Despite recognition of aldehyde dehydrogenase (Aldh) as a surrogate marker in isolating primitive hematopoietic stem and progenitor cells (HSPC) [1], its role in HSPC regulation, particularly during embryonic development, remains unclear. In this study, we examined the role of Aldh during embryonic hematopoiesis in zebrafish, which has emerged as a model for hematopoietic studies. [2] Wild--?type and transgenic [Tg(gata1:gfp),Tg(fli1:gfp)] zebrafish embryos were microinjected with anti--?sense morpholinos (MO) at 1--?cell to 4--?cell stage and evaluated by morphology, flow cytometry, in situ hybridization (ISH) and Q-RT-PCR. In addition, human CD34+ cells, which were enriched with hematopoietic stem cells (HSC), were isolated from umbilical cord blood samples for analysis of ALDH16A1 expression. It was subsequently compared with CD34- cells which were devoid of HSC activity. When aldh16a1 was knocked down by anti-sense morpholino (the embryos were referred herewith aldh16a1MO embryos), gene expression associated with erythropoiesis was significantly reduced at 18hpf .(gata1:0.70±0.03fold; p=0.002) (α-embryonic hemoglobin: 0.48±0.04fold; p=0.003) (β-embryonic hemoglobin: 0.56±0.03fold; p=0.001). Angiogenesis was also perturbed at 48 and 72hpf. Furthermore, human ALDH16A1 was significantly upregulated (4.79±1.00fold; p=0.00006) in CD34+ (enriched with HSC) as compared to CD34- (devoid of HSC) populations in umbilical cord blood. Aldh16a1 is important for the maintenance of primitive hematopoiesis at early (18hpf) and angiogenesis at later (48,72 hpf) embryonic stages. As angiogenesis plays an important role in pathophysiology of malignancies, novel therapy against ALDH16A1 might be exploited in therapeutic intervention in cancer treatment. Moreover, a specific role of zebrafish aldh16a1 in primitive erythropoiesis and a higher level of ALDH16A1 expression in human HSC-enriched cells suggested a conserved mechanism whereby ALDH regulates hematopoiesis. / published_or_final_version / Medicine / Master / Master of Research in Medicine

Zebrafish has emerged as an important vertebrate model for studying hematopoiesis and its genetic and chemical modifiers. The zebrafish embryos are unique in their optical transparency, ease of maintenance and high fecundity. They are also amendable to genetic and pharmacological perturbation at high throughput. As a result, the embryos are suitable for various experimental techniques and have a high efficiency in large-scale drug screening. Recently, zebrafish has also emerged as a model for the study of human disease. In this model organism, primitive hematopoiesis is transitory and it occurs in the intermediate cells mass and comprises primarily erythroid cells. Definitive hematopoiesis arises from the ventral wall of dorsal aorta and moves to the caudal hematopoietic tissues, thence the kidney, where life-long and multi-lineage differentiation occurs. The chemical screening platform comprises O-dianisidine staining for hemoglobin containing cells (erythroid) during primitive hematopoiesis. Positive hits were validated based on flow cytometry of dissociated transgenic Tg(gata1:GFP) embryos and whole-mount in-situ hybridization (WISH) for hematopoietic genes. Gene knock-down was conducted by morpholinos (MO) injected into zebrafish embryos at 1-4 cell stage and the effects on hematopoietic development evaluated by WISH and quantitative real-time PCR. Chemical screening of 74 compounds has been performed. These compounds were obtained from a chemical library comprising 879 compounds from NIH (National Institutes of Health) and pre-screened by their effects on cancer cell lines. Four compounds (Tin(IV), chlorotriphenyl [1-(4-ethoxyphenyl)- 3-cyanoureato]-hydrogen,triethylamine, Nogamycin, N,N-Dibenzyldaunorubicin hydrochloride and Allyl 2,3,4-tri-O-benzyl-6-O-(tert-butyldimethylsilyl)-α- D-gluco-Pyranoside) which significantly reduced O-dianisidine staining were identified of which one (Allyl 2,3,4-tri-O-benzyl-6-O-(tert-butyldimethylsilyl) -α-D-glucopyranoside was shown to reduce GFP+ population in Tg(gata1:GFP) population Another chemical (2-Propanol,1,1'-[(1-methylethylidene)bis(4, 1-phenyleneoxy)]bis[3-[(1,1,3,3-tetramethylbutyl)amino]-,dihydrochloride]) was shown to reduce c-myb (marker of definitive hematopoiesis) expression in the ventral wall of dorsal aorta. I also attempted gene knock in zebrafish embryos based on anti-sense morpholino microinjection. A gene encoding for arl4ab was examined, as it was shown to be expressed in hematopoietic tissue in zebrafish embryos but its function is entirely unknown. Knock-down of arl4ab significantly reduced c-myb and runx1 expression in the ventral wall of dorsal aorta and it can be reversed by co-injecting arl4ab mRNA. scl and gata1 expression as well as GFP expression in transgenic Tg(flk1:GFP) embryos that represented vascular development was unaffected. In summary, a zebrafish platform for the study of chemical and genetic modifiers was established. The results have provided important leads for further study into the mechanisms whereby these modifiers regulate hematopoiesis in the zebrafish embryos. / published_or_final_version / Medicine / Master / Master of Medical Sciences

