Molecular Mechanisms of Hematopoietic Stem Cell Development- The Role of Retinoic Acid Signaling Bhaskar Chanda For the Doctor of Philosophy Medical Biophysics University of Toronto 2013 Abstract During mouse embryonic development, the formation of blood or hematopoiesis occurs in multiple phases. The first phase or primitive hematopoiesis generates a restricted subset of blood cell lineages but is devoid of lymphoid and hematopoietic stem cell (HSC) potential. The next phase of hematopoiesis, also known as definitive hematopoiesis, is characterized by its ability to generate multilineage hematopoietic progenitors and HSCs from a specialized population of endothelial cells known as hemogenic endothelium (HE). Such endothelial to hematopoietic transitions (EHT) have been recently observed at a clonal level, however, molecular mechanisms that underlie EHT leading to the specification of HSCs have remained poorly understood. Here we show that retinoic acid (RA) signaling plays a pivotal role in embryonic hematopoiesis and HSC development. RA signaling inhibits primitive hematopoiesis, and promotes definitive hematopoiesis. This inductive effect of RA signaling extends to the specification of HSCs. Activation of the RA signaling pathway ex vivo in AGM-derived HE dramatically enhanced the repopulating potential, whereas its conditional inhibition in vivo abrogated HSC development. These repressive and inductive effects of RA signaling were mediated primarily via retinoic acid receptor (RAR)- α. We further analyzed the mechanistic basis of RA signaling with a combined use of cellular, molecular and biochemical assays, and show that β-catenin dependent Wnt signaling is the downstream mediator of RA signaling. Collectively, this thesis provides new insight into molecular mechanisms that control embryonic hematopoiesis and identify the RA pathway as a key regulator of definitive hematopoiesis and HSC specification.

The promyelocytic leukaemia protein (PML) is a tumour suppressor initially identified in acute promyelocytic leukaemia (APL). In APL, PML and the retinoic acid receptor alpha (RARalpha) genes are fused as a consequence of the translocation t(15;17). The product of the chimeric gene is the oncogenic PML-RARalpha protein. The PML gene encodes multiple nuclear and cytoplasmic isoforms. PML nuclear isoforms (nPML) are the main components of the PML nuclear bodies (PML-NBs), sub-nuclear structures involved in the modulation of essential cellular players including the tumour suppressor p53. Nuclear PML has been intensively studied, while, the role of cytoplasmic PML remains poorly understood. Increasing evidence indicates that PML could bear cytoplasmic functions in both physiological and pathological settings. This study aims to gain more insights into the function of PML and PML-RARalpha cytoplasmic pool of proteins. Recently, two missense mutations resulting in truncated PML cytoplasmic protein (Mut PML) have been identified in aggressive APL cases. We found that Mut PML alters the structure and the function of the PML-NB mainly through the cytoplasmic relocation of nPML. Remarkably, Mut PML inhibits p53 transcriptional, growth suppressive and apoptotic functions. In the cytoplasm, Mut PML interacts and stabilizes PML-RARalpha, thus potentiating its block of RA-induced transcription and differentiation. A mutant of PML-RARalpha (Delta2) accumulating in the cytoplasm is able to inhibit RA-dependent transcription and differentiation, suggesting that cytoplasmic localization of PML-RARalpha may contribute to transformation. Finally, we found that Delta2 expression blocks G-CSF-dependent myeloid differentiation and causes partial transformation of primary haematopoietic progenitor cells.

