scholarly journals Development of High-Content Assays for Kidney Progenitor Cell Expansion in Transgenic Zebrafish

2013 ◽  
Vol 18 (10) ◽  
pp. 1193-1202 ◽  
Author(s):  
Subramaniam Sanker ◽  
Maria Cecilia Cirio ◽  
Laura L. Vollmer ◽  
Natasha D. Goldberg ◽  
Lee A. McDermott ◽  
...  
Keyword(s):  
Artificial Intelligence ◽  
Image Analysis ◽  
Time Course ◽  
Kidney Development ◽  
Egfp Expression ◽  
Transgenic Zebrafish ◽  
Zebrafish Embryos ◽  
Kidney Regeneration ◽  
Kidney Organogenesis

Reactivation of genes normally expressed during organogenesis is a characteristic of kidney regeneration. Enhancing this reactivation could potentially be a therapeutic target to augment kidney regeneration. The inductive events that drive kidney organogenesis in zebrafish are similar to the initial steps in mammalian kidney organogenesis. Therefore, quantifying embryonic signals that drive zebrafish kidney development is an attractive strategy for the discovery of potential novel therapeutic modalities that accelerate kidney regeneration. The Lim1 homeobox protein, Lhx1, is a marker of kidney development that is also expressed in the regenerating kidneys after injury. Using a fluorescent Lhx1a-EGFP transgene whose phenotype faithfully recapitulates that of the endogenous protein, we developed a high-content assay for Lhx1a-EGFP expression in transgenic zebrafish embryos employing an artificial intelligence–based image analysis method termed cognition network technology (CNT). Implementation of the CNT assay on high-content readers enabled automated real-time in vivo time-course, dose-response, and variability studies in the developing embryo. The Lhx1a assay was complemented with a kidney-specific secondary CNT assay that enables direct measurements of the embryonic renal tubule cell population. The integration of fluorescent transgenic zebrafish embryos with automated imaging and artificial intelligence–based image analysis provides an in vivo analysis system for structure-activity relationship studies and de novo discovery of novel agents that augment innate regenerative processes.

scholarly journals Cardioluminescence in transgenic zebrafish embryos: a calcium imaging tool to study drug effects and pathological modeling

2021 ◽  
Author(s):  
Manuel Vicente ◽  
Jussep Salgado-Almario ◽  
Michelle M. Collins ◽  
Antonio Martinez-Sielva ◽  
Masafumi Minoshima ◽  
...  
Keyword(s):  
Signal To Noise Ratio ◽  
Light Output ◽  
Drug Effects ◽  
Study Drug ◽  
Transgenic Zebrafish ◽  
Zebrafish Embryos ◽  
Cardiovascular Research ◽  
Imaging Tool ◽  
Adrenergic Blocker

The zebrafish embryo has emerged as an excellent model in cardiovascular research. The existing techniques to monitor Ca2+ in the heart based on fluorescent Ca2+ biosensors are limited due to phototoxicity and photobleaching. To overcome these issues, we have used bioluminescence. We generated a transgenic line expressing GFP-Aequorin in the heart, Tg(cmlc2:GA), and optimized an in vivo aequorin reconstitution protocol to improve the luminescence capacity. This allowed imaging Ca2+ in long duration recordings in embryos of 3 to 5 days post-fertilization. The analogs diacetyl h-coelenterazine and f-coelenterazine enhanced the light output and signal-to-noise ratio from the embryos. With this cardioluminescence model, we monitored the time-averaged Ca2+ levels and beat-to-beat Ca2+ oscillations. Changes in Ca2+ levels were observed by incubation with BayK8644, an L-type Ca2+ channel agonist, the channel blocker nifedipine, and β-adrenergic blocker propranolol. Treatment of zebrafish embryos with terfenadine for 24 hours has been proposed as a model of heart failure. Tg(cmlc2:GA) embryos treated with terfenadine showed a 2:1 atrioventricular block and a decrease in the ventricular Ca2+ levels.

