scholarly journals In vivo myosin step-size from zebrafish skeletal muscle

Open Biology ◽  
2016 ◽  
Vol 6 (5) ◽  
pp. 160075 ◽  
Author(s):  
Thomas P. Burghardt ◽  
Katalin Ajtai ◽  
Xiaojing Sun ◽  
Naoko Takubo ◽  
Yihua Wang
Keyword(s):  
Skeletal Muscle ◽  
Fluorescent Protein ◽  
Angular Displacement ◽  
Transgenic Zebrafish ◽  
Top Down ◽  
Step Size ◽  
Bottom Up ◽  
Lever Arm ◽  
Single Lever

Muscle myosins transduce ATP free energy into actin displacement to power contraction. In vivo , myosin side chains are modified post-translationally under native conditions, potentially impacting function. Single myosin detection provides the ‘bottom-up’ myosin characterization probing basic mechanisms without ambiguities inherent to ensemble observation. Macroscopic muscle physiological experimentation provides the definitive ‘top-down’ phenotype characterizations that are the concerns in translational medicine. In vivo single myosin detection in muscle from zebrafish embryo models for human muscle fulfils ambitions for both bottom-up and top-down experimentation. A photoactivatable green fluorescent protein (GFP)-tagged myosin light chain expressed in transgenic zebrafish skeletal muscle specifically modifies the myosin lever-arm. Strychnine induces the simultaneous contraction of the bilateral tail muscles in a live embryo, causing them to be isometric while active. Highly inclined thin illumination excites the GFP tag of single lever-arms and its super-resolution orientation is measured from an active isometric muscle over a time sequence covering many transduction cycles. Consecutive frame lever-arm angular displacement converts to step-size by its product with the estimated lever-arm length. About 17% of the active myosin steps that fall between 2 and 7 nm are implicated as powerstrokes because they are beyond displacements detected from either relaxed or ATP-depleted (rigor) muscle.

Live imaging of Runx1 expression in the dorsal aorta tracks the emergence of blood progenitors from endothelial cells

Blood ◽  
2010 ◽  
Vol 116 (6) ◽  
pp. 909-914 ◽  
Author(s):  
Enid Yi Ni Lam ◽  
Christopher J. Hall ◽  
Philip S. Crosier ◽  
Kathryn E. Crosier ◽  
Maria Vega Flores
Keyword(s):  
Endothelial Cells ◽  
Transcription Factor ◽  
Fluorescent Protein ◽  
Living Organism ◽  
Time Lapse ◽  
Dorsal Aorta ◽  
Transgenic Zebrafish ◽  
Hematopoietic Stem ◽  
Green Fluorescent

Abstract Blood cells of an adult vertebrate are continuously generated by hematopoietic stem cells (HSCs) that originate during embryonic life within the aorta-gonad-mesonephros region. There is now compelling in vivo evidence that HSCs are generated from aortic endothelial cells and that this process is critically regulated by the transcription factor Runx1. By time-lapse microscopy of Runx1-enhanced green fluorescent protein transgenic zebrafish embryos, we were able to capture a subset of cells within the ventral endothelium of the dorsal aorta, as they acquire hemogenic properties and directly emerge as presumptive HSCs. These nascent hematopoietic cells assume a rounded morphology, transiently occupy the subaortic space, and eventually enter the circulation via the caudal vein. Cell tracing showed that these cells subsequently populated the sites of definitive hematopoiesis (thymus and kidney), consistent with an HSC identity. HSC numbers depended on activity of the transcription factor Runx1, on blood flow, and on proper development of the dorsal aorta (features in common with mammals). This study captures the earliest events of the transition of endothelial cells to a hemogenic endothelium and demonstrates that embryonic hematopoietic progenitors directly differentiate from endothelial cells within a living organism.

