scholarly journals Endogenous pioneer neutrophils release NETs during the swarming response in zebrafish

2019 ◽  
Author(s):  
Hannah M. Isles ◽  
Catherine A. Loynes ◽  
Noémie Hamilton ◽  
Clare F. Muir ◽  
Anastasia Kadochnikova ◽  
...  
Keyword(s):  
Tissue Damage ◽  
Damage Model ◽  
Neutrophil Migration ◽  
Transgenic Zebrafish ◽  
Histone H2a ◽  
Zebrafish Model ◽  
Extracellular Traps ◽  
Sequence Of Events ◽  
Phase Sequence

AbstractNeutrophils are rapidly recruited to inflammatory sites where they coordinate their migration to form clusters, a process termed neutrophil swarming. The factors which modulate neutrophil swarming during its early stages are not fully understood, requiring the development of new in vivo models. Using transgenic zebrafish larvae to study endogenous neutrophil migration in a tissue damage model, we demonstrate that neutrophil swarming is a conserved process in zebrafish immunity, sharing essential features with mammalian systems. We show that neutrophil swarms initially develop around a pioneer neutrophil, in a three-phase sequence of events. By adopting a high-resolution confocal microscopy approach, we observed the release of cell fragments by early swarming neutrophils. We developed a neutrophil specific histone H2A transgenic reporter line TgBAC(mpx:GFP)i114;Tg(lyz:H2A-mCherry)sh530 to study neutrophil extracellular traps (NETs), and found that endogenous neutrophils recruited to sites of tissue damage released NETs at the start of the swarming process. The optical transparency achieved using the zebrafish model has provided some of the highest resolution imaging of NET release in vivo to date. Using a combination of transgenic reporter lines and DNA intercalating agents, we demonstrate that pioneer neutrophils release extracellular traps during the swarming response, suggesting that cell death signalling via NETosis might be important in driving the swarming response.

scholarly journals Pioneer neutrophils release chromatin within in vivo swarms

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Hannah M Isles ◽  
Catherine A Loynes ◽  
Sultan Alasmari ◽  
Fu Chuen Kon ◽  
Katherine M Henry ◽  
...  
Keyword(s):  
Tissue Injury ◽  
Damage Model ◽  
Neutrophil Migration ◽  
Initial Step ◽  
Transgenic Zebrafish ◽  
In Vivo Models ◽  
Essential Components ◽  
Cellular Components

Neutrophils are rapidly recruited to inflammatory sites where their coordinated migration forms clusters, a process termed neutrophil swarming. The factors that modulate early stages of neutrophil swarming are not fully understood, requiring the development of new in vivo models. Using transgenic zebrafish larvae to study endogenous neutrophil migration in a tissue damage model, we demonstrate that neutrophil swarming is a conserved process in zebrafish immunity, sharing essential features with mammalian systems. We show that neutrophil swarms initially develop around an individual pioneer neutrophil. We observed the violent release of extracellular cytoplasmic and nuclear fragments by the pioneer and early swarming neutrophils. By combining in vitro and in vivo approaches to study essential components of neutrophil extracellular traps (NETs), we provide in-depth characterisation and high-resolution imaging of the composition and morphology of these release events. Using a photoconversion approach to track neutrophils within developing swarms, we identify that the fate of swarm-initiating pioneer neutrophils involves extracellular chromatin release and that the key NET components gasdermin, neutrophil elastase, and myeloperoxidase are required for the swarming process. Together our findings demonstrate that release of cellular components by pioneer neutrophils is an initial step in neutrophil swarming at sites of tissue injury.

scholarly journals Development of BCR-ABL1 Transgenic Zebrafish Model Reproducing Chronic Myeloid Leukemia (CML) Like-Disease and Providing a New Insight into CML Mechanisms

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 445
Author(s):  
Daniela Zizioli ◽  
Simona Bernardi ◽  
Marco Varinelli ◽  
Mirko Farina ◽  
Luca Mignani ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Fusion Gene ◽  
Philadelphia Chromosome ◽  
Leukemic Cells ◽  
Transgenic Zebrafish ◽  
Hematopoietic Tissue ◽  
Zebrafish Model ◽  
Altered Expression

