutinize signaling pathways in myogenic development in view of developing therapies for muscle related diseases.

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.

Hämatopoetische Stammzellen besitzen die Fähigkeit, die dauerhafte Erhaltung ihrer eigenen Population im Knochenmark zu gewährleisten und gleichzeitig zur Neubildung der verschiedenen Zelltypen des peripheren Blutes beizutragen. Die Sequenz von Entscheidungsprozessen, die den Übergang einer undifferenzierten Stammzelle in eine funktionale ausgereifte Zelle beschreibt, wird als Linienspezifikation bezeichnet. Obwohl viele Details zu den molekularen Mechanismen dieser Entscheidungsprozesse mittlerweile erforscht sind, bestehen noch immer große Unklarheiten, wie die komplexen phänotypischen Veränderungen hervorgerufen und reguliert werden. Im Rahmen dieser Dissertation wird ein geeignetes mathematisches Modell der Linienspezifikation hämatopoetischer Stammzellen entwickelt, welches dann in ein bestehendes Modell der hämatopoetischen Stammzellorganisation auf Gewebsebene integriert wird. Zur Verifizierung des theoretischen Modells werden Simulationsergebnisse mit verschiedenen experimentellen Daten verglichen. Der zweite Teil dieser Arbeit konzentriert sich auf die Beschreibung und Analyse der Entwick- lungsprozesse von Einzelzellen, die aus diesem integrierten Modell hervorgehen. Aufbauend auf den entsprechenden Modellsimulationen wird dazu eine topologische Charakterisierung der resultierenden zellulären Genealogien etabliert, welche durch verschiedener Maße für deren Quantifizierung ergänzt wird. Das vorgestellte mathematische Modell stellt eine neuartige Verknüpfung der intrazellulären Linienspezifikation mit der Beschreibung der hämatopoetischen Stammzellorganisation auf Populationsebene her. Dadurch wird das Stammzellm- odell von Röder und Löffler um die wichtige Dimension der Linienspezifikation ergänzt und damit in seinem Anwendungsbereich deutlich ausgedehnt. Durch die Analyse von Einzelzellverläufen trägt das Modell zu einem grundlegenden Verständnis der inhärenten Heterogenität hämatopoetischer Stammzellen bei.

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