Mitoferrin1 Transgenic Zebrafish Line Serves as a Model to Study Erythroid Cell Fate during Hematopoiesis

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3576-3576
Author(s):  
Julio Amigo ◽  
Ming Yu ◽  
Camila O. dos Santos ◽  
Prasad N. Paradkar ◽  
Jeffrey D. Cooney ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Site Directed Mutagenesis ◽  
Developmental Expression ◽  
Luciferase Reporter ◽  
Erythroid Cell ◽  
Egfp Expression ◽  
Transgenic Zebrafish ◽  
Zebrafish Embryos ◽  
Erythroid Cells ◽  
Egfp Reporter

Abstract We previously identified the mitochondrial solute carrier, Mitoferrin1 (Slc25a37, Mfrn1) as the principal mitochondrial iron importer essential for heme and iron-sulfur (Fe-S) cluster synthesis in developing erythroblasts. Its closely related paralog, Mitoferrin2 (Slc25a28, Mfrn2) functions in an analogous role in non-erythroid cells. Zebrafish with mutations in Mfrn1 have defects in hemoglobinization and maturation of erythroid cells caused by defective acquisition of iron into the mitochondria (GC Shaw, et al., 2006 Nature 440:96–100). Mfrn1 is highly expressed in embryonic and definitive sites of hematopoiesis in zebrafish and mouse, such as the developing blood island (ICM), fetal liver and adult bone marrow. In contrast, Mfrn2 is ubiquitously expressed, including at very low levels in erythroid cells. To understand the transcriptional regulation of Mfrn1 and Mfrn2, we used bioinformatics tools to identify potential cis-regulatory motifs (CRM) within each gene from mouse. The Gateway-modified Tol-2 vector was used to rapidly clone these conserved, minimal CRM fragments from mouse upstream of the basal promoter and an EGFP-reporter. Each construct was then introduced into zebrafish embryos for transient and stable expression. Using this strategy, we identified CRM’s that recapitulate the endogenous mRNA expression pattern of Mfrn genes during zebrafish development. Germ line stable transmission of the murine Tg(Mfrn1:EGFP) reporter in zebrafish showed robust EGFP expression in erythroid progenitors and mature erythrocytes and the remarkable conservation of function for CRM’s across species. In contrast, the mouse Tg(Mfrn2:EGFP) was expressed in skeletal muscle, heart, liver, and pronephros. The Mfrn1 enhancer is located ~35 kb upstream of the transcription start site and contains two GATA consensus motifs, which bind GATA-1 by chromatin immunoprecipitation analysis. Moreover, the ~150 bp Mfrn1 enhancer fragment exhibits transcriptional activation when coupled to the minimal γ-globin promoter driving expression of a luciferase reporter in K562 cells. Site-directed mutagenesis revealed that both GATA motifs are required for robust erythroid expression. In a complementary approach, transient knockdown of GATA-1 in zebrafish embryos using anti-sense morpholinos selectively ablated Mfrn1 mRNA expression in the ICM, consistent with the epistatic relationship of GATA-1 and Mfrn1. The zebrafish transgenic lines harboring the two murine Mfrn enhancers have proven useful in studying the regulatory and developmental expression in the Mfrn genes in erythroid and non-hematopoietic organs, such as heart and liver. Our results show that the combined use of bioinformatics, Gateway-mediated cloning, and Tol-2 mediated transgenesis in zebrafish embryos is an effective approach to functionally interrogate the transcriptional activity of putative CRM’s in vivo. The conservation and faithful expression of mouse CRM’s in zebrafish demonstrate the utility of this functional approach for analyzing mammalian CRM’s.

scholarly journals Real-Time Analysis of Endogenous Wnt Signalling in 3D Mesenchymal Stromal Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-9 ◽  
Author(s):  
Fatima Saleh ◽  
Alice Carstairs ◽  
S. Leah Etheridge ◽  
Paul Genever
Keyword(s):  
Time Course ◽  
Molecular Mechanisms ◽  
Adipogenic Differentiation ◽  
Wnt Signalling ◽  
Egfp Expression ◽  
Culture Techniques ◽  
Real Time Analysis ◽  
Pathway Activation

Wnt signalling has been implicated in the regulation of stem cell self-renewal and differentiation; however, the majority of in vitro studies are carried out using monolayer 2D culture techniques. Here, we used mesenchymal stromal cell (MSC) EGFP reporter lines responsive to Wnt pathway activation in a 3D spheroid culture system to mimic better the in vivo environment. Endogenous Wnt signalling was then investigated under basal conditions and when MSCs were induced to undergo osteogenic and adipogenic differentiation. Interestingly, endogenous Wnt signalling was only active during 3D differentiation whereas 2D cultures showed no EGFP expression throughout an extended differentiation time-course. Furthermore, exogenous Wnt signalling in 3D adipogenic conditions inhibited differentiation compared to unstimulated controls. In addition, suppressing Wnt signalling by Dkk-1 restored and facilitated adipogenic differentiation in MSC spheroids. Our findings indicate that endogenous Wnt signalling is active and can be tracked in 3D MSC cultures where it may act as a molecular switch in adipogenesis. The identification of the signalling pathways that regulate MSCs in a 3D in vivo-like environment will advance our understanding of the molecular mechanisms that control MSC fate.