Resumo: O objetivo deste estudo foi avaliar o papel do ácido retinóico na remodelação cardíaca induzida pela exposição à fumaça do cigarro. Os ratos foram divididos em três grupos: controle (C, n = 8); expostos animais expostos à fumaça do cigarro (EFC, n = 9); animais expostos à fumaça de cigarro e suplementados com ácido retinóico (EFC-AR, n = 9). Após dois meses, foram submetidos ao ecocardiograma e análise morfométrica. Os dados estão expressos em média ± desvio padrão ou medianas e intervalo interquartílico (Q1-Q3). Não houve diferença na pressão sistólica caudal entre os grupos (C = 116 ± 27 mmHg; EFC = 129 ± 17 mmHg; EFC-AR = 139 ± 19 mmHg; p = 0,102). O grupo EFC mostrou maior diâmetro diastólico do ventrículo esquerdo ajustado ao peso corporal (C = 18,42 ± 3,57 mm/kg; EFC = 23 ± 1,85 mm/kg; EFC-AR = 19,51 ± 0,99 mm/kg; p = 0,001) e sistólico ajustado ao peso corporal (C = 8,25 ± 2,16 mm/kg; EFC = 11,53 ± 1,31 mm/kg; EFC-AR = 8,25 ± 0,71 mm/kg; p = <0,001) quando comparado com C grupo e EFC-AR. Por outro lado, não houve diferença entre C e EFC-AR. A espessura da parede do ventrículo esquerdo ajustado ao diâmetro diastólico do ventrículo esquerdo foi maior no grupo C do que nos EFC (C = 0,20 (0,18-0,23); EFC = 0,15 (0,14-0,18); EFC-AR = 0,19 (0,17-0,22; p = 0,003). A fração de ejeção (C = 0,91 ± 0,02; EFC = 0,87 ± 0,03; EFC-AR = 0,92 ± 0,03; p = 0,003) e a fração de encurtamento (C = 55,77 ± 4,41%; EFC = 49,73 ± 4,43%; EFC-AR = 57,60 ± 5,15%; p = 0,005) foram mais elevados nos animais do grupo C e EFC-AR em relação aos animais do grupo EFC. Por outro lado, não houve diferenças entre C e EFC-AR. Além disso, o tabagismo está associado com um aumento significativo da área seccional do miócito (C = 294 ± 21 μm2; EFC = 347 ± 44 μm2; EFC-AR = 310 ± 37 μm2; p = 0,016). Por outro lado... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The objective of this study was to evaluate the role of retinoic acid in the myocardial remodeling induced by tobacco smoke exposure. Rats were allocated into three groups: control (C, n=8); exposed to tobacco smoke (ETS, n=9); exposed to tobacco smoke and all-transretinoic acid (ETS-AR, n = 9). After two months, were submitted to echocardiogram, and morphometric analyses. Data are expressed as mean ± SD or medians (including the lower quartile and upper quartile). There were no differences in the tail systolic pressure among the groups (C = 116 ± 27 mmHg, ETS = 129 ± 17 mmHg, ETS-AR = 139 ± 19 mmHg; p = 0,102). ETS showed higher normalized left ventricular diastolic (C = 18,42 ± 3,57 mm/kg, ETS = 23 ± 1,85 mm/kg, ETS-AR = 19,51 ± 0,99 mm/kg; p = 0,001) and systolic (C = 8,25 ± 2,16 mm/kg, ETS = 11,53 ± 1,31 mm/kg, ETS-AR = 8,25 ± 0,71mm/kg; p = <0,001) diameters than C and ETS-AR. On the other hand, there were no differences between C and ETS-RA. LVWT/LVEDD was higher in C than ETS (C = 0,20 (0,18-0,23), ETS = 0,15 (0,14-0,18), ETS-RA = 0,19 (0,17-0,22; p = 0,030). The ejection fraction (C = 0,91 ± 0,02, ETS = 0,87 ± 0,03, ETS-AR = 0,92 ± 0,03; p = 0,003) and fractional shortening (C = 55,77 ± 4,41 %, ETS = 49,73 ± 4,43 %, ETS-AR = 57,60 ± 5,15 %; p = 0,005) were higher in C and ETS-RA animals than ETS animals. On the other hand, there were no differences between C and ETS-RA. Also, smoking was associated with significantly increased myocyte cross-sectional area (C = 294 ± 21 μm2, ETS = 347 ± 44 μm2, ETS-RA = 310 ± 37 μm2; p = 0,016). On the other hand, there were no differences between C and ETS-RA. Retinoic acid did not affect other functional or morphological variables. In conclusion, ...(Complete abstract click electronic access below) / Orientador: Leonardo A. M. Zornoff / Coorientador: Sergio A. R. de Paiva / Banca: Paula Schmidt Azevedo Gaiolla / Banca: Wilson Nadruz Junior / Mestre

