Small molecule screen in embryonic zebrafish using modular variations to target segmentation

Richter, Sandra; Schulze, Ulrike; Tomančák, Pavel; Oates, Andrew C.

doi:10.1038/s41467-017-01469-5

Cited by 21 publications

(19 citation statements)

References 73 publications

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“…We used an in situ hybridisation screen to analyse gene expression changes, as this has the advantages of being scalable to different sized projects and relatively inexpensive to perform—with results that are stable and reproducible. Spatial resolution of staining patterns can be accurately scored: expression pattern screens have recently been used to identify small molecules that can induce subtle differences in gene expression domains along the pronephros (Poureetezadi et al, 2016) and in the somites (Richter et al, 2017). Although quantification of gene expression levels is less reliable with an enzymatic reaction compared with a fluorescent signal, we utilised the strong contrast between the high vcanb expression in the ear of adgrg6 mutant fish compared with the low expression in a small dorsal region of the wild-type ear at four dpf to produce a robust scoring system for our phenotype rescue.…”

Section: Discussionmentioning

confidence: 99%

“…A promising alternative approach lies in the potential of unbiased whole-animal screening of small molecules. In recent years, zebrafish have emerged as an important tool for in vivo phenotypic screening for new therapeutics (Brady et al, 2016) and for understanding biological mechanisms (Baxendale et al, 2017; Richter et al, 2017). Zebrafish have many advantages for drug discovery: they are a vertebrate species whose embryos can fit into individual wells of a multiwell plate, facilitating high-throughput analysis; they generate large numbers of offspring; they can absorb compounds directly added to the water, and whole-organism screening enables toxicity, absorption, metabolism and excretion of compounds to be assayed early in the screening pipeline.…”

Section: Introductionmentioning

confidence: 99%

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Identification of compounds that rescue otic and myelination defects in the zebrafish adgrg6 (gpr126) mutant

Diamantopoulou

Baxendale

Léon

et al. 2019

eLife

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Adgrg6 (Gpr126) is an adhesion class G protein-coupled receptor with a conserved role in myelination of the peripheral nervous system. In the zebrafish, mutation of adgrg6 also results in defects in the inner ear: otic tissue fails to down-regulate versican gene expression and morphogenesis is disrupted. We have designed a whole-animal screen that tests for rescue of both up- and down-regulated gene expression in mutant embryos, together with analysis of weak and strong alleles. From a screen of 3120 structurally diverse compounds, we have identified 68 that reduce versican b expression in the adgrg6 mutant ear, 41 of which also restore myelin basic protein gene expression in Schwann cells of mutant embryos. Nineteen compounds unable to rescue a strong adgrg6 allele provide candidates for molecules that may interact directly with the Adgrg6 receptor. Our pipeline provides a powerful approach for identifying compounds that modulate GPCR activity, with potential impact for future drug design.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Identification of compounds that rescue otic and myelination defects in the zebrafish adgrg6 (gpr126) mutant

Diamantopoulou

Baxendale

Léon

et al. 2019

eLife

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“…As a model system, zebrafish embryos are well suitable for this kind of screening strategy. Several small molecule screens in zebrafish embryos have been performed to uncover novel regulators of stem cell homeostasis, embryonic development, inflammation or behavioral states (Peterson et al, 2000;North et al, 2007;Kokel et al, 2010;Rihel et al, 2010;Robertson et al, 2014;Richter et al, 2017). In these screens, embryos or larvae are incubated in solutions of the active compounds, which are assumed to be taken up passively through skin and gills, often facilitated by including non-lethal concentrations of organic solvents like DMSO.…”