Zebrafish has proven to be a versatile and reliable experimental in vivo tool to study human hematopoiesis and model hematological malignancies. Transgenic technologies enable the generation of specific leukemia types by the expression of human oncogenes under specific promoters. Using this technology, a variety of myeloid and lymphoid malignancies zebrafish models have been described. Chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasia characterized by the BCR-ABL1 fusion gene, derived from the t (9;22) translocation causing the Philadelphia Chromosome (Ph). The BCR-ABL1 protein is a constitutively activated tyrosine kinas inducing the leukemogenesis and resulting in an accumulation of immature leukemic cells into bone marrow and peripheral blood. To model Ph+ CML, a transgenic zebrafish line expressing the human BCR-ABL1 was generated by the Gal4/UAS system, and then crossed with the hsp70-Gal4 transgenic line. The new line named (BCR-ABL1pUAS:CFP/hsp70-Gal4), presented altered expression of hematopoietic markers during embryonic development compared to controls and transgenic larvae showed proliferating hematopoietic cells in the caudal hematopoietic tissue (CHT). The present transgenic zebrafish would be a robust CML model and a high-throughput drug screening tool.

scholarly journals Perturbations of BMP/TGF-β and VEGF/VEGFR signalling pathways in non-syndromic sporadic brain arteriovenous malformations (BAVM)

2018 ◽  
Vol 55 (10) ◽  
pp. 675-684 ◽  
Author(s):  
Kun Wang ◽  
Sen Zhao ◽  
Bowen Liu ◽  
Qianqian Zhang ◽  
Yaqi Li ◽  
...  
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Arteriovenous Malformations ◽  
Transforming Growth Factor ◽  
Transgenic Zebrafish ◽  
Zebrafish Model ◽  
Brain Arteriovenous Malformations ◽  
Pathogenic Variants ◽  
Uncertain Significance

BackgroundBrain arteriovenous malformations (BAVM) represent a congenital anomaly of the cerebral vessels with a prevalence of 10–18/100 000. BAVM is the leading aetiology of intracranial haemorrhage in children. Our objective was to identify gene variants potentially contributing to disease and to better define the molecular aetiology underlying non-syndromic sporadic BAVM.MethodsWe performed whole-exome trio sequencing of 100 unrelated families with a clinically uniform BAVM phenotype. Pathogenic variants were then studied in vivo using a transgenic zebrafish model.ResultsWe identified four pathogenic heterozygous variants in four patients, including one in the established BAVM-related gene, ENG, and three damaging variants in novel candidate genes: PITPNM3, SARS and LEMD3, which we then functionally validated in zebrafish. In addition, eight likely pathogenic heterozygous variants (TIMP3, SCUBE2, MAP4K4, CDH2, IL17RD, PREX2, ZFYVE16 and EGFR) were identified in eight patients, and 16 patients carried one or more variants of uncertain significance. Potential oligogenic inheritance (MAP4K4 with ENG, RASA1 with TIMP3 and SCUBE2 with ENG) was identified in three patients. Regulation of sma- and mad-related proteins (SMADs) (involved in bone morphogenic protein (BMP)/transforming growth factor beta (TGF-β) signalling) and vascular endothelial growth factor (VEGF)/vascular endotheliual growth factor recepter 2 (VEGFR2) binding and activity (affecting the VEGF signalling pathway) were the most significantly affected biological process involved in the pathogenesis of BAVM.ConclusionsOur study highlights the specific role of BMP/TGF-β and VEGF/VEGFR signalling in the aetiology of BAVM and the efficiency of intensive parallel sequencing in the challenging context of genetically heterogeneous paradigm.

scholarly journals Live imaging of neutrophil motility in a zebrafish model of WHIM syndrome