A novel regulatory element between the human FGA and FGG genes

2012 ◽  
Vol 108 (09) ◽  
pp. 427-434 ◽  
Author(s):  
Richard J. Fish ◽  
Marguerite Neerman-Arbez
Keyword(s):  
Gene Cluster ◽  
Fluorescent Protein ◽  
Regulatory Element ◽  
Luciferase Reporter ◽  
Transgenic Zebrafish ◽  
Regulatory Sequences ◽  
Gene Promoters ◽  
Cvd Risk ◽  
Enhancer Activity

SummaryHigh circulating fibrinogen levels correlate with cardiovascular disease (CVD) risk. Fibrinogen levels vary between people and also change in response to physiological and environmental stimuli. A modest proportion of the variation in fibrinogen levels can be explained by genotype, inferring that variation in genomic sequences that regulate the fibri-nogen genes (FGA, FGB and FGG) may affect hepatic fibrinogen production and perhaps CVD risk. We previously identified a conserved liver enhancer in the fibrinogen gene cluster (CNC12), between FGB and FGA. Genome-wide Chromatin immunoprecipitation-sequencing (ChIP-seq) demonstrated that transcription factors which bind fibrinogen gene promoters also interact with CNC12, as well as two potential fibrinogen enhancers (PFE), between FGA and FGG. Here we show that one of the PFE sequences has potent hepatocyte enhancer activity. Using a luciferase reporter gene system, we found that PFE2 enhances minimal promoter- and FGA promoter-driven gene expression in hepatoma cells, regardless of its orientation with respect to the promoters. A region within PFE2 bears a short series of conserved nucleotides which maintain enhancer activity without flanking sequence. We also demonstrate that PFE2 is a liver enhancer in vivo, driving enhanced green fluorescent protein expression in transgenic zebrafish larval livers. Our study shows that combining public domain ChIP-seq data with in vitro and in vivo functional tests can identify novel fibrinogen gene cluster regulatory sequences. Variation in such elements could affect fibrinogen production and influence CVD risk.

Laser-induced gene expression in specific cells of transgenic zebrafish

Development ◽  
2000 ◽  
Vol 127 (9) ◽  
pp. 1953-1960 ◽  
Author(s):  
M.C. Halloran ◽  
M. Sato-Maeda ◽  
J.T. Warren ◽  
F. Su ◽  
Z. Lele ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Green Fluorescent Protein Gene ◽  
Transgenic Zebrafish ◽  
Hsp70 Promoter ◽  
Expression Of Genes ◽  
Transgenic Lines ◽  
Transgenic Embryos ◽  
Green Fluorescent

Over the past few years, a number of studies have described the generation of transgenic lines of zebrafish in which expression of reporters was driven by a variety of promoters. These lines opened up the real possibility that transgenics could be used to complement the genetic analysis of zebrafish development. Transgenic lines in which the expression of genes can be regulated both in space and time would be especially useful. Therefore, we have cloned the zebrafish promoter for the inducible hsp70 gene and made stable transgenic lines of zebrafish that express the reporter green fluorescent protein gene under the control of a hsp70 promoter. At normal temperatures, green fluorescent protein is not detectable in transgenic embryos with the exception of the lens, but is robustly expressed throughout the embryo following an increase in ambient temperature. Furthermore, we have taken advantage of the accessibility and optical clarity of the embryos to express green fluorescent protein in individual cells by focussing a sublethal laser microbeam onto them. The targeted cells appear to develop normally: cells migrate normally, neurons project axons that follow normal pathways, and progenitor cells divide and give rise to normal progeny cells. By generating other transgenic lines in which the hsp70 promoter regulates genes of interest, it should be possible to examine the in vivo activity of the gene products by laser-inducing specific cells to express them in zebrafish embryos. As a first test, we laser-induced single muscle cells to make zebrafish Sema3A1, a semaphorin that is repulsive for specific growth cones, in a hsp70-sema3A1 transgenic line of zebrafish and found that extension by the motor axons was retarded by the induced muscle.