scholarly journals Transcriptional activation of zebrafish cyp11a1 promoter is dependent on the nuclear receptor Ff1b

2009 ◽  
Vol 43 (3) ◽  
pp. 121-130 ◽  
Author(s):  
Sue Ing Quek ◽  
Woon Khiong Chan
Keyword(s):  
Transcriptional Activation ◽  
Bioinformatic Analysis ◽  
Regulatory Elements ◽  
Luciferase Reporter ◽  
Egfp Expression ◽  
Synthesis Rate ◽  
Zebrafish Embryos ◽  
Mobility Shift ◽  
Cytochrome P450scc

The cytochrome P450scc (cholesterol side-chain cleavage enzyme) encoded by CYP11A1 catalyzes the first step in steroidogenesis by converting cholesterol to pregnenolone, and thus, controls the synthesis rate of steroid hormones. In mammals, steroidogenic factor 1 (SF1) has been implicated in the cAMP-mediated transcriptional activation of CYP11A1 promoter. In zebrafish, Ff1b has been established as the homolog of SF1. To assess the dependency of cyp11a1 expression on Ff1b, the putative promoter of zebrafish cyp11a1, spanning 1.7 kb, was isolated and bioinformatic analysis revealed two conserved FF1 response elements (FREs) that potentially bind Ff1b. Transfection studies in cell lines of different lineages confirmed that this promoter fragment contained the necessary regulatory elements required for its basal transcription. Truncation and mutagenesis studies performed in Y1 adrenocortical cells revealed that only the proximal FRE was essential for transcriptional activation. Electrophoretic mobility shift assay, however, indicated that Ff1b bound to both FREs, while their in vivo occupancy was confirmed using a chromatin immunoprecipitation assay. Lastly, the cyp11a1 promoter was able to direct EGFP expression specifically to the interrenal gland and genital ridge when transiently expressed in microinjected zebrafish embryos, and the promoter activity is potentiated by ff1b overexpression as measured from luciferase reporter activity in zebrafish embryos.

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.

scholarly journals Opportunities for Understanding MS Mechanisms and Progression With MRI Using Large-Scale Data Sharing and Artificial Intelligence

Neurology ◽  
2021 ◽  
pp. 10.1212/WNL.0000000000012884
Author(s):  
Hugo Vrenken ◽  
Mark Jenkinson ◽  
Dzung Pham ◽  
Charles R.G. Guttmann ◽  
Deborah Pareto ◽  
...  
Keyword(s):  
Artificial Intelligence ◽  
Image Analysis ◽  
Data Sharing ◽  
Large Scale ◽  
Personal Data ◽  
Human Observer ◽  
Imaging Data ◽  
Large Scale Data ◽  
Scale Data

Multiple sclerosis (MS) patients have heterogeneous clinical presentations, symptoms and progression over time, making MS difficult to assess and comprehend in vivo. The combination of large-scale data-sharing and artificial intelligence creates new opportunities for monitoring and understanding MS using magnetic resonance imaging (MRI).First, development of validated MS-specific image analysis methods can be boosted by verified reference, test and benchmark imaging data. Using detailed expert annotations, artificial intelligence algorithms can be trained on such MS-specific data. Second, understanding disease processes could be greatly advanced through shared data of large MS cohorts with clinical, demographic and treatment information. Relevant patterns in such data that may be imperceptible to a human observer could be detected through artificial intelligence techniques. This applies from image analysis (lesions, atrophy or functional network changes) to large multi-domain datasets (imaging, cognition, clinical disability, genetics, etc.).After reviewing data-sharing and artificial intelligence, this paper highlights three areas that offer strong opportunities for making advances in the next few years: crowdsourcing, personal data protection, and organized analysis challenges. Difficulties as well as specific recommendations to overcome them are discussed, in order to best leverage data sharing and artificial intelligence to improve image analysis, imaging and the understanding of MS.

scholarly journals scRNA Transcription Profile of Adult Zebrafish Podocytes Using a Novel Reporter Strain

2021 ◽  
Vol 55 (S4) ◽  
pp. 35-47
Keyword(s):  
Calcium Binding ◽  
Kidney Development ◽  
Light Sheet ◽  
Transgenic Zebrafish ◽  
Fluorescent Reporter ◽  
Single Plane ◽  
Fluorescent Reporters ◽  
Injury And Repair ◽  
Gene Switching

