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Haematopoiesis during embryonic development
S4
Invited Speakers/ Experimental Hematology 41 (2013) S2–S9
S1009 - THE INTRINSIC APOPTOSIS CASPASE CASCADE REGULATES HEMATOPOIETIC STEM CELL HOMEOSTASIS AND FUNCTION Benjamin Kile Cancer and Hematology Division, The Walter and Eliza Hall Institute, Parkville, Victoria, Australia Since the demonstration that overexpression of pro-survival Bcl-2 causes an expansion of hematopoietic stem cell (HSC) number (Domen et al. 2000 J Exp Med), it has become accepted that apoptotic death is a common fate for HSCs. Apoptosis is believed to be critical for regulating the size of the HSC pool. Consistent with this, mice lacking Caspase-3 were reported to have increased numbers of HSCs (Janzen et al. 2008 Cell Stem Cell). This, however, was ascribed to perturbations in cytokine signaling, rather than impaired HSC apoptosis. To examine the role of the intrinsic apoptosis pathway in HSCs in more detail, we generated bone marrow chimeras lacking the pro-apoptotic proteins Bak/ Bax, Apaf-1, Caspase-9 or Caspase-3/7. Surprisingly, loss of Bak and Bax - the essential mediators of the intrinsic pathway - had little impact on HSC number. In contrast (but consistent with Janzen et al.) bone marrow chimeras lacking the downstream effectors Apaf-1, Caspase-9 or Caspase-3/7 exhibited a 10-fold expansion of the HSC compartment. Casp9-/- bone marrow exhibited an impaired ability to reconstitute irradiated recipients. Thus, apoptosis mediated by Bak and Bax is dispensable for HSC homeostasis, whereas, the function of the apoptotic caspase cascade is not. This raised the questions 1) Is the HSC expansion in Caspase-9-deficient mice intrinsic to HSCs? 2) Is the function of the apoptotic caspase cascade in HSCs to promote cell death? To answer them, we firstly generated bone marrow chimeras containing 50% wild-type and 50% Casp9-/- cells. In these animals, wild-type HSCs exhibited the same expansion and proliferation as Casp9-/- HSCs. Thus, the defect in Caspase-9 deficient mice is the result of HSC extrinsic factors. We then tested the relationship between Caspase-9-deficient HSC expansion and Bak/Bax-mediated apoptosis by generating Bak-/- Bax-/- Casp9-/- mice. Deletion of Bak/ Bax rescued the HSC phenotype in Caspase-9 deficient mice. Together, these data suggest that the intrinsic caspase cascade is essential for normal Bak/Bax-mediated cell death in the hematopoietic system, and in its absence, aberrant cell death feeds back to drive HSC expansion and dysfunction. In support of this notion, we found evidence of up-regulated type I interferon signaling in the HSC compartment. Our study demonstrates that Bak/Bax-mediated cell death of HSCs is dispensable for the maintenance of the HSC pool at steady state. This raises the question of whether HSCs die via alternative cell death pathways, or, whether their death is less frequent than previously believed.
S1011 - ASYMMETRIC CELL DIVISION AND SPINDLE ORIENTATION IN NEURAL STEM CELLS - FROM DROSOPHILA TO HUMANS J€ urgen Knoblich IMBA, Vienna, Austria When we think of mitosis, we commonly have a process in mind where a cell gives rise to two identical daughter cells. In whole organisms, however, many cell divisions are actually asymmetric and give rise to two daughter cells of different size, shape or developmental fate. Asymmetric cell divisions are particularly important in stem cells, as they allow those cells to generate both self-renewing and differentiating daughter cells, an ability that is common to all stem cells. We therefore use stem cells in the developing brain of both fruitflies and mice as a model to understand the principle mechanisms that regulate and orient asymmetric cell divisions. More recently, we have extended our efforts to mammalian model systems, where mutations in regulators of basic cell biological processes like the orientation of the mitotic spindle are known to cause strong brain malformations resulting in severe mental retardation. As recent experiments have shown striking differences between human and mouse brain development, we have made an effort to establish experimental strategies where those regulators and their effects on brain development can be studied in a human setting.
