Ribosomal proteins in zebrafish haematopoiesis and human disease

PhDSeveral congenital disorders of human haematopoiesis including Diamond- Blackfan anaemia result from heterozygous loss of genes involved in ribosome biogenesis. Further, hemizygosity for ribosomal protein gene RPS14 has been implicated in the pathogenesis of myelodysplastic syndrome with loss of 5q, suggesting that genes involved in ribosome biogenesis may act as both haploinsufficient tumour suppressors and regulators of normal haematopoiesis. Ribosome biogenesis is highly conserved through evolution and readily studied in simple organisms such as yeasts. However the zebrafish provides a wellestablished genetic model system which is ideally suited to rapid assessment of vertebrate haematopoiesis. I have therefore used the zebrafish to study genes involved in ribosome biogenesis and their effects on developmental haematopoiesis relevant to human disease. Presented in this work is investigation of the effect of disruption of 4 genes known to be involved in ribosome biogenesis on zebrafish haematopoiesis. Firstly, I describe a gene, Dead-box 18 (ddx18), identified in a forward genetic screen, whose disruption results in defective haematopoiesis and embryonic lethality. Secondly, I have studied the effects of loss of zebrafish orthologues of the human nucleophosmin gene (NPM1), the most frequently mutated gene in human acute myeloid leukaemia. Loss of Npm1 resulted in aberrant numbers of myeloid cells. Heterologous overexpression of mutated NPM1(NPMc+) resulted in increased production of haematopoietic stem cells suggesting a role for NPMc+ in pathogenesis of AML. Finally, I have shown that loss of Rps14 and Rps19 result in anaemia in developing zebrafish and have investigated p53-independent mechanisms for this effect. The findings described herein demonstrate that disruption of normal ribosome biogenesis frequently results in abnormal developmental haematopoiesis. Further genetic assessment of these tissue-specific pathways deregulated by loss of normal ribosome function may represent an important common mechanism underlying the pathogenesis of congenital and acquired disorders of haematopoiesis, and may provide novel pathways for therapeutic targeting

Technologies bringing young Zebrafish from a niche field to the limelight

Fundamental life science and pharmaceutical research are continually striving to provide physiologically relevant context for their biological studies. Zebrafish present an opportunity for high-content screening (HCS) to bring a true in vivo model system to screening studies. Zebrafish embryos and young larvae are an economical, human-relevant model organism that are amenable to both genetic engineering and modification, and direct inspection via microscopy. The use of these organisms entails unique challenges that new technologies are overcoming, including artificial intelligence (AI). In this perspective article, we describe the state-of-the-art in terms of automated sample handling, imaging, and data analysis with zebrafish during early developmental stages. We highlight advances in orienting the embryos, including the use of robots, microfluidics, and creative multi-well plate solutions. Analyzing the micrographs in a fast, reliable fashion that maintains the anatomical context of the fluorescently labeled cells is a crucial step. Existing software solutions range from AI-driven commercial solutions to bespoke analysis algorithms. Deep learning appears to be a critical tool that researchers are only beginning to apply, but already facilitates many automated steps in the experimental workflow. Currently, such work has permitted the cellular quantification of multiple cell types in vivo, including stem cell responses to stress and drugs, neuronal myelination and macrophage behavior during inflammation and infection. We evaluate pro and cons of proprietary versus open-source methodologies for combining technologies into fully automated workflows of zebrafish studies. Zebrafish are poised to charge into HCS with ever-greater presence, bringing a new level of physiological context

Differential Requirement of Gata2a and Gata2b for Primitive and Definitive Myeloid Development in Zebrafish

Germline loss or mutation of one copy of the transcription factor GATA2 in humans leads to a range of clinical phenotypes affecting hematopoietic, lymphatic and vascular systems. GATA2 heterozygous mice show only a limited repertoire of the features observed in humans. Zebrafish have two copies of the Gata2 gene as a result of an additional round of ancestral whole genome duplication. These genes, Gata2a and Gata2b, show distinct but overlapping expression patterns, and between them, highlight a significantly broader range of the phenotypes observed in GATA2 deficient syndromes, than each one alone. In this manuscript, we use mutants for Gata2a and Gata2b to interrogate the effects on hematopoiesis of these two ohnologs, alone and in combination, during development in order to further define the role of GATA2 in developmental hematopoiesis. We define unique roles for each ohnolog at different stages of developmental myelopoiesis and for the emergence of hematopoietic stem and progenitor cells. These effects are not additive in the haploinsufficient state suggesting a redundancy between these two genes in hematopoietic stem and progenitor cells. Rescue studies additionally support that Gata2b can compensate for the effects of Gata2a loss. Finally we show that adults with loss of combined heterozygosity show defects in the myeloid compartment consistent with GATA2 loss in humans. These results build on existing knowledge from other models of GATA2 deficiency and refine our understanding of the early developmental effects of GATA2. In addition, these studies shed light on the complexity and potential structure-function relationships as well as sub-functionalization of Gata2 genes in the zebrafish model

TLR7 ligation augments hematopoiesis in Rps14 (uS11) deficiency via paradoxical suppression of inflammatory signaling