A common characteristic in neurodegenerative diseases of the brain is death of specific neuronal populations. The lack of neuron proliferation and axon extension in most parts of the central nervous system leads to chronic loss of neurons in the case of injury or disease. Therefore it is essential to identify signals involved in neurogenesis and neuronal survival. A favorable model in which to study these events is the olfactory sensory neurons in the main olfactory epithelium and their target in the glomeruli of the olfactory bulb. In spite of constant regeneration, each olfactory sensory neuron maintain expression of one particular odorant receptor and the specificity of their axonal projections to the glomeruli. Most mammals also have an accessory olfactory system consisting of the sensory neurons in the vomeronasal epithelium and their target area the accessory olfactory bulb. Differential expression of receptors and other genes divides the olfactory and vomeronasal epithelium into zones, but the function and mechanisms underlying the establishment of these zones are still elusive. We identified four genes with graded expression patterns that correlated with the zones of the olfactory epithelium. One of the identified genes encodes a retinoic acid synthesizing enzyme, RALDH-2. We showed that RALDH-2 was expressed in a gradient in cells of the lamina propria underneath the olfactory epithelium, suggesting a possible retinoic acid regulation of zonally expressed genes in the olfactory epithelium. To investigate the role of retinoic acid in the olfactory systems, we generated a transgenic mouse strain that selectively expressed a dominant negative retinoic acid receptor in mature olfactory and vomeronasal neurons. We found that subsequent to the establishment of axonal projections, the neurons of both olfactory systems died prematurely by retrograde caspase-3 activation. In the main olfactory system the onset of apoptosis was associated with the appearance of incorrect heterogenous glomeruli with axons of more than one OR identity. Additionally, the activity regulated cell adhesion molecule kirrel-2 was down regulated suggesting an additional regulation of this gene by retinoic acid. Deficient retinoic acid signaling in olfactory sensory neurons could thus induce apoptosis by changing the parameters for axonal competition by neural activity and kirrel-2 expression. We found evidence for a selective neuronal death in the accessory olfactory system of the dnRAR mice, where only vomeronasal sensory neurons belonging to the basal zone died by retrograde caspase-3 activation. This implies that the two populations of sensory neurons in the vomeronasal epithelium differently depend on retinoic acid for their survival.

Although numerous transcription factors (TFs) are expressed by oligodendrocytes (OGs), the role(s) of most of these TFs in oligodendrogenesis remains to be elucidated. One such TF is Hoxa2, which was recently shown to be expressed by O4-positive (+) pro-OGs. Hence, the main objectives of this thesis were to determine the expression profile and function(s) of Hoxa2 during OG development. Immunocytochemical analysis of primary mixed glial cultures demonstrated that Hoxa2 is expressed throughout oligodendrogenesis, diminishing only with the acquisition of a myelinating phenotype. Subsequently, immunohistochemical analysis suggested that Hoxa2 is expressed by migratory oligodendroglial cells in the embryonic spinal cord. However, double immunofluorescent analysis of Hoxa2 transgenic knockout mice showed that OG specification and early maturation proceed normally in the absence of Hoxa2 in the spinal cord. As Hoxa2 is one of 39 murine Hox genes, which exhibit extensive overlapping expression profiles in the spinal cord, we decided to examine the expression of an additional Hox TF, Hoxb4, during OG development. Immunocytochemical analysis of primary mixed glial cultures demonstrated that Hoxb4 is also expressed throughout OG development. Furthermore, comparison of the expression profiles of Hoxb4 and Olig2 suggested that Hoxb4 is expressed by oligodendroglial cells in the spinal cord. Hence, Hoxb4, as well as other Hox TFs could compensate for Hoxa2 in the spinal cord in its absence. As the anterior boundary of most Hox genes has been found to be in the hindbrain or spinal cord, we decided to look at the telencephalon which would be less likely to have compensatory mechanisms. Our results showed that similar to the spinal cord, Hoxa2 is expressed by oligodendroglial cells in the telencephalon. Subsequently, it was found that over-expressing Hoxa2 in CG4 cells, an