P0053A METHOD FOR THE ISOLATION OF FLUORESCENT GLOMERULI FROM ZEBRAFISH LARVAE

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Anna Iervolino ◽  
Tim Lange ◽  
Florian Siegerist ◽  
Maximilian Schindler ◽  
Giovambattista Capasso ◽  
...  
Keyword(s):  
Glomerular Filtration ◽  
Fluorescent Protein ◽  
Transgenic Zebrafish ◽  
Renal Tubules ◽  
Glomerular Filtration Barrier ◽  
Podocyte Injury ◽  
Zebrafish Larvae ◽  
Filtration Barrier ◽  
Glomerular Damage

Abstract Background and Aims The zebrafish is a powerful animal model to study the glomerular morphology and the function of the permselectivity of the glomerular filtration barrier. Since zebrafish larvae develop quickly and can be bred to transparency, in vivo observation of these animals is possible. At 48 hours post fertilization (dpf), zebrafish develop a single filtering glomerulus which is attached to a pair of renal tubules. Like in mammals, the glomerular filtration barrier consists of a fenestrated endothelium, the glomerular basement membrane (GBM) and interdigitating podocyte foot processes bridged by a molecularly conserved slit diaphragm. By the use of genetically modified zebrafish strains with fluorescently labeled podocytes, it is possible to study alterations of the glomerulus during the development of renal disease directly in vivo and in vitro. As an injury model we used the nitroreductase/metronidazole (NTR/MTZ) zebrafish line to induce podocyte apoptosis and detachment from the GBM. Moreover, treatment of these larvae with MTZ induces glomerular injury that mimics focal segmental glomerulosclerosis (FSGS). The aim of our study was to establish a glomeruli isolation method which allows us to identify deregulation of miRNAs and mRNAs in the injured glomeruli by sequencing. Method The transgenic zebrafish strain Cherry (Tg(nphs2:Eco.nfsB-mCherry); mitfaw2/w2; mpv17a9/a9) which expresses the prokaryotic enzyme nitroreductase (NTR) fused to mCherry, a red fluorescent protein, under the control of the podocyte-specific podocin (nphs2) promoter in a transparent zebrafish strain, was used. The NTR/MTZ is a model of cell ablation to mimic podocyte injury. The prodrug MTZ (80 µM) is converted into a cytotoxin by NTR leading to a dose-dependent apoptosis exclusively in NTR-expressing podocytes. To induce podocyte injury, we treated Cherry larvae at 4 days post fertilization with MTZ (80 µM) freshly dissolved in 0.1% DMSO-E3 medium for 48 hours. Control larvae were treated with 0.1% DMSO-E3 medium. The treatment was stopped by a MTZ washout at 6 dpf. In order to perform the miRNA and mRNA sequencing on glomeruli isolated from MTZ-treated and control larvae we tried to establish a method to obtain total RNA samples of good quality. For this purpose, three different approaches were tested and validated: 1) Sieving method, 2) Fluorescence-Activated Cell Sorting method (FACS), and 3) manual isolation of glomeruli by using a micropipette. Results Zebrafish larvae developed a glomerular damage similar to FSGS after MTZ-treatment. MTZ-treated larvae showed severe pericardial edema, a reduction of the nephrin and podocin expression, proteinuria and an increased mortality rate at 8 dpf. After many tests we showed that glomeruli isolation using the sieving method and FACS were not efficient due to contaminations with other organs (sieving) and a loss of a large amount of cells per sample (FACS), respectively. Samples of the required quality for sequencing resulted only from the manual glomeruli isolation. Conclusion Here we describe methods to isolate fluorescent glomeruli from transgenic zebrafish larvae. For our studies, we used the NTZ/MTR kidney disease model in order to identify mRNAs and miRNAs regulated in response to glomerular damage. This technique will further allow to screen for healing drugs in high-throughput experiments.

(Invited) In Vitro and In Vivo Near-Infrared Imaging with Biocompatible Bottom-up and Top-Down-Synthesized Graphene Quantum Dots

2020 ◽  
Vol MA2020-01 (6) ◽  
pp. 648-648
Author(s):  
Anton V Naumov ◽  
Md Tanvir Hasan ◽  
Elizabeth Campbell ◽  
Ching-Wei Lin ◽  
Angela M. Belcher
Keyword(s):  
Quantum Dots ◽  
Near Infrared ◽  
Infrared Imaging ◽  
Graphene Quantum Dots ◽  
Top Down ◽  
Near Infrared Imaging ◽  
Bottom Up

scholarly journals Physiological and histological changes in skeletal muscle following in vivo gene transfer by electroporation