S1010 - IN VIVO IMAGING OF QUIESCENT AND PHYSIOLOGICALLY ACTIVATED HAEMATOPOIETIC STEM CELLS Cristina Lo Celso Life Sciences, Imperial College London, London, UK
S1012 - HAEMATOPOIESIS DURING EMBRYONIC DEVELOPMENT Samir Taoudi1,2 1 Molecular Medicine, Walter and Eliza Hall Institute, Melbourne, Victoria, Australia; 2 Medical Biology, University of Melbourne, Melbourne, Victoria, Australia
Understanding the mechanisms linking stem cell-niche interaction and stem cell fate is critical for developing regenerative medicine approaches. The nature of such interactions between hematopoietic stem cells (HSC) and the bone marrow (BM) microenvironment has long been elusive due to the difficulty of penetrating bones for direct observation and the fluid nature of the hematopoietic tissue itself. Several functional studies based on ablating or over-expressing specific genes in the hematopoietic or distinct BM stroma compartments have highlighted the presence of an intricate and dynamic network of regulatory signals responsible for the crosstalk between HSC and the BM microenvironment. The question, however, remains open as to whether multiple, molecularly and functionally distinct HSC niches exist within the bone marrow and whether HSC trafficking between them may be necessary to switch fate between quiescence and proliferation, self-renewal and differentiation. To address this question, we developed an imaging technique combining two photon and confocal microscopy that allows in-vivo imaging of live transplanted hematopoietic stem and progenitor cells (HSPC) in mouse BM with single cell resolution. Using this technique we showed that engrafting long-term repopulating HSC (LT-HSC) localize near osteoblastic cells, while their progeny are more distal. Our results also highlight that localization of LT-HSC and their progeny near osteoblasts correlates with improved engraftment outcomes. Studies based on single time-point observations demonstrated that asynchronous HSPC proliferation initiates BM reconstitution, however did not provide information about long-term interactions between HSC and their BM niche (or niches), which are responsible for maintenance of balanced haematopoiesis. We therefore developed a new in vivo imaging experimental protocol, allowing time-lapse imaging of HSPC, leading us to uncover their differential abilities to engage with the BM microenvironment over time. Moreover, using a physiological model of HSC activation, we observed that changes in the nature of the interactions between stem cells and the BM microenvironment accompany switches in fate choice.
Mature blood cells have multiple functions ranging from transporting oxygen around the body to protecting us from infection. The ability to continue producing enough blood cells throughout life is critical to our health. This process is known as haematopoiesis. At the foundation of blood cell production is the haematopoietic progenitor/ stem cell (HSCs); these cell types are not specialised for any function except to ensure that when necessary large mature blood cells can be readily generated. In the adult, the ability to continuously generate mature blood whilst preserving sufficient numbers of progenitor/stem cells is ensured by a process known as self-renewal - a question that remains unanswered is how these cells are produced in the first place. Haematopoietic development in the embryo occurs in a sequential process, during which primitive erythropoiesis and progenitor formation occurs in the yolk sac. Once this first wave of haematopoiesis is established HSCs are then formed; this is a complex multi-site process involving the AGM region, yolk sac and placenta. During foetal life the majority of HSCs reside within the liver, where HSCs continue to expand and initiate definitive haematopoiesis (a sustained haematopoietic system driven by a self-renewing HSC). The embryo provides an invaluable resource for tackling the problems central to haematopoiesis research, particularly the complex problem of how concomitant HSC self-renewal and hematopoietic differentiation can be achieved. I will be discussing how our laboratory is teasing apart these processes, with particular focus on identifying when specification of early hematopoietic lineages is first detectable. Biography (250 word limit): In 2002 Samir joined the laboratory of Professor Alexander Medvinsky at the Institute for Stem Cell Research (Edinburgh), initially as a PhD student and then as a post-doctoral researcher. During this time Samir investigated the process of haematopoietic stem cell formation during mouse embryogenesis