Blood ◽  
2010 ◽  
Vol 116 (15) ◽  
pp. 2803-2811 ◽  
Author(s):  
Kevin B. Walters ◽  
Julie M. Green ◽  
Jill C. Surfus ◽  
Sa Kan Yoo ◽  
Anna Huttenlocher
Keyword(s):  
Primary Immunodeficiency ◽  
Transgenic Zebrafish ◽  
Hematopoietic Tissue ◽  
Primary Immunodeficiency Disorder ◽  
Zebrafish Model ◽  
Whim Syndrome ◽  
Stromal Cell Derived Factor ◽  
Morpholino Oligonucleotides ◽  
Guanosine Triphosphatase

Abstract CXCR4 is a G protein–coupled chemokine receptor that has been implicated in the pathogenesis of primary immunodeficiency disorders and cancer. Autosomal dominant gain-of-function truncations of CXCR4 are associated with warts, hypo-gammaglobulinemia, infections, and myelokathexis (WHIM) syndrome, a primary immunodeficiency disorder characterized by neutropenia and recurrent infections. Recent progress has implicated CXCR4-SDF1 (stromal cell-derived factor 1) signaling in regulating neutrophil homeostasis, but the precise role of CXCR4-SDF1 interactions in regulating neutrophil motility in vivo is not known. Here, we use the optical transparency of zebrafish to visualize neutrophil trafficking in vivo in a zebrafish model of WHIM syndrome. We demonstrate that expression of WHIM mutations in zebrafish neutrophils induces neutrophil retention in hematopoietic tissue, impairing neutrophil motility and wound recruitment. The neutrophil retention signal induced by WHIM truncation mutations is SDF1 dependent, because depletion of SDF1 with the use of morpholino oligonucleotides restores neutrophil chemotaxis to wounds. Moreover, localized activation of a genetically encoded, photoactivatable Rac guanosine triphosphatase is sufficient to direct migration of neutrophils that express the WHIM mutation. The findings suggest that this transgenic zebrafish model of WHIM syndrome may provide a valuable tool to screen for agents that modify CXCR4-SDF1 retention signals.

scholarly journals The CXCL12/CXCR4 signalling axis retains neutrophils at inflammatory sites in zebrafish

2019 ◽  
Author(s):  
Hannah M. Isles ◽  
Kimberly Herman ◽  
Anne L. Robertson ◽  
Catherine A. Loynes ◽  
Lynne R. Prince ◽  
...  
Keyword(s):  
Inflammatory Disease ◽  
Tissue Damage ◽  
Tissue Injury ◽  
Transgenic Zebrafish ◽  
Sequencing Data ◽  
Cxcr4 Antagonist ◽  
Reverse Migration ◽  
Zebrafish Larvae ◽  
Inflammation Resolution

AbstractThe inappropriate retention of neutrophils in the lung is a major driver of the excessive tissue damage characteristic of respiratory inflammatory diseases including COPD, ARDS and cystic fibrosis. The molecular programmes which orchestrate neutrophil recruitment to inflammatory sites through chemotactic guidance have been well studied. However, how neutrophil sensitivity to these cues is modulated during inflammation resolution is not understood. The identification of neutrophil reverse migration as a mechanism of inflammation resolution and the ability to modulate this therapeutically has identified a new target to treat inflammatory disease. Here we investigate the role of the CXCL12/CXCR4 signalling axis in modulating neutrophil retention at inflammatory sites. We used an in vivo tissue injury model to study inflammation using transgenic zebrafish larvae. Expression of cxcl12a and cxcr4b during the tissue damage response was assessed using in situ hybridisation and analysis of RNA sequencing data. CRISPR/Cas9 was used to knockdown cxcl12a and cxcr4b in zebrafish larvae. The CXCR4 antagonist AMD3100 was used to block the Cxcl12/Cxcr4 signalling axis pharmacologically. We identified that cxcr4b and cxcl12a are expressed at the wound site in zebrafish larvae during the inflammatory response. Following tail-fin transection, removal of neutrophils from inflammatory sites is significantly increased in cxcr4b and cxcl12a CRISPR knockdown larvae. Pharmacological inhibition of the Cxcl12/Cxcr4 signalling axis accelerates inflammation resolution, an effect caused by an increase in neutrophil reverse migration. The findings of this study suggest that CXCR4/CXCL12 signalling may play an important role in neutrophil retention at inflammatory sites, identifying a potential new target for the therapeutic removal of neutrophils from the lung in chronic inflammatory disease.