2011 ◽  
Vol 301 (5) ◽  
pp. C1239-C1250 ◽  
Author(s):  
Joseph A. Roche ◽  
Diana L. Ford-Speelman ◽  
Lisa W. Ru ◽  
Allison L. Densmore ◽  
Renuka Roche ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Green Fluorescent Protein ◽  
Intramuscular Injection ◽  
Fluorescent Protein ◽  
Contractile Function ◽  
Complete Recovery ◽  
Secondary Damage ◽  
The Individual ◽  
Green Fluorescent

Electroporation (EP) is used to transfect skeletal muscle fibers in vivo, but its effects on the structure and function of skeletal muscle tissue have not yet been documented in detail. We studied the changes in contractile function and histology after EP and the influence of the individual steps involved to determine the mechanism of recovery, the extent of myofiber damage, and the efficiency of expression of a green fluorescent protein (GFP) transgene in the tibialis anterior (TA) muscle of adult male C57Bl/6J mice. Immediately after EP, contractile torque decreased by ∼80% from pre-EP levels. Within 3 h, torque recovered to ∼50% but stayed low until day 3. Functional recovery progressed slowly and was complete at day 28. In muscles that were depleted of satellite cells by X-irradiation, torque remained low after day 3, suggesting that myogenesis is necessary for complete recovery. In unirradiated muscle, myogenic activity after EP was confirmed by an increase in fibers with central nuclei or developmental myosin. Damage after EP was confirmed by the presence of necrotic myofibers infiltrated by CD68+ macrophages, which persisted in electroporated muscle for 42 days. Expression of GFP was detected at day 3 after EP and peaked on day 7, with ∼25% of fibers transfected. The number of fibers expressing green fluorescent protein (GFP), the distribution of GFP+ fibers, and the intensity of fluorescence in GFP+ fibers were highly variable. After intramuscular injection alone, or application of the electroporating current without injection, torque decreased by ∼20% and ∼70%, respectively, but secondary damage at D3 and later was minimal. We conclude that EP of murine TA muscles produces variable and modest levels of transgene expression, causes myofiber damage due to the interaction of intramuscular injection with the permeabilizing current, and that full recovery requires myogenesis.

scholarly journals An in vivo translation-reporter system for the study of protein synthesis in zebrafish embryos

2018 ◽  
Author(s):  
Inês Garcez Palha ◽  
Isabelle Anselme ◽  
Sylvie Schneider-Maunoury ◽  
François Giudicelli
Keyword(s):  
Protein Synthesis ◽  
Protease Inhibitor ◽  
Fluorescent Protein ◽  
Mrna Translation ◽  
Transgenic Zebrafish ◽  
Zebrafish Embryos ◽  
Reporter System ◽  
Control Of Gene Expression ◽  
A Cell

ABSTRACTControl of gene expression at the translation level is increasingly regarded as a key feature in many biological processes. Simple, inexpensive, and reliable procedures to visualise sites of protein production are required to allow observation of the spatiotemporal patterns of mRNA translation at subcellular resolution. We present a method, named SPoT (for Subcellular Patterns of Translation), developed upon the original TimeStamp technique (Lin et al., 2008), consisting in the expression of a fluorescent protein fused to a tagged, self-cleavable protease domain. Addition of a cell-permeable protease inhibitor instantly stabilizes newly produced, tagged protein allowing to distinguish recently synthesized protein from more ancient one. After a brief protease inhibitor treatment, the ratio of tagged vs non-tagged forms is highest at sites where proteins are the most recent, i.e. sites of synthesis. Therefore, by comparing tagged and non-tagged protein it is possible to spotlight sites of translation. By specifically expressing the SPoT cassette in neurons of transgenic zebrafish embryos, we reveal sites of neuronal protein synthesis in diverse cellular compartments during early development.