oligodendroglial cell line derived from the perinatal rat cerebral cortex, impairs their differentiation. In an attempt to determine the mechanism by which it accomplishes this, we examined the expression of polysialylated neural cell adhesion molecule (PSA-NCAM), which has been implicated in this process. Contrary to our expectations, however, it was found that over-expressing Hoxa2 in CG4 cells results in significantly fewer PSA-NCAM+ cells. Hence, the results suggest that Hoxa2s effect on OG differentiation is independent of its effect on PSA-NCAM expression. The expression of Hox genes is enhanced by retinoic acid (RA), which, in turn, both inhibits, as well as promotes OG differentiation. Although the reason for these opposing roles is uncertain, examination of the experimental protocols utilized by different research groups reveals disparities in age, CNS region, as well as RA concentration. As a result, RAs effect on oligodendrogenesis could be stage- and/or concentration-dependent. In order to determine which of these factors could impact RAs effect on OG differentiation we treated CG4 cells with two different concentrations of RA at two distinct time points. The results showed that both factors (concentration and time/stage) can impact RAs effect on CG4 cell differentiation. In an attempt to determine the mechanism by which it accomplishes this, we examined the expression of PSA-NCAM. Contrary to our expectations, the results suggest that RAs effect on CG4 differentiation is independent of its effect on PSA-NCAM expression. The results of this thesis suggest that Hoxa2 and RA could play multiple roles in OG development. Although these roles appear to be similar, further research will be needed to determine whether Hoxa2 acts a downstream effector in the RA signaling pathway in oligodendroglial cells.

Epigenetics continues to be redefined and new discoveries are likely to revolutionise the field still further. This thesis explores different aspects of how epigenetic regulation of gene expression contributes to human disease. Paper I explores the function of the IKKα kinase in regulating gene expression through the nuclear retinoic acid receptor (RAR). We define a set of genes requiring IKKα for their expression and found recruitment of IKKα to the RAR dependent on structural motifs in its protein sequence. This interplay between the NFκB pathway and nuclear receptor regulated transcription is important to consider when designing therapeutic strategies. Papers II and III focus on the plasma cell malignancy multiple myeloma (MM) and define a gene regulatory circuit defining an underexpressed gene profile in MM dependent on the Polycomb proteins. We provide proof-of-principle that the use of small chemical inhibitors may be operational in reactivating genes silenced by H3K27me3 and that this leads to decreased tumour load and increased survival in the 5T33 in vivo model of MM. We explored the genome-wide distribution of H3K27me3 and H3K4me3, and defined their association with gene expression in freshly-isolated malignant plasma cells from MM patients. Importantly, H3K27me3-marked genes in MM associated with more aggressive stages of the disease and less favourable survival. We present evidence that gene targeting by H3K27me3 is likely to not only involve a small population of tumour cells, but rather represent a common MM profile and further provide a rationale for evaluating epigenetic therapeutics in MM. Paper IV shows that pro-inflammatory gene expression in monocytes of psychiatric patients can be induced in vitro by sodium pump inhibitors, as the steroid hormone ouabain. We suggest that the ouabain-induced gene expression is regulated by an intricate network involving microRNAs, Polycomb and the H3K27me3 demethylase JMJD3. Our data indicates that epigenetic regulators play a role in transmitting cues between intrinsic and/extrinsic stimuli and gene expression in psychiatric illness. This thesis provides novel insights on how seemingly unrelated pathways may converge on transcriptional regulation and evidence that epigenetic modifiers contribute to the pathogenesis of human complex diseases such as multiple myeloma and mood disorders.