Myelodysplastic syndrome (MDS) is a hematological malignancy characterized by blood cytopenias and predisposition to acute myeloid leukemia (AML). Therapies for MDS are lacking, particularly those that have an impact in the early stages of disease. We developed a model of MDS in zebrafish with knockout of Rps14, the primary mediator of the anemia associated with del(5q) MDS. These mutant animals display dose- and age-dependent abnormalities in hematopoiesis, culminating in bone marrow failure with dysplastic features. We used Rps14 knockdown to undertake an in vivo small-molecule screening, to identify compounds that ameliorate the MDS phenotype, and we identified imiquimod, an agonist of Toll-like receptor-7 (TLR7) and TLR8. Imiquimod alleviates anemia by promoting hematopoietic stem and progenitor cell expansion and erythroid differentiation, the mechanism of which is dependent on TLR7 ligation and Myd88. TLR7 activation in this setting paradoxically promoted an anti-inflammatory gene signature, indicating cross talk via TLR7 between proinflammatory pathways endogenous to Rps14 loss and the NF-κB pathway. Finally, in highly purified human bone marrow samples from anemic patients, imiquimod led to an increase in erythroid output from myeloerythroid progenitors and common myeloid progenitors. Our findings have both specific implications for the development of targeted therapeutics for del(5q) MDS and wider significance identifying a potential role for TLR7 ligation in modifying anemia

DNAJC21 Mutations Link a Cancer-Prone Bone Marrow Failure Syndrome to Corruption in 60S Ribosome Subunit Maturation

This work was funded by The Medical Research Council UK (MR/K000292/1), Children with Cancer UK (2013/144), Barts and The London Charity (845/1796), and Leukaemia Lymphoma Research/Bloodwise (14032)

SARS-CoV-2-specific immune responses and clinical outcomes after COVID-19 vaccination in patients with immune-suppressive disease

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immune responses and infection outcomes were evaluated in 2,686 patients with varying immune-suppressive disease states after administration of two Coronavirus Disease 2019 (COVID-19) vaccines. Overall, 255 of 2,204 (12%) patients failed to develop anti-spike antibodies, with an additional 600 of 2,204 (27%) patients generating low levels (<380 AU ml−1). Vaccine failure rates were highest in ANCA-associated vasculitis on rituximab (21/29, 72%), hemodialysis on immunosuppressive therapy (6/30, 20%) and solid organ transplant recipients (20/81, 25% and 141/458, 31%). SARS-CoV-2-specific T cell responses were detected in 513 of 580 (88%) patients, with lower T cell magnitude or proportion in hemodialysis, allogeneic hematopoietic stem cell transplantation and liver transplant recipients (versus healthy controls). Humoral responses against Omicron (BA.1) were reduced, although cross-reactive T cell responses were sustained in all participants for whom these data were available. BNT162b2 was associated with higher antibody but lower cellular responses compared to ChAdOx1 nCoV-19 vaccination. We report 474 SARS-CoV-2 infection episodes, including 48 individuals with hospitalization or death from COVID-19. Decreased magnitude of both the serological and the T cell response was associated with severe COVID-19. Overall, we identified clinical phenotypes that may benefit from targeted COVID-19 therapeutic strategies

Ribosomal proteins in zebrafish haematopoiesis and human disease

Several congenital disorders of human haematopoiesis including Diamond- Blackfan anaemia result from heterozygous loss of genes involved in ribosome biogenesis. Further, hemizygosity for ribosomal protein gene RPS14 has been implicated in the pathogenesis of myelodysplastic syndrome with loss of 5q, suggesting that genes involved in ribosome biogenesis may act as both haploinsufficient tumour suppressors and regulators of normal haematopoiesis. Ribosome biogenesis is highly conserved through evolution and readily studied in simple organisms such as yeasts. However the zebrafish provides a wellestablished genetic model system which is ideally suited to rapid assessment of vertebrate haematopoiesis. I have therefore used the zebrafish to study genes involved in ribosome biogenesis and their effects on developmental haematopoiesis relevant to human disease. Presented in this work is investigation of the effect of disruption of 4 genes known to be involved in ribosome biogenesis on zebrafish haematopoiesis. Firstly, I describe a gene, Dead-box 18 (ddx18), identified in a forward genetic screen, whose disruption results in defective haematopoiesis and embryonic lethality. Secondly, I have studied the effects of loss of zebrafish orthologues of the human nucleophosmin gene (NPM1), the most frequently mutated gene in human acute myeloid leukaemia. Loss of Npm1 resulted in aberrant numbers of myeloid cells. Heterologous overexpression of mutated NPM1(NPMc+) resulted in increased production of haematopoietic stem cells suggesting a role for NPMc+ in pathogenesis of AML. Finally, I have shown that loss of Rps14 and Rps19 result in anaemia in developing zebrafish and have investigated p53-independent mechanisms for this effect. The findings described herein demonstrate that disruption of normal ribosome biogenesis frequently results in abnormal developmental haematopoiesis. Further genetic assessment of these tissue-specific pathways deregulated by loss of normal ribosome function may represent an important common mechanism underlying the pathogenesis of congenital and acquired disorders of haematopoiesis, and may provide novel pathways for therapeutic targeting.EThOS - Electronic Theses Online ServiceGBUnited Kingdo