scholarly journals Epigallocatechin-3-gallate suppresses neutrophil migration speed in a transgenic zebrafish model accompanied by reduced inflammatory mediators

2019 ◽  
Vol Volume 12 ◽  
pp. 231-239
Author(s):  
Thao Nguyen ◽  
Brittany Payan ◽  
Amarayca Zambrano ◽  
Yong Du ◽  
Maria Bondesson ◽  
...  
Keyword(s):  
Inflammatory Mediators ◽  
Neutrophil Migration ◽  
Transgenic Zebrafish ◽  
Migration Speed ◽  
Zebrafish Model

scholarly journals Live cell imaging of Salmonella Typhimurium interaction with zebrafish larvae after injection and immersion delivery methods

2016 ◽  
Author(s):  
Macarena A Varas ◽  
Alonso Fariña ◽  
Francisco Díaz-Pascual ◽  
Javiera Ortíz-Severín ◽  
Andrés E Marcoleta ◽  
...  
Keyword(s):  
Immune Response ◽  
Bacterial Infections ◽  
Inoculum Size ◽  
Live Cell ◽  
Transgenic Zebrafish ◽  
Human Pathogens ◽  
Zebrafish Model ◽  
Zebrafish Larvae ◽  
Serovar Typhimurium

Surrogate host models have been employed to study bacterial virulence mechanisms of important human pathogens. Particularly, zebrafish (Danio rerio) has been used to determine the role of vertebrate innate immunity during bacterial infections. The easy-to-obtain large number of embryos and optical transparency of larvae allow live cell imaging of the infection progress and the major cellular types of the innate immune system that develop during the first days of embryogenesis. In zebrafish model, microinjecting bacteria into embryos and/or larvae can cause infection. Alternatively, an infection can be generated by static immersion of larvae on a microbial suspension. Both methods differ in the mode and time of infection, inoculum size and host response. In this work, we compare the in vivo immune response induced by Salmonella enterica serovar Typhimurium (S. Typhimurium) inoculated by immersion and microinjection in zebrafish larvae. To this end, an immersion protocol using transgenic zebrafish larvae was developed for in vivo monitoring of GFP-tagged S. Typhimurium infection progress and immune response during 72 h. The infection progress was compared to that of zebrafish larvae inoculated by microinjection. Our results in zebrafish corroborate previous Salmonella virulence studies in murine models and reveal that host-pathogen interaction not only depends on the virulence of the strain, but also on the inoculation method and host conditions.

Fishing with a Transgenic Line: Using Zebrafish to Elucidate Mechanisms and Therapeutics in NUP98-NSD1 AML

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1638-1638
Author(s):  
Corey Filiaggi ◽  
Adam P Deveau ◽  
Sergey Prykhozhij ◽  
Graham Dellaire ◽  
Jason N. Berman
Keyword(s):  
Fluorescent Protein ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasm ◽  
Homeobox Gene ◽  
Transgenic Zebrafish ◽  
Gfp Expression ◽  
Zebrafish Model ◽  
Dismal Prognosis ◽  
Marker Expression