Although numerous transcription factors (TFs) are expressed by oligodendrocytes (OGs), the role(s) of most of these TFs in oligodendrogenesis remains to be elucidated. One such TF is Hoxa2, which was recently shown to be expressed by O4-positive (+) pro-OGs. Hence, the main objectives of this thesis were to determine the expression profile and function(s) of Hoxa2 during OG development. Immunocytochemical analysis of primary mixed glial cultures demonstrated that Hoxa2 is expressed throughout oligodendrogenesis, diminishing only with the acquisition of a myelinating phenotype. Subsequently, immunohistochemical analysis suggested that Hoxa2 is expressed by migratory oligodendroglial cells in the embryonic spinal cord. However, double immunofluorescent analysis of Hoxa2 transgenic knockout mice showed that OG specification and early maturation proceed normally in the absence of Hoxa2 in the spinal cord. As Hoxa2 is one of 39 murine Hox genes, which exhibit extensive overlapping expression profiles in the spinal cord, we decided to examine the expression of an additional Hox TF, Hoxb4, during OG development. Immunocytochemical analysis of primary mixed glial cultures demonstrated that Hoxb4 is also expressed throughout OG development. Furthermore, comparison of the expression profiles of Hoxb4 and Olig2 suggested that Hoxb4 is expressed by oligodendroglial cells in the spinal cord. Hence, Hoxb4, as well as other Hox TFs could compensate for Hoxa2 in the spinal cord in its absence. As the anterior boundary of most Hox genes has been found to be in the hindbrain or spinal cord, we decided to look at the telencephalon which would be less likely to have compensatory mechanisms. Our results showed that similar to the spinal cord, Hoxa2 is expressed by oligodendroglial cells in the telencephalon. Subsequently, it was found that over-expressing Hoxa2 in CG4 cells, an oligodendroglial cell line derived from the perinatal rat cerebral cortex, impairs their differentiation. In an attempt to determine the mechanism by which it accomplishes this, we examined the expression of polysialylated neural cell adhesion molecule (PSA-NCAM), which has been implicated in this process. Contrary to our expectations, however, it was found that over-expressing Hoxa2 in CG4 cells results in significantly fewer PSA-NCAM+ cells. Hence, the results suggest that Hoxa2s effect on OG differentiation is independent of its effect on PSA-NCAM expression. The expression of Hox genes is enhanced by retinoic acid (RA), which, in turn, both inhibits, as well as promotes OG differentiation. Although the reason for these opposing roles is uncertain, examination of the experimental protocols utilized by different research groups reveals disparities in age, CNS region, as well as RA concentration. As a result, RAs effect on oligodendrogenesis could be stage- and/or concentration-dependent. In order to determine which of these factors could impact RAs effect on OG differentiation we treated CG4 cells with two different concentrations of RA at two distinct time points. The results showed that both factors (concentration and time/stage) can impact RAs effect on CG4 cell differentiation. In an attempt to determine the mechanism by which it accomplishes this, we examined the expression of PSA-NCAM. Contrary to our expectations, the results suggest that RAs effect on CG4 differentiation is independent of its effect on PSA-NCAM expression. The results of this thesis suggest that Hoxa2 and RA could play multiple roles in OG development. Although these roles appear to be similar, further research will be needed to determine whether Hoxa2 acts a downstream effector in the RA signaling pathway in oligodendroglial cells.

Dendritic cells (DCs) in different compartments can affect tolerance via distinct mechanisms. Thus, retinoid acid (RA) and integrins expressed by CD103+ dendritic cells in the gut play important roles in regional regulatory T cell induction and trafficking, while IL-10 expression by lung-associated tolerogenic dendritic cells is integral to tolerance in that compartment. Whether RA- and IL-10-differentiated DC (DCRA and DC10, respectively) can reciprocally induce tolerance in either compartment remains largely unexplored. We have shown that DC10 induce asthma tolerance in part by activating CD25+Foxp3+ Treg, but also by recruiting other cells (e.g., endogenous pulmonary DC) into an infectious tolerance pathway. Herein we began to assess whether DCRA can be equally tolerogenic, and whether they employ similar mechanisms, in an OVA/alum mouse model of asthma. On FACS analysis, we found that DCRA expressed significantly higher levels of CD40, CD86 and MHC II than DC10 (i.e., at levels equivalent to fully mature DC). DCRA secreted higher levels of TGF-β1 and IL-27 than DC10, but equivalent levels of IL-10. DCRA and DC10 suppressed in vitro Th2 response, but DCRA were more effective than DC10 at suppressing proliferation. Both DCRA and DC10 increased expression of Foxp3+ on effector T cells. DCRA promoted little expansion of Foxp3+ T cells. In contrast, DC10 promoted expansion of Foxp3+ T cells. Treatment of asthmatic mice with DC10 and DCRA reduced airway hyperresponsiveness and serum allergen-specific IgE and IgG1 levels. We previously showed that DC10-induced tolerance is critically dependent on their expression of IL-10. The results of this study showed that both DCRA and DC10 induce tolerance in asthmatic mice through different mechanisms.