BACKGROUND/AIMS: The role of podocytes is well conserved across species from drosophila to teleosts, and mammals. Identifying the molecular markers that actively maintain the integrity of the podocyte will enable a greater understanding of the changes that lead to damage. METHODS: We generated transgenic zebrafish, expressing fluorescent reporters driven by the podocin promoter, for the visualization and isolation of podocytes. We have conducted single cell RNA sequencing (scRNA-seq) on isolated podocytes from a zebrafish reporter line. RESULTS: We demonstrated that the LifeAct-TagRFP-T fluorescent reporter faithfully replicated podocin expression in vivo. We were also able to show spontaneous GCaMP6s fluorescence using light sheet (single plane illumination) microscopy. We identified many podocyte transcripts, encoding proteins related to calcium-binding and actin filament assembly, in common with those expressed in human and mouse mature podocytes. CONCLUSION: We describe the establishment of novel transgenic zebrafish and their use to identify and isolate podocyte cells for the preparation of a scRNA-seq library from normal podocytes. The scRNA-seq data identifies distinct populations of cells and potential gene switching between clusters. These data provide a foundation for future comparative studies and for exploiting the zebrafish as a model for kidney development, disease, injury and repair.

scholarly journals Overexpression of Lifeact-GFP Disrupts F-Actin Organization in Cardiomyocytes and Impairs Cardiac Function

2021 ◽  
Vol 9 ◽  
Author(s):  
Rui Xu ◽  
Shaojun Du
Keyword(s):  
Actin Filament ◽  
Heart Development ◽  
Molecular Probe ◽  
Actin Dynamics ◽  
Transgenic Zebrafish ◽  
Zebrafish Embryos ◽  
Gfp Expression ◽  
Actin Organization ◽  
Cardiac Muscles

Lifeact-GFP is a frequently used molecular probe to study F-actin structure and dynamic assembly in living cells. In this study, we generated transgenic zebrafish models expressing Lifeact-GFP specifically in cardiac muscles to investigate the effect of Lifeact-GFP on heart development and its application to study cardiomyopathy. The data showed that transgenic zebrafish with low to moderate levels of Lifeact-GFP expression could be used as a good model to study contractile dynamics of actin filaments in cardiac muscles in vivo. Using this model, we demonstrated that loss of Smyd1b, a lysine methyltransferase, disrupted F-actin filament organization in cardiomyocytes of zebrafish embryos. Our studies, however, also demonstrated that strong Lifeact-GFP expression in cardiomyocytes was detrimental to actin filament organization in cardiomyocytes that led to pericardial edema and early embryonic lethality of zebrafish embryos. Collectively, these data suggest that although Lifeact-GFP is a good probe for visualizing F-actin dynamics, transgenic models need to be carefully evaluated to avoid artifacts induced by Lifeact-GFP overexpression.

scholarly journals Kidney Regeneration in Later-Stage Mouse Embryos via Transplanted Renal Progenitor Cells

2019 ◽  
Vol 30 (12) ◽  
pp. 2293-2305 ◽  
Author(s):  
Shuichiro Yamanaka ◽  
Yatsumu Saito ◽  
Toshinari Fujimoto ◽  
Tsuyoshi Takamura ◽  
Susumu Tajiri ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Cell Transplantation ◽  
Progenitor Cell ◽  
Kidney Development ◽  
Fluorescent Protein ◽  
Mouse Embryos ◽  
Kidney Regeneration ◽  
Renal Progenitor Cells ◽  
Renal Progenitor

BackgroundThe limited availability of donor kidneys for transplantation has spurred interest in investigating alternative strategies, such as regenerating organs from stem cells transplanted into animal embryos. However, there is no known method for transplanting cells into later-stage embryos, which may be the most suitable host stages for organogenesis, particularly into regions useful for kidney regeneration.MethodsWe demonstrated accurate transplantation of renal progenitor cells expressing green fluorescent protein to the fetal kidney development area by incising the opaque uterine muscle layer but not the transparent amniotic membrane. We allowed renal progenitor cell–transplanted fetuses to develop for 6 days postoperatively before removal for analysis. We also transplanted renal progenitor cells into conditional kidney-deficient mouse embryos. We determined growth and differentiation of transplanted cells in all cases.ResultsRenal progenitor cell transplantation into the retroperitoneal cavity of fetuses at E13–E14 produced transplant-derived, vascularized glomeruli with filtration function and did not affect fetal growth or survival. Cells transplanted to the nephrogenic zone produced a chimera in the cap mesenchyme of donor and host nephron progenitor cells. Renal progenitor cells transplanted to conditional kidney-deficient fetuses induced the formation of a new nephron in the fetus that is connected to the host ureteric bud.ConclusionsWe developed a cell transplantation method for midstage to late-stage fetuses. In vivo kidney regeneration from renal progenitor cells using the renal developmental environment of the fetus shows promise. Our findings suggest that fetal transplantation methods may contribute to organ regeneration and developmental research.

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