Zebrafish has emerged as an important model for the study of embryonic hematopoiesis. It is a well-characterized model with numerous advantages. Large amount of embryos can be produced by a single pair of zebrafish and the optically transparent embryos allow direct visualization and manipulation of embryonic processes. Large-scale chemical screening using zebrafish embryos can be developed for robust screening of chemical libraries. The zebrafish hematopoiesis resembles that of mammals and occurs in two successive waves, primitive and definitive hematopoiesis. High-throughput read-outs are available to study the effects of different chemicals and genetic modifications on hematopoiesis. In first part of this study, an initial screening using O-dianisidine staining and whole-mount in-situ hybridization as read-out for chemicals that might perturb the regulation of hematopoiesis was conducted. Positive hit was further evaluated by flow cytometry of dissociated transgenic Tg(gata1:GFP) zebrafish embryos. A total of 50 compounds were screened from the "Mechanistic set" chemical libraries obtained from Developmental Therapeutics Program of the National Cancer Institute. One compound, "NSC 643834" was shown to reduce O-dianisidine staining at different concentrations tested. The second part of this study was performed to investigate the role of inca2 in zebrafish hematopoiesis. inca2 was found to be upregulated in chordin morphant zebrafish in which primitive hematopoiesis was expanded. The spatial expression of inca2 was examined by whole mount in-situ hybridization of embryos at different developmental stages. Furthermore the function of inca2 was investigated by gene knockdown using inca2 anti-sense morpholino. Primitive hematopoiesis was perturbed, suggesting that inca2 might play an important role in the regulation of this process. In conclusion, the present study demonstrated the distinct advantages and feasibility of using zebrafish as a platform of high throughput chemical screening and genetic manipulation. The result provided important ground to investigate the regulatory mechanisms of embryonic hematopoiesis. / published_or_final_version / Medicine / Master / Master of Research in Medicine

Epigenetics, or the reversible and heritable marks of gene regulation not including DNA sequence, encompasses modifications on both the DNA and histones and is as important as the DNA sequence itself. Gene transcription, DNA repair, DNA replication, and the cell cycle are each impacted by the chromatin structure. A variety of enzymes modulate these modifications, and a suite of factors interacts with them to aid in promoting or inhibiting cellular functions. Many of these chromatin-modifying factors are deregulated in cancer, making them novel therapeutic targets. This dissertation describes the identification of an H3K9 histone methyltransferase, SUV39H1, as a suppressor of rhabdomyosarcoma formation in zebrafish. This suppressor is dependent on the methyltransferase domain of the enzyme, ruling out any scaffold effects since this enzyme is a part of a multiprotein complex. SUV39H1-overexpressing and control tumors share many of the same characteristics, including proliferation rate, muscle differentiation state, and tumor growth rate. The tumor suppressive phenotype cannot be rescued by alterations in the downstream muscle program alone. However, SUV39H1-overexpressing fish initiate fewer tumors, which results in the observed suppressive phenotype. This initiation defect occurs between 5 and 7 days of life in the zebrafish, likely by impacting cyclin B1 expression. This dissertation also describes the development of a novel F1 transgenic screening strategy in the zebrafish. This approach was utilized to screen a variety of chromatin-modifying factors for their effects on hematopoietic development. The developed strategy will have future applications as a zebrafish screening tool. Our data suggest that chromatin-modifying factors play an important role in rhabdomyosarcoma and illustrate the use of the zebrafish in discovering genes involved in tumorigenesis and hematopoiesis.