Abstract The NUP98-NSD1 (NND1) translocation is a fusion oncogene recently identified in pediatric acute myeloid leukemia (AML), where it occurs in approximately 16% of patients. NND1 predicts a dismal prognosis, with a 4-year event-free survival <10%. The mechanism of action of NND1 may be through the activation of the posterior homeobox gene, HOXA9. NND1 patients often harbour an internal tandem duplication of fms-like tyrosine kinase 3 (FLT3-ITD), another genetic lesion associated with poor prognosis. Co-expression of NND1 and FLT3-ITD results in worse survival than either aberration in isolation. NND1 may be sufficient to produce a myeloproliferative phenotype, but the interaction with FLT3-ITD activates essential downstream signaling pathways necessary for AML pathogenesis. A better understanding of the mechanisms by which NND1 dysregulates hematopoiesis and interacts with FLT3-ITD is fundamental to developing targeted therapies to improve the outcome in this disease. The zebrafish has been established as a robust and reliable model of hematologic malignancies, with conserved genetics and ease of genetic interrogation. Our group previously generated a transgenic zebrafish model expressing the related fusion oncogene, NUP98-HOXA9, in which embryos had anemia and expansion of myeloid cells, and adult fish exhibited a myeloproliferative neoplasm (MPN). Using this model, we discovered novel downstream epigenetic regulators that could be targeted therapeutically and restore normal embryonic hematopoiesis. Moreover, the up-regulated genes that we identified correlated with features of high-risk AML in human datasets, highlighting the translational relevance of this human disease model and justifying the employment of this approach to investigate NND1-driven AML (Deveau et al, Leukemia 2015). Plasmid constructs have been generated that incorporate human NND1 into the zebrafish using the Tol2 system, with detection by green fluorescent protein (GFP) expression. Injection of CMV-NND1-sGFP revealed strong GFP expression from 24-48 hours post fertilization (hpf) ubiquitously and in hematopoietic cells. Whole-mount in situ hybridization experiments of plasmid-injected embryos have shown that, similar to the NUP98-HOXA9 model, embryos expressing NND1 develop a pre-leukemic state, with a decrease in red blood cell marker expression (gata1) and an increase in myeloid marker expression (l-plastin). Currently no animal models exist for NND1 AML. Our initial studies have revealed a myeloproliferative phenotype in zebrafish embryos, providing an in vivo tool for further genetic and epigenetic interrogation, as well as a preclinical platform for novel drug discovery in this disease. Disclosures No relevant conflicts of interest to declare.

scholarly journals Cxcl8b and Cxcr2 Regulate Neutrophil Migration through Bloodstream in Zebrafish

2017 ◽  
Vol 2017 ◽  
pp. 1-11 ◽  
Author(s):  
Constanza Zuñiga-Traslaviña ◽  
Karina Bravo ◽  
Ariel E. Reyes ◽  
Carmen G. Feijóo
Keyword(s):  
Blood Circulation ◽  
Damage Model ◽  
Neutrophil Migration ◽  
Suitable Model ◽  
Mrna Levels ◽  
Damage Models ◽  
Transgenic Embryos ◽  
Molecular Signals

Neutrophils play an essential role during an inflammatory response, which is dependent on their rapid recruitment from the bone marrow to the vasculature. However, there is no information about the molecular signals that regulate neutrophil entry to circulation during an inflammatory process in humans. This is mainly due to the lack of a suitable model of study that contains similar set of molecules and that allows in vivo analyses. In this study, we used the zebrafish to assess the role of Cxcl8a, Cxcl8b, and Cxcr2 in neutrophil migration to blood circulation after injury. Using Tg(BACmpx:GFP)i114 transgenic embryos and two damage models (severe and mild), we developed in vivo lack of function assays. We found that the transcription levels of cxcl8a, cxcl8b, and cxcr2 were upregulated in the severe damage model. In contrast, only cxcr2 and cxcl8a mRNA levels were increased during mild damage. After knocking down Cxcl8a, neutrophil quantity decreased at the injury site, while Cxcl8b decreased neutrophils in circulation. When inhibiting Cxcr2, we observed a decrease in neutrophil entry to the bloodstream. In conclusion, we identified different functions for both Cxcl8 paralogues, being the Cxcl8b/Cxcr2 axis that regulates neutrophil entry to the bloodstream, while Cxcl8a/Cxcr2 regulates the migration to the affected area.

scholarly journals A transgenic zebrafish model for in vivo long-term imaging of retinotectal synaptogenesis

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Xu-fei Du ◽  
Bing Xu ◽  
Yu Zhang ◽  
Min-jia Chen ◽  
Jiu-lin Du
Keyword(s):  
Transgenic Zebrafish ◽  
Zebrafish Model
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