Invited Speakers/ Experimental Hematology 41 (2013) S2–S9
S1009 - THE INTRINSIC APOPTOSIS CASPASE CASCADE REGULATES HEMATOPOIETIC STEM CELL HOMEOSTASIS AND FUNCTION Benjamin Kile Cancer and Hematology Division, The Walter and Eliza Hall Institute, Parkville, Victoria, Australia Since the demonstration that overexpression of pro-survival Bcl-2 causes an expansion of hematopoietic stem cell (HSC) number (Domen et al. 2000 J Exp Med), it has become accepted that apoptotic death is a common fate for HSCs. Apoptosis is believed to be critical for regulating the size of the HSC pool. Consistent with this, mice lacking Caspase-3 were reported to have increased numbers of HSCs (Janzen et al. 2008 Cell Stem Cell). This, however, was ascribed to perturbations in cytokine signaling, rather than impaired HSC apoptosis. To examine the role of the intrinsic apoptosis pathway in HSCs in more detail, we generated bone marrow chimeras lacking the pro-apoptotic proteins Bak/ Bax, Apaf-1, Caspase-9 or Caspase-3/7. Surprisingly, loss of Bak and Bax - the essential mediators of the intrinsic pathway - had little impact on HSC number. In contrast (but consistent with Janzen et al.) bone marrow chimeras lacking the downstream effectors Apaf-1, Caspase-9 or Caspase-3/7 exhibited a 10-fold expansion of the HSC compartment. Casp9-/- bone marrow exhibited an impaired ability to reconstitute irradiated recipients. Thus, apoptosis mediated by Bak and Bax is dispensable for HSC homeostasis, whereas, the function of the apoptotic caspase cascade is not. This raised the questions 1) Is the HSC expansion in Caspase-9-deficient mice intrinsic to HSCs? 2) Is the function of the apoptotic caspase cascade in HSCs to promote cell death? To answer them, we firstly generated bone marrow chimeras containing 50% wild-type and 50% Casp9-/- cells. In these animals, wild-type HSCs exhibited the same expansion and proliferation as Casp9-/- HSCs. Thus, the defect in Caspase-9 deficient mice is the result of HSC extrinsic factors. We then tested the relationship between Caspase-9-deficient HSC expansion and Bak/Bax-mediated apoptosis by generating Bak-/- Bax-/- Casp9-/- mice. Deletion of Bak/ Bax rescued the HSC phenotype in Caspase-9 deficient mice. Together, these data suggest that the intrinsic caspase cascade is essential for normal Bak/Bax-mediated cell death in the hematopoietic system, and in its absence, aberrant cell death feeds back to drive HSC expansion and dysfunction. In support of this notion, we found evidence of up-regulated type I interferon signaling in the HSC compartment. Our study demonstrates that Bak/Bax-mediated cell death of HSCs is dispensable for the maintenance of the HSC pool at steady state. This raises the question of whether HSCs die via alternative cell death pathways, or, whether their death is less frequent than previously believed.
S1011 - ASYMMETRIC CELL DIVISION AND SPINDLE ORIENTATION IN NEURAL STEM CELLS - FROM DROSOPHILA TO HUMANS J€ urgen Knoblich IMBA, Vienna, Austria When we think of mitosis, we commonly have a process in mind where a cell gives rise to two identical daughter cells. In whole organisms, however, many cell divisions are actually asymmetric and give rise to two daughter cells of different size, shape or developmental fate. Asymmetric cell divisions are particularly important in stem cells, as they allow those cells to generate both self-renewing and differentiating daughter cells, an ability that is common to all stem cells. We therefore use stem cells in the developing brain of both fruitflies and mice as a model to understand the principle mechanisms that regulate and orient asymmetric cell divisions. More recently, we have extended our efforts to mammalian model systems, where mutations in regulators of basic cell biological processes like the orientation of the mitotic spindle are known to cause strong brain malformations resulting in severe mental retardation. As recent experiments have shown striking differences between human and mouse brain development, we have made an effort to establish experimental strategies where those regulators and their effects on brain development can be studied in a human setting.