The focus of this thesis is the role of ribosomal proteins in hematopoiesis and development. Ribosomal proteins are mutated in patients with Diamond Blackfan anemia (DBA). These mutations primarily affect blood tissues, as DBA patients have a macrocytic anemia. We have identified hematopoietic defects in zebrafish with a mutation in ribosomal protein S29 (rps29). \(Rps29^{-/-}\) embryos have defects in hematopoietic stem cell formation, aorta specification, and hemoglobinization. Embryos also have increased numbers of apoptotic cells, and microarray analysis reveals up-regulation of a p53 gene signature. All of the hematopoietic phenotypes are rescued by p53 mutation, demonstrating that p53 activation induced by ribosomal protein knockdown is mediating the \(rps29^{-/-}\) mutant phenotype. In addition, polysome profiles of mutant embryos identify a decrease in 80s monosome and polysome fractions. Preliminary RNA sequencing analysis of the polysome fractions suggested a shift in genes being translated in the mutant. We performed a chemical screen on rps29 embryos. Using embryo morphology and vascular expression patterns as read-outs, 600 compounds of known bioactivity were screened. One compound, A-3, improves embryo morphology, and a structurally related compound, W-7, rescues the vasculature defect. These compounds are calmodulin inhibitors, and A-3 can also rescue the hemoglobin defect in \(rps29^{-/-}\) embryos. To elucidate the compounds’ mechanism of action, A549 and \(CD34^+\) cells with RPS19 knocked down by shRNA were treated with chemical hits. In these cells, calmodulin inhibitors cause a decrease of p21 even with p53 induction. These data support a model where calmodulin inhibition can inhibit the p53 pathway upon ribosomal protein knockdown. In parallel to our zebrafish studies, we generated induced pluripotent stem (iPS) cells from DBA patient fibroblasts as a part of a large-scale collaboration. Three iPS lines are validated, and a total of 27 lines will be generated from patients with mutations in RPS19, RPL5, and RPL11. Testing for defects in blood differentiation and determining the role of p53 in these lines will enable validation of this system as a model of DBA. The iPS lines can subsequently be used for chemical and genetic screening to identify novel DBA pathways and potential therapies.

Most currently defined hematopoietic progenitor pools are heterogeneous, contributing to uncertainty regarding the development of certain blood cells. The origins of plasmacytoid dendritic cells, for instance, have long been controversial and progenitors exclusively committed to this lineage have never been described. We show here that the fate of hematopoietic progenitors is determined in part by their surface levels of 9-O-acetyl sialic acid. Pro-plasmacytoid dendritic cells were identified as lineage negative 9-O-acetyl sialic acid low progenitors that lack myeloid and lymphoid potential but differentiate into pre-plasmacytoid dendritic cells. The latter cells are also lineage negative, 9-O-acetyl sialic acid low cells but are exclusively committed to the plasmacytoid dendritic cell lineage. Levels of 9-O-acetyl sialic acid provide a distinct way to define progenitors and thus facilitate the study of hematopoietic differentiation.

The use of both multipotent progenitors and fully differentiated cells has been demonstrated to be effective for cell-based immunotherapy. The goal of this thesis was to establish an in vitro hematopoietic differentiation system to generate hematopoietic progenitor cells (HPCs) and functional T cells from pluripotent stem cells. Generation of progenitor T cells by co-culturing stem cells on Notch ligand-expressing OP9 stromal cells (OP9-DL1) had been successfully employed previously. However, further differentiation of these cells in vitro into mature, antigen-specific, functional T cells, without retroviral transduction of T cell receptors (TcRs), had not been achieved. In the thymic niche, differentiation of T cells to a state of antigen specificity is controlled by the interaction of their developing TcRs with the Major Histocompatibility Complex (MHC) on thymic stromal cells. We hypothesized that, by providing exogenous antigen-specific MHC/TcR signals, stem and progenitor cells could be engineered into functional effector T cells specific for the same antigen. In Chapter 3 and 4, we demonstrate that both thymus-derived double positive (DP: CD4+CD8+) immature T cells and mouse Embryonic Stem (ES) cells can be efficiently differentiated into antigen-specific CD8+ T cells using either MHC tetramers or peptide-loaded stromal cells. DP cells, following MHC/TcR signaling, retained elevated RAG1 levels, suggesting continuing TcR gene rearrangement. Both DP and ES cell-derived CD8+ T cells showed significant Cytotoxic T Lymphocyte (CTL) activity against antigen-loaded target cells, indicating that these cells are functional. This directed differentiation strategy could provide an efficient method for generating functional, antigen-specific CTLs from stem cells for potential use in adoptive T cell therapies. The use of ES cells in the clinic has been hindered by the unavailability of patient-specific ES cells and the ethical issues surrounding the use of human embryos. Induced pluripotent stem (iPS) cells offer great hope to regenerative medicine as their use can circumvent both the patient-specific and ethical issues associated with ES cells. In Chapter 5, we have developed a feeder cell-free suspension culture system supplemented with OP9-DL1 secretary factors to efficiently generated HPCs from iPS and ES cells. The differentiation potential of these HPCs was demonstrated by generation of DCs in the presence of GM-CSF and IL-3. The DCs express the activation molecules, CD86 and CD80 in response to LPS stimulation and are able to stimulate T cell proliferation in a mixed lymphocyte reaction. We employed extensive quantitative RT-PCR analysis to identify a number of differentially expressed genes in HPCs generated from the feeder-free culture. / text

