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

Electrical cardioversion for atrial fibrillation: outcomes in 'real-life' clinical practice

Background: There is currently considerable debate with regard to the optimal management of atrial fibrillation/flutter (AF), including the long-term success of electrical cardioversion and the duration of anti-coagulation thereafter. The aim of this study was to investigate the current management and outcomes of electrical cardioversion in unselected patients in ordinary clinical practice. Methods: A prospective, observational study of 111 consecutive patients with AF who had been referred for electrical cardioversion was undertaken in a large teaching hospital. After cardioversion, patients were followed-up for 12 months or until death if this occurred earlier. Results: Sinus rhythm was restored immediately in 96 of 111 (86%) patients. Only 54 of 88 (61%) patients in sinus rhythm at discharge remained in this rhythm at 1 month. Of these 54, a further 21 (39%) had relapsed into AF by 12 months. Independent predictors of sinus rhythm at discharge were younger age (for a difference of 5 years, odds ratio=1.54; 95% confidence interval 1.04 to 1.16; P=0.002) and absence of hypertension (1.73, 1.22–1.91; P=0.015). The presence of sinus rhythm at discharge (6.4, 1.6–25.3; P=0.007) was an independent predictor of sinus rhythm at 1 month, whereas older age was a negative predictor (0.96, 0.92–1.0; P=0.05). Health-related quality of life improved at 1 and 12 months in those patients who remained in sinus rhythm compared to those who remained in AF. Conclusions: Though electrical cardioversion for AF has a high initial success rate only a minority of patients remained in sinus rhythm 1 year. The common practice of discontinuing anticoagulant treatment in patients in sinus rhythm at 1 month may be unsafe. Long-term maintenance of sinus rhythm is, however, associated with better health-related quality of life

Expression of the cytoplasmic NPM1 mutant (NPMc+) causes the expansion of hematopoietic cells in zebrafish

Mutations in the human nucleophosmin (NPM1) gene are the most frequent genetic alteration in adult acute myeloid leukemias (AMLs) and result in aberrant cytoplasmic translocation of this nucleolar phosphoprotein (NPMc+). However, underlying mechanisms leading to leukemogenesis remain unknown. To address this issue, we took advantage of the zebrafish model organism, which expresses 2 genes orthologous to human NPM1, referred to as npm1a and npm1b. Both genes are ubiquitously expressed, and their knockdown produces a reduction in myeloid cell numbers that is specifically rescued by NPM1 expression. In zebrafish, wild-type human NPM1 is nucleolar while NPMc+ is cytoplasmic, as in human AML, and both interact with endogenous zebrafish Npm1a and Npm1b. Forced NPMc+ expression in zebrafish causes an increase in pu.1+ primitive early myeloid cells. A more marked perturbation of myelopoiesis occurs in p53m/m embryos expressing NPMc+, where mpx+ and csf1r+ cell numbers are also expanded. Importantly, NPMc+ expression results in increased numbers of definitive hematopoietic cells, including erythromyeloid progenitors in the posterior blood island and c-myb/cd41+ cells in the ventral wall of the aorta. These results are likely to be relevant to human NPMc+ AML, where the observed NPMc+ multilineage expression pattern implies transformation of a multipotent stem or progenitor cell

Endothelial von Willebrand factor regulates angiogenesis

The regulation of blood vessel formation is of fundamental importance to many physiological processes, and angiogenesis is a major area for novel therapeutic approaches to diseases from ischemia to cancer. A poorly understood clinical manifestation of pathological angiogenesis is angiodysplasia, vascular malformations that cause severe gastrointestinal bleeding. Angiodysplasia can be associated with von Willebrand disease (VWD), the most common bleeding disorder in man. VWD is caused by a defect or deficiency in von Willebrand factor (VWF), a glycoprotein essential for normal hemostasis that is involved in inflammation. We hypothesized that VWF regulates angiogenesis. Inhibition of VWF expression by short interfering RNA (siRNA) in endothelial cells (ECs) caused increased in vitro angiogenesis and increased vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2)–dependent proliferation and migration, coupled to decreased integrin αvβ3 levels and increased angiopoietin (Ang)–2 release. ECs expanded from blood-derived endothelial progenitor cells of VWD patients confirmed these results. Finally, 2 different approaches, in situ and in vivo, showed increased vascularization in VWF-deficient mice. We therefore identify a new function of VWF in ECs, which confirms VWF as a protein with multiple vascular roles and defines a novel link between hemostasis and angiogenesis. These results may have important consequences for the management of VWD, with potential therapeutic implications for vascular diseases