S1010 - IN VIVO IMAGING OF QUIESCENT AND PHYSIOLOGICALLY ACTIVATED HAEMATOPOIETIC STEM CELLS Cristina Lo Celso Life Sciences, Imperial College London, London, UK
S1012 - HAEMATOPOIESIS DURING EMBRYONIC DEVELOPMENT Samir Taoudi1,2 1 Molecular Medicine, Walter and Eliza Hall Institute, Melbourne, Victoria, Australia; 2 Medical Biology, University of Melbourne, Melbourne, Victoria, Australia
Understanding the mechanisms linking stem cell-niche interaction and stem cell fate is critical for developing regenerative medicine approaches. The nature of such interactions between hematopoietic stem cells (HSC) and the bone marrow (BM) microenvironment has long been elusive due to the difficulty of penetrating bones for direct observation and the fluid nature of the hematopoietic tissue itself. Several functional studies based on ablating or over-expressing specific genes in the hematopoietic or distinct BM stroma compartments have highlighted the presence of an intricate and dynamic network of regulatory signals responsible for the crosstalk between HSC and the BM microenvironment. The question, however, remains open as to whether multiple, molecularly and functionally distinct HSC niches exist within the bone marrow and whether HSC trafficking between them may be necessary to switch fate between quiescence and proliferation, self-renewal and differentiation. To address this question, we developed an imaging technique combining two photon and confocal microscopy that allows in-vivo imaging of live transplanted hematopoietic stem and progenitor cells (HSPC) in mouse BM with single cell resolution. Using this technique we showed that engrafting long-term repopulating HSC (LT-HSC) localize near osteoblastic cells, while their progeny are more distal. Our results also highlight that localization of LT-HSC and their progeny near osteoblasts correlates with improved engraftment outcomes. Studies based on single time-point observations demonstrated that asynchronous HSPC proliferation initiates BM reconstitution, however did not provide information about long-term interactions between HSC and their BM niche (or niches), which are responsible for maintenance of balanced haematopoiesis. We therefore developed a new in vivo imaging experimental protocol, allowing time-lapse imaging of HSPC, leading us to uncover their differential abilities to engage with the BM microenvironment over time. Moreover, using a physiological model of HSC activation, we observed that changes in the nature of the interactions between stem cells and the BM microenvironment accompany switches in fate choice.
Mature blood cells have multiple functions ranging from transporting oxygen around the body to protecting us from infection. The ability to continue producing enough blood cells throughout life is critical to our health. This process is known as haematopoiesis. At the foundation of blood cell production is the haematopoietic progenitor/ stem cell (HSCs); these cell types are not specialised for any function except to ensure that when necessary large mature blood cells can be readily generated. In the adult, the ability to continuously generate mature blood whilst preserving sufficient numbers of progenitor/stem cells is ensured by a process known as self-renewal - a question that remains unanswered is how these cells are produced in the first place. Haematopoietic development in the embryo occurs in a sequential process, during which primitive erythropoiesis and progenitor formation occurs in the yolk sac. Once this first wave of haematopoiesis is established HSCs are then formed; this is a complex multi-site process involving the AGM region, yolk sac and placenta. During foetal life the majority of HSCs reside within the liver, where HSCs continue to expand and initiate definitive haematopoiesis (a sustained haematopoietic system driven by a self-renewing HSC). The embryo provides an invaluable resource for tackling the problems central to haematopoiesis research, particularly the complex problem of how concomitant HSC self-renewal and hematopoietic differentiation can be achieved. I will be discussing how our laboratory is teasing apart these processes, with particular focus on identifying when specification of early hematopoietic lineages is first detectable. Biography (250 word limit): In 2002 Samir joined the laboratory of Professor Alexander Medvinsky at the Institute for Stem Cell Research (Edinburgh), initially as a PhD student and then as a post-doctoral researcher. During this time Samir investigated the process of haematopoietic stem cell formation during mouse embryogenesis