Human embryonic stem cells (hESC) present a potentially unlimited supply of hematopoietic progenitors for cell-based therapies. However, current protocols for generating these progenitors typically also generate undesired cell types due to imprecise control of the hESC microenvironment and poor understanding of the signalling networks regulating mesoderm differentiation (the germ layer from which hematopoietic cells emerge). This report demonstrates that activation of the downstream effectors of Activin/Nodal and bone morphogenetic protein (BMP) signalling (Smad2 (composite of Sma (smaller) and Mad (mothers against decapentaplegic) and Smad1, respectively) are both required for mesoderm differentiation. It is further shown that microcontact printing-mediated control of hESC colony size creates local microenvironments that guide differentiation, via a Smad1-dependent mechanism, preferentially towards the mesoderm lineage. These findings demonstrate the need for precise control of the microenvironment in order to effectively guide hESC differentiation to produce specific cell types for potential therapeutic applications.

Human embryonic stem cells (hESC) present a potentially unlimited supply of hematopoietic progenitors for cell-based therapies. However, current protocols for generating these progenitors typically also generate undesired cell types due to imprecise control of the hESC microenvironment and poor understanding of the signalling networks regulating mesoderm differentiation (the germ layer from which hematopoietic cells emerge). This report demonstrates that activation of the downstream effectors of Activin/Nodal and bone morphogenetic protein (BMP) signalling (Smad2 (composite of Sma (smaller) and Mad (mothers against decapentaplegic) and Smad1, respectively) are both required for mesoderm differentiation. It is further shown that microcontact printing-mediated control of hESC colony size creates local microenvironments that guide differentiation, via a Smad1-dependent mechanism, preferentially towards the mesoderm lineage. These findings demonstrate the need for precise control of the microenvironment in order to effectively guide hESC differentiation to produce specific cell types for potential therapeutic applications.

Negative regulation of cytokine signalling is essential for maintaining hematopoietic homeostasis. We investigated the role of SH2B1 and CBL in the negative regulation of EPO and GM-CSF signaling, respectively. Erythropoiesis is driven by the cytokine erythropoietin (EPO), which mediates its signal by binding to its cognate receptor, the erythropoietin receptor (EPO-R). Murine knock-in studies have demonstrated EPO-R Tyr343 to play an important role in EPO mediated signalling. We have utilized a Cloning of Ligand Target (COLT) screen to identify the adaptor protein SH2B1 as an interactor of EPO-R pTyr343. We have demonstrated that SH2B1 binds to EPO-R via two mechanisms. The amino-terminus of SH2B1 and the membrane proximal region of EPO-R mediate SH2B1 constitutive binding to EPO-R. SH2B1 binds to EPO-R pTyr343 and pTyr 401 in an SH2 domain-dependent manner. SH2B1 displayed dose- and time- dependent Serine/Threonine phosphorylation in response to EPO stimulation. Knockdown of SH2B1 resulted in enhanced activation of Jak2 and EPO-R. These studies demonstrate SH2B1 as a novel negative regulator of EPO signalling. Mutations in the linker region and the RING finger of CBL have been identified in a number of myeloid malignancies, including juvenile myelomonocytic leukemia. We investigated how linker region mutant, CBL-Y371H, and RING finger mutant, CBL-C384R lead to GM-CSF hypersensitivity. Expression of these CBL mutants in the human hematopoietic cell line, TF-1, showed enhanced stimulation induced phosphorylation of GM-CSFR βc. We also demonstrated that the loss of E3 ligase activity of these CBL mutants results in increased expression of JAK2 and LYN kinases. Assessment of the effects of CBL mutants on downstream signalling revealed enhanced phosphorylation of SHP2, CBL and S6. Dasatinib induced inhibition of SRC family kinases abolished the elevated phosphorylation of CBL mutants, and equalized the phosphorylation of GM-CSFR βc in the wild type and CBL mutant cells.