Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Romano, Shannon N.; Gorelick, Daniel A.

doi:10.3791/51533

Cited by 6 publications

(4 citation statements)

References 10 publications

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“…To study the effects of estrogen-like EEDs on development, zebrafish embryos are commonly used to screen for toxicity and to study the effects of estrogens on organ formation and function (Bouwmeester et al, 2016;Carroll et al, 2014;Gorelick and Halpern, 2011;Gorelick et al, 2014;Hao et al, 2013;Kinch et al, 2015;Namdaran et al, 2012;Padilla et al, 2012;Romano and Gorelick, 2014;Sun et al, 2010;Tal et al, 2016;Truong et al, 2014). Embryos are often exposed to micromolar concentrations of E2 in fish water, despite the fact that the EC 50 of E2 for the zebrafish estrogen receptors alpha, beta 1 and beta 2 (ERa, ERb1, ERb2) is 77, 39, and 118 pM, respectively (Pinto et al, 2014).…”

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confidence: 99%

Quantification of Estradiol Uptake in Zebrafish Embryos and Larvae

Souder

Gorelick

2017

Toxicological Sciences

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Zebrafish are a powerful model system to assess the molecular and cellular effects of exposure to toxic chemicals during embryonic development. To study the effects of environmental endocrine disruptors, embryos and larvae are commonly exposed to supraphysiologic concentrations of these compounds in the water, but their bioavailability in zebrafish is largely unknown. One hypothesis is that supraphysiologic concentrations of estrogens in the water are required to achieve physiologic levels in vivo; however, this has not been directly tested. To test this hypothesis, we developed an assay using radiolabeled estradiol ([3H]E2) to measure uptake from water at multiple concentrations and exposure durations in developing zebrafish from 0 to 5 days postfertilization (dpf). We found that [3H]E2 uptake increased with increasing concentration, duration, and developmental stage. Percent uptake from the total volume of treatment solution increased with increasing exposure duration and developmental stage, but remained constant with increasing concentration. We also found that the chorion, an acellular envelope surrounding embryos through 3 dpf, did not substantially affect [3H]E2 uptake. Finally, we found that at 1 dpf, E2 was preferentially taken up by the yolk at multiple exposure durations, while at 2 dpf E2 was preferentially taken up into the embryonic body. Our results support the hypothesis that exposing zebrafish embryos and larvae to supraphysiologic concentrations of estrogens is required to achieve physiologically relevant doses in vivo. The isotopic assay reported here will provide a foundation for determining the uptake of other compounds for teratogenicity, toxicology and drug discovery studies.

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confidence: 99%

Quantification of Estradiol Uptake in Zebrafish Embryos and Larvae

Souder

Gorelick

2017

Toxicological Sciences

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“…Several zebrafish imaging screens have been published previously, but they were mostly carried out in 96-well plates and the resolution was low to allow imaging of the entire well. [22][23][24] Screening in 384-well plates at higher magnification with automated image analysis is rare and particularly challenging. 25,26 First, as zebrafish are nonadherent, robotic wash steps (e.g., after fixation) are at risk of washing the embryos away.…”

Section: Discussionmentioning

confidence: 99%

In Vivo Chemical Screen in Zebrafish Embryos Identifies Regulators of Hematopoiesis Using a Semiautomated Imaging Assay

et al. 2016

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Hematopoietic stem and progenitor cells (HSPCs) generate all cell types of the blood and are crucial for homeostasis of all blood lineages in vertebrates. Hematopoietic stem cell transplantation (HSCT) is a rapidly evolving technique that offers potential cure for hematologic cancers, such as leukemia or lymphoma. HSCT may be autologous or allogenic. Successful HSCT depends critically on the abundance of engraftment-competent HSPCs, which are currently difficult to obtain in large numbers. Therefore, finding compounds that enhance either the number or the activity of HSPCs could improve prognosis for patients undergoing HSCT and is of great clinical interest. We developed a semiautomated screening method for whole zebrafish larvae using conventional liquid handling equipment and confocal microscopy. Applying this pipeline, we screened 550 compounds in triplicate for proliferation of HSPCs in vivo and identified several modulators of hematopoietic stem cell activity. One identified hit was valproic acid (VPA), which was further validated as a compound that expands and maintains the population of HSPCs isolated from human peripheral blood ex vivo. In summary, our in vivo zebrafish imaging screen identified several potential drug candidates with clinical relevance and could easily be further expanded to screen more compounds.

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“…One approach has been to use small glass capillaries for imaging embryos, as in the VAST Bioimager which allows for automated imaging of zebrafish embryos in a chosen orientation (Pulak, 2016, Pardo-Martin et al, 2010, Pardo-Martin et al, 2013, Chang et al, 2012, Haney et al, 2020. Zebrafish embryos can also be imaged in a semi-automated format by standard microscopes in 96-well plates (Romano and Gorelick, 2014), although there is limited control over the orientation of the fish without manual manipulation or inspection of the images.…”

Section: Introductionmentioning

confidence: 99%

A versatile, automated and high-throughput drug screening platform for zebrafish embryos

et al. 2021

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Zebrafish provide a unique opportunity for drug screening in living animals, with the fast developing, transparent embryos allowing for relatively high-throughput, microscopy-based screens. However, the limited availability of rapid, flexible imaging and analysis platforms has limited the use of zebrafish in drug screens. We have developed an easy-to-use, customisable automated screening procedure suitable for high-throughput phenotype-based screens of live zebrafish. We utilised the WiScan® Hermes High Content Imaging System to rapidly acquire brightfield and fluorescent images of embryos, and the WiSoft® Athena Zebrafish Application for analysis, which harnesses an Artificial Intelligence-driven algorithm to automatically detect fish in brightfield images, identify anatomical structures, partition the animal into regions, and exclusively select the desired side-oriented fish. Our initial validation combined structural analysis with fluorescence images to enumerate GFP-tagged haematopoietic stem and progenitor cells in the tails of embryos, which correlated with manual counts. We further validated this system to assess the effects of genetic mutations and x-ray irradiation in high content using a wide range of assays. Further, we performed simultaneous analysis of multiple cell types using dual fluorophores in high throughput. In summary, we demonstrate a broadly applicable and rapidly customisable platform for high-content screening in zebrafish.

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Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Cited by 6 publications

References 10 publications

Quantification of Estradiol Uptake in Zebrafish Embryos and Larvae

Quantification of Estradiol Uptake in Zebrafish Embryos and Larvae

In Vivo Chemical Screen in Zebrafish Embryos Identifies Regulators of Hematopoiesis Using a Semiautomated Imaging Assay

A versatile, automated and high-throughput drug screening platform for zebrafish embryos

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