Abstract P303: Development Of Casper/myl7/annexin-5 Transparent Transgenic In-vivo Zebrafish Model To Study The Cardiomyocyte Function

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Surendra K Rajpurohit ◽  
Aaron Gopal ◽  
May Y Mon ◽  
Nikhil Patel ◽  
Vishal Arora
Keyword(s):  
Regulatory Gene ◽  
Fluorescent Microscopy ◽  
Confocal Imaging ◽  
Transgenic Zebrafish ◽  
Heart Cells ◽  
Zebrafish Model ◽  
Transgenic Expression ◽  
Transgenic Progeny ◽  
Zebrafish Heart

The Zebrafish provided an excellent platform to study the genetic and molecular approach ofcardiac research. Zebrafish heart cells similar to human heart cells at the molecular level anddetermine gene functions that control cardiac function and dysfunction. In zebrafish heart, myl7is myosin 7 gene and identified as a regulatory gene orthologs to human MYL7. In the heart,Annexin5 activities contribute to cardiomyocyte dedifferentiation, proliferation and epicardial injuryresponses which leads to cardiac cell death by apoptosis and narcosis pathways. We aredeveloping annexin-5 activity in the cardiovascular function under normal and in metabolicaberration by generating homozygous Casper/ myl7:RFP; annexin-5:YFP transgenic zebrafish.By developing Casper/myl7/Annexin-5 transparent transgenic zebrafish model, we establish time-lapse in-vivo confocal microscopy to study of cellular phenotype/pathologies of thecardiomyocytes over time in newly developed strain to quantify changes in cardiomyocytemorphology and function overtime, comparing control and cardiac injury and cardio-oncologymodels. Transgenic zebrafish has normal type skin pigmentation background. In zebrafish,tracking of transgenic reporter activity in in-vivo is only possible in transparent stage. To maintaintransparency throughout the life, these strains crossbred with the skin transparent mutant Casper.Casper contributes to the study by integrating a transparent characteristic in adult zebrafish thatallows for simpler transparent visualization and observation. We develop casper transgenicprogenies through cross breeding with the transgenic strain of myl7:RFP;annexin-5:YFP .Confocal and fluorescent microscopy used to get accurate, precise imaging and to determinefluorescent protein being activated. 1.1: Generation of homozygous casper / myl7:RFP;annexin-5:YFP zebrafish (Generation F01-F05). 1.2: Screening and sorting the transgenic progeny andIn vivo imaging to validate cardiac morphology through in-vivo confocal imaging. Generation ofhomozygous casper / myl7:RFP;annexin-5:YFP zebrafish: Casper-Annexin5 homozygous stain:Cross breed casper and myl7/Annexin5 fish; F01: Generate the eggs from breeder and grow theembryo to attenuate larvae to screen for transgenic expression. F01 generation, larvae showtransgenic expression (47%). F02: transgenic expression larvae (39%). F02 heterozygous shownormal skin pattern; F03, larval show transgenic expression (43%). F04, transgenic larvae(90%).F04; 100% fishes are phenotypically casper; F05: heterozygous transgenic progeny togrow and continue to generate until achieve 100% homozygous casper-myl7-Annexin5 strain.These novel results provide in-vivo whole organism-based platform to design high throughputscreening and establish new horizon for drug discovery in the Cardiac Disease and Cardio-oncology.

scholarly journals Development of Tg(UAS:SEC-Hsa.ANXA5-YFP,myl7:RFP); Casper(roy−/−,nacre−/−) Transparent Transgenic In Vivo Zebrafish Model to Study the Cardiomyocyte Function

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1963
Author(s):  
Surendra K. Rajpurohit ◽  
Aaron Gopal ◽  
May Ye Mon ◽  
Nikhil G. Patel ◽  
Vishal Arora
Keyword(s):  
In Vivo Imaging ◽  
High Throughput Screening ◽  
Skin Pigmentation ◽  
Fluorescent Microscopy ◽  
Confocal Imaging ◽  
Transgenic Zebrafish ◽  
Cellular Phenotype ◽  
Zebrafish Model ◽  
Over Time

The zebrafish provided an excellent platform to study the genetic and molecular approach of cellular phenotype-based cardiac research. We designed a novel protocol to develop the transparent transgenic zebrafish model to study annexin-5 activity in the cardiovascular function by generating homozygous transparent skin Casper (roy−/−,nacre−/−); myl7:RFP; annexin-5:YFP transgenic zebrafish. The skin pigmentation background of any vertebrate model organism is a major obstruction for in vivo confocal imaging to study the transgenic cellular phenotype-based study. By developing Casper (roy−/−,nacre−/−); myl7; annexin-5 transparent transgenic zebrafish strain, we established time-lapse in vivo confocal microscopy to study cellular phenotype/pathologies of cardiomyocytes over time to quantify changes in cardiomyocyte morphology and function over time, comparing control and cardiac injury and cardio-oncology. Casper contributes to the study by integrating a transparent characteristic in adult zebrafish that allows for simpler transparent visualization and observation. The Casper (roy−/−,nacre−/−) transgenic progenies developed through cross-breeding with the transgenic strain of Tg (UAS:SEC-Hsa.ANXA5-YFP,myl7:RFP). Confocal and fluorescent microscopy were being used to obtain accurate, precise imaging and to determine fluorescent protein being activated. This study protocol was conducted under two sections; 1.1: Generation of homozygous Tg (UAS:SEC-Hsa.ANXA5-YFP,myl7:RFP); Casper (roy−/−,nacre−/−) zebrafish (generation F01-F06) and 1.2: Screening and sorting the transparent transgenic progeny and in vivo imaging to validate cardiac morphology through in vivo confocal imaging. We coined the newly developed strain as Tg (UAS:SEC-Hsa.ANXA5-YFP,myl7:RFP); Casper (roy−/−,nacre−/−) gmc1. Thus, the newly developed strain maintains transparency of the skin throughout the entire life of zebrafish and is capable of application of a non-invasive in vivo imaging process. These novel results provide an in vivo whole organism-based platform to design high-throughput screening and establish a new horizon for drug discovery in cardiac cell death and cardio-oncology therapeutics and treatment.

scholarly journals Evaluation of CML TKI Induced Cardiovascular Toxicity and Development of Potential Rescue Strategies in a Zebrafish Model

2021 ◽  
Vol 12 ◽  
Author(s):  
Shan Cheng ◽  
Pan Jin ◽  
Heying Li ◽  
Duanqing Pei ◽  
Xiaodong Shu
Keyword(s):  
Adverse Events ◽  
Kinase Inhibitors ◽  
Angiotensin Receptor Blockers ◽  
Clinical Stage ◽  
Confocal Imaging ◽  
Transgenic Zebrafish ◽  
Cardiovascular Toxicity ◽  
Zebrafish Model ◽  
T315i Mutation

Tyrosine kinase inhibitors (TKIs) to BCR-ABL1 have been successfully used to treat chronic myeloid leukemia (CML), however, multiple TKI-associated adverse events have been reported and become an emerging problem in patients. The mechanisms of TKI-induced toxicity are not fully understood and it remains challenging to predict potential cardiovascular toxicity of a compound. In this study, we established a zebrafish model to evaluate potential in vivo cardiovascular toxicity of TKIs. We treated the endothelium labeled Tg(kdrl:EGFP) transgenic zebrafish embryos with TKIs then performed confocal imaging to evaluate their vascular structure and function. We found that among FDA approved CML TKIs, ponatinib (the only approved TKI that is efficacious to T315I mutation) is the most toxic one. We then evaluated safety profiles of several clinical stage kinase inhibitors that can target T315I and found that HQP1351 treatment leads to vasculopathies similar to those induced by ponatinib while the allosteric ABL inhibitor asciminib does not induce noticeable cardiovascular defects, indicating it could be a promising therapeutic reagent for patients with T315I mutation. We then performed proof-of-principle study to rescue those TKI-induced cardiovascular toxicities and found that, among commonly used anti-hypertensive drugs, angiotensin receptor blockers such as azilsartan and valsartan are able to reduce ponatinib or HQP1351 induced cardiovascular toxicities. Together, this study establishes a zebrafish model that can be useful to evaluate cardiovascular toxicity of TKIs as well as to develop strategies to minimize TKI-induced adverse events.

MYST3/NCOA2-Induced Acute Myeloid Leukemia in Transgenic Fish

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5329-5329
Author(s):  
Julia Zhuravleva ◽  
Jérôme Paggetti ◽  
Laurent Martin ◽  
Arlette Hammann ◽  
Eric Solary ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Fusion Gene ◽  
Lymphoblastic Leukemia ◽  
Transgenic Zebrafish ◽  
Zebrafish Model ◽  
Transgenic Expression ◽  
Acute Myeloid

Abstract The MYST3/NCOA2 (MOZ/TIF2) fusion gene generated by the inv(8)(p11q13) chromosomal abnormality was described in a specific subgroup of acute myeloid leukemias (AML) that represents less than 5% of AML4/5. This abnormality fuses MYST3 (MOZ), a member of the MYST family of histone acetyl-transferases (HAT) to NCOA2 (TIF2), a member of the p160 HAT family. The transforming properties of MYST3/NCOA2 were demonstrated in mouse committed myeloid progenitors in vitro and in vivo. Hematopoiesis is very similar in zebrafish and in higher vertebrates. Homologues of a large number of genes involved in mammalian myelopoiesis were identified in this animal model. We have recently shown that ncoa2 (tif2) played a role in zebrafish primitive hematopoiesis. This animal also represents a model for investigating leukemogenesis. Transgenic expression of rag2-EGFP-mMyc or rag2-ICN1-EGFP induces a T-cell acute lymphoblastic leukemia (ALL) whereas transgenic expression of the ETV6/RUNX1 fusion gene induces a B-cell type ALL. We generated a transgenic zebrafish in which the MYST3/NCOA2 fusion gene was expressed under control of the spi1 (pu.1) promoter. An AML developed in two of 180 MYST3/NCOA2-EGFP-expressing embryos, 14 and 26 months after injection of the fusion gene in a one cell embryo, respectively. This leukemia was characterized by an extensive invasion of kidneys by myeloid blast cells. This model, which is the first zebrafish model of acute myeloid leukemia, demonstrates the oncogenic potency of MYST3/NCOA2 fusion gene.

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.

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 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

scholarly journals A transgenic zebrafish model for monitoring xbp1 splicing and endoplasmic reticulum stress in vivo

2015 ◽  
Vol 137 ◽  
pp. 33-44 ◽  
Author(s):  
Junling Li ◽  
Zhiliang Chen ◽  
Lian-Yong Gao ◽  
Angelo Colorni ◽  
Michal Ucko ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Endoplasmic Reticulum Stress ◽  
Transgenic Zebrafish ◽  
Zebrafish Model

scholarly journals Generation of a Novel Transgenic Zebrafish for Studying Adipocyte Development and Metabolic Control

2021 ◽  
Vol 22 (8) ◽  
pp. 3994
Author(s):  
Yousheng Mao ◽  
Kwang-Heum Hong ◽  
Weifang Liao ◽  
Li Li ◽  
Seong-Jin Kim ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Metabolic Diseases ◽  
Fluorescent Protein ◽  
Therapeutic Interventions ◽  
Transgenic Zebrafish ◽  
Zebrafish Model ◽  
Adipose Tissues ◽  
Green Fluorescent

Zebrafish have become a popular animal model for studying various biological processes and human diseases. The metabolic pathways and players conserved among zebrafish and mammals facilitate the use of zebrafish to understand the pathological mechanisms underlying various metabolic disorders in humans. Adipocytes play an important role in metabolic homeostasis, and zebrafish adipocytes have been characterized. However, a versatile and reliable zebrafish model for long-term monitoring of adipose tissues has not been reported. In this study, we generated stable transgenic zebrafish expressing enhanced green fluorescent protein (EGFP) in adipocytes. The transgenic zebrafish harbored adipose tissues that could be detected using GFP fluorescence and the morphology of single adipocyte could be investigated in vivo. In addition, we demonstrated the applicability of this model to the long-term in vivo imaging of adipose tissue development and regulation based on nutrition. The transgenic zebrafish established in this study may serve as an excellent tool to advance the characterization of white adipose tissue in zebrafish, thereby aiding the development of therapeutic interventions to treat metabolic diseases in humans.

Sign in / Sign up

Export Citation Format

Share Document