Section: Discussionmentioning

confidence: 99%

Chemical Genetics Screen Identifies Epigenetic Mechanisms Involved in Dopaminergic and Noradrenergic Neurogenesis in Zebrafish

et al. 2020

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The cell type diversity and complexity of the nervous system is generated by a network of signaling events, transcription factors, and epigenetic regulators. Signaling and transcriptional control have been easily amenable to forward genetic screens in model organisms like zebrafish. In contrast, epigenetic mechanisms have been somewhat elusive in genetic screens, likely caused by broad action in multiple developmental pathways that masks specific phenotypes, but also by genetic redundancies of epigenetic factors. Here, we performed a screen using small molecule inhibitors of epigenetic mechanisms to reveal contributions to specific aspects of neurogenesis in zebrafish. We chose development of dopaminergic and noradrenergic neurons from neural progenitors as target of epigenetic regulation. We performed the screen in two phases: First, we tested a small molecule inhibitor library that targets a broad range of epigenetic protein classes and mechanisms, using expression of the dopaminergic and noradrenergic marker tyrosine hydroxylase as readout. We identified 10 compounds, including HDAC, Bromodomain and HAT inhibitors, which interfered with dopaminergic and noradrenergic development in larval zebrafish. In the second screening phase, we aimed to identify neurogenesis stages affected by these 10 inhibitors. We analyzed treated embryos for effects on neural stem cells, growth progression of the retina, and apoptosis in neural tissues. In addition, we analyzed effects on islet1 expressing neuronal populations to determine potential selectivity of compounds for transmitter phenotypes. In summary, our targeted screen of epigenetic inhibitors identified specific compounds, which reveal chromatin regulator classes that contribute to dopaminergic and noradrenergic neurogenesis in vivo.

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“…3D). The quality of the 3D bioprinted spheroids was tested with the statistically robust standardized mean difference (SSMD) analysis (30,31). We conducted this analysis on all high-throughput screening experiments, with the results indicating that 95% of our printed spheroids, and hence the assays, were of good or greater quality, when compared to the acceptable criteria of the SSMD analysis ( Fig.…”

Section: D Bioprinted Spheroids As a High-throughput Tool For Evaluamentioning

confidence: 99%

Precise, high-throughput production of multicellular spheroids with a bespoke 3D bioprinter

Utama

Atapattu

O’Mahony³

et al. 2020

Preprint

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KeywordsBioprinting | 3d cell culture | high-throughput screening | multicellular spheroids | biomaterials Abstract 3D in vitro cancer models are important therapeutic and biological discovery tools, yet formation of multicellular spheroids in a throughput and highly controlled manner to achieve robust and statistically relevant data, remains challenging. Here, we developed an enabling technology consisting of a bespoke drop-on-demand 3D bioprinter capable of high-throughput printing of 96well plates of spheroids. 3D-multicellular spheroids are embedded inside a tissue-like matrix with precise control over size and cell number. Application of 3D bioprinting for high-throughput drug screening was demonstrated with doxorubicin. Measurements showed that IC50 values were sensitive to spheroid size, embedding and how spheroids conform to the embedding, revealing parameters shaping biological responses in these models. Our study demonstrates the potential of 3D bioprinting as a robust high-throughput platform to screen biological and therapeutic parameters. Significance StatementIn vitro 3D cell cultures serve as more realistic models, compared to 2D cell culture, for understanding diverse biology and for drug discovery. Preparing 3D cell cultures with defined parameters is challenging, with significant failure rates when embedding 3D multicellular spheroids into extracellular mimics. Here, we report a new 3D bioprinter we developed in conjunction with bioinks to allow 3D-multicellular spheroids to be produced in a high-throughput manner. Highthroughput production of embedded multicellular spheroids allowed entire drug-dose responses to be performed in 96-well plate format with statistically relevant numbers of data points. We have deconvoluted important parameters in drug responses including the impact of spheroid size and embedding in an extracellular matrix mimic on IC50 values.

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Small molecule screen in embryonic zebrafish using modular variations to target segmentation

Cited by 21 publications

References 73 publications

Identification of compounds that rescue otic and myelination defects in the zebrafish adgrg6 (gpr126) mutant

Identification of compounds that rescue otic and myelination defects in the zebrafish adgrg6 (gpr126) mutant

Chemical Genetics Screen Identifies Epigenetic Mechanisms Involved in Dopaminergic and Noradrenergic Neurogenesis in Zebrafish

Precise, high-throughput production of multicellular spheroids with a bespoke 3D bioprinter

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