The Role of rap1b in Hoxb4 Transgenic Zebrafish Line,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3413-3413
Author(s):  
Zhixu He ◽  
Liping Shu ◽  
Xiaoyan Yang ◽  
Min Deng ◽  
Tingxi Liu
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Fluorescent Protein ◽  
Conflicts Of Interest ◽  
Control Group ◽  
Transgenic Zebrafish ◽  
Gene Expressions ◽  
Hematopoietic Stem ◽  
Experimental Group

Abstract Abstract 3413 Self-renewal – the generation of daughter cells having the same regenerative properties as the parental cell – is the defining feature of hematopoietic stem cells (HSCs). Homeobox b4 (Hoxb4) is the key gene shown capable of expanding hematopoietic stem cells (HSCs) in vivo and in vitro. It is imperative to establish a stable Hoxb4 overexpressing transgenic live animal model. Here, using a Cre-loxP system, we established a transgenic zebrafish line that stably overexpressing enhanced green fluorescent protein (EGFP) – tagged hoxb4 protein under the control of hemangioblast-specific lmo2 promoter. The results indicate that Hoxb4 favors hematopoietic stem cell development of hemangioblasts in vivo. The mechanisms of Hoxb4-mediated HSC proliferation and the reason why Hoxb4 overexpression can cause retardation of specific and terminal blood cells in vivo are still unknown. Rap1 belongs to the Ras subfamily of small GTP-binding proteins. Rap1 can regulate cell proliferation, differentiation, and adhesion through distinct mechanisms. So we want to know whether Hoxb4 overexpression can influence Rap1b expression. The expression difference of Rap1b in blank control group(Tubegin Wild type), experimental control group (Tg(zLmo2:LDL-EGFP)×Tg(zLmo2:Cre)), experimental group (Tg(zLmo2:LDL-Hoxb4-EGFP) ×Tg(zLmo2:Cre)) zebrafish embryo were detected by whole embryo in situ hybridization (WISH) with Rap1b antisense mRNA probe. It was found that Rap1b expression was extended from 30 to 36 hour postfertilization(hpf) when Hoxb4 was overexpressed. In the Hoxb4 expressed lines, rap1b was upregulated and strongly expressed in the caudal hematopoietic tissue (CHT) at 36 hpf. Then, the EGFP labeled hematopoietic cell in transgenic line were collected with the fluorescence activated cell sorter (FACS). The Rap1b mRNA expression of sorting cell were compared by SqRT-PCR (Semi-quantitative Reverse Transcription and Polymerase Chain Reaction). The gene expressions of Rap1b was upregulated in Hoxb4 experimental group. These results indicate that Rap1b gene maybe is downstream target gene of Hoxb4 gene in hematopoietic system of zebrafish. Disclosures: No relevant conflicts of interest to declare.

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 Pituitary Corticotroph Ontogeny and Regulation in Transgenic Zebrafish

2003 ◽  
Vol 17 (5) ◽  
pp. 959-966 ◽  
Author(s):  
Ning-Ai Liu ◽  
Haigen Huang ◽  
Zhongan Yang ◽  
Wiebke Herzog ◽  
Matthias Hammerschmidt ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Cell Mass ◽  
Gene Promoter ◽  
Genetic System ◽  
Regulatory Elements ◽  
Transgenic Zebrafish ◽  
Posterior Pituitary ◽  
Gfp Expression ◽  
Green Fluorescent

Abstract We characterized zebrafish proopiomelanocortin (POMC) gene promoter, and sequence analysis revealed that the promoter contains regulatory elements conserved among vertebrate species. To monitor the ontogeny of the pituitary POMC lineage in living vertebrates, we generated transgenic zebrafish expressing green fluorescent protein (GFP) driven by the POMC promoter. Zebrafish POMC-GFP is first expressed asymmetrically as two bilateral groups of cells most anterior to the neural ridge midline at 18–20 h post fertilization (hpf). POMC-GFP-positive cells then fuse into a single-cell mass within the pituitary anlage after 24 hpf and subsequently organize as distinct anterior and posterior domains between 48 and 64 hpf. Immunohistochemical studies with ACTH and αMSH antisera showed that POMC-GFP was mainly targeted to both anterior and posterior pituitary corticotrophs, whereas posterior pituitary region melanotrophs did not express GFP. To determine in vivo zebrafish corticotroph responses, dexamethasone (10−5m) was added to live embryos, which selectively suppressed POMC-GFP expression in the anterior group of corticotrophs, suggesting a distinct domain that is responsive to glucocorticoid feedback. Transgenic zebrafish with specific POMC-GFP expression in pituitary corticotrophs offers a powerful genetic system for in vivo study of vertebrate corticotroph lineage development.

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