Molecular Mechanisms of Hematopoietic Stem Cell Development- The Role of Retinoic Acid Signaling Bhaskar Chanda For the Doctor of Philosophy Medical Biophysics University of Toronto 2013 Abstract During mouse embryonic development, the formation of blood or hematopoiesis occurs in multiple phases. The first phase or primitive hematopoiesis generates a restricted subset of blood cell lineages but is devoid of lymphoid and hematopoietic stem cell (HSC) potential. The next phase of hematopoiesis, also known as definitive hematopoiesis, is characterized by its ability to generate multilineage hematopoietic progenitors and HSCs from a specialized population of endothelial cells known as hemogenic endothelium (HE). Such endothelial to hematopoietic transitions (EHT) have been recently observed at a clonal level, however, molecular mechanisms that underlie EHT leading to the specification of HSCs have remained poorly understood. Here we show that retinoic acid (RA) signaling plays a pivotal role in embryonic hematopoiesis and HSC development. RA signaling inhibits primitive hematopoiesis, and promotes definitive hematopoiesis. This inductive effect of RA signaling extends to the specification of HSCs. Activation of the RA signaling pathway ex vivo in AGM-derived HE dramatically enhanced the repopulating potential, whereas its conditional inhibition in vivo abrogated HSC development. These repressive and inductive effects of RA signaling were mediated primarily via retinoic acid receptor (RAR)- &alpha;. We further analyzed the mechanistic basis of RA signaling with a combined use of cellular, molecular and biochemical assays, and show that &beta;-catenin dependent Wnt signaling is the downstream mediator of RA signaling. Collectively, this thesis provides new insight into molecular mechanisms that control embryonic hematopoiesis and identify the RA pathway as a key regulator of definitive hematopoiesis and HSC specification.

Increased amyloid-β (Aβ) load in the brain, neurite degeneration, neuronal loss, and decreased levels of several neurotrophins are among the characteristics of Alzheimer’s disease (AD). Generation of Aβ occurs when the amyloid precursor protein (APP) is proteolytically processed by β- and γ-secretases in the amyloidogenic pathway. However, Aβ formation is prevented if APP is cleaved by α- and γ- secretases in the non-amyloidogenic pathway. The normal function of APP is still not fully known. It seems clear that the different fragments that are produced during proteolytic processing have different bioactive properties. APP and its metabolites have been implicated in neurite outgrowth, synaptogenesis, cell adhesion, neuroprotection and apoptosis. The aim of this thesis was to investigate how neurotrophic factors affect the synthesis and processing of APP and its two mammalian paralogues the APP-like protein-1 and-2 (APLP1 and APLP2). We also wanted to determine how the expression levels of α- and β- secretases were affected in response to these factors. In addition, we wanted to analyze if the levels and function of the most well characterized APP adaptor protein, Fe65, was regulated during neuronal differentiation. Our results show that retinoic acid (RA), insulin-like growth factor-1 (IGF-1), and brain derived neurotrophic factor (BDNF) all regulate expression levels and processing of the APP protein family. Interestingly, the increased processing of the APP family involves different signaling pathways. The PI3-K/Akt pathway is involved in IGF-1-induced APP and APLP1, but not APLP2, processing. In addition, RA-induced expression of the α-secretase, a disintegrin and metalloproteinase (ADAM) 10 is dependent on PI3-K, whereas PKC is involved in RA-induced expression of another α-secretase, ADAM17/TACE. Furthermore, we present evidence that maturation of the adaptor protein Fe65, as well as its docking to APP, increases concomitant with neuronal differentiation. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript.