ZebrafishMiner: an open source software for interactive evaluation of domain-specific fluorescence in zebrafish

Abstract High-throughput microscopy makes it possible to observe the morphology of zebrafish on large scale to quantify genetic, toxic or drug effects. The image acquisition is done by automated microscopy, images are evaluated automatically by image processing pipelines, tailored specifically to the requirements of the scientific question. The transfer of such algorithms to other projects, however, is complex due to missing guidelines and lack of mathematical or programming knowledge. In this work, we implement an image processing pipeline for automatic fluorescence quantification in user-defined domains of zebrafish embryos and larvae of different age. The pipeline is capable of detecting embryos and larvae in image stacks and quantifying domain activity. To make this protocol available to the community, we developed an open source software package called „ZebrafishMiner“ which guides the user through all steps of the processing pipeline and makes the algorithms available and easy to handle. We implemented all routines in an MATLAB-based graphical user interface (GUI) that gives the user control over all image processing parameters. The software is shipped with a manual of 30 pages and three tutorial datasets, which guide the user through the manual step by step. It can be downloaded at https://sourceforge.net/projects/scixminer/.</jats:p

Novel cargo-binding site in the β and δ subunits of coatomer

Arginine (R)-based ER localization signals are sorting motifs that confer transient ER localization to unassembled subunits of multimeric membrane proteins. The COPI vesicle coat binds R-based signals but the molecular details remain unknown. Here, we use reporter membrane proteins based on the proteolipid Pmp2 fused to GFP and allele swapping of COPI subunits to map the recognition site for R-based signals. We show that two highly conserved stretches—in the β- and δ-COPI subunits—are required to maintain Pmp2GFP reporters exposing R-based signals in the ER. Combining a deletion of 21 residues in δ-COP together with the mutation of three residues in β-COP gave rise to a COPI coat that had lost its ability to recognize R-based signals, whilst the recognition of C-terminal di-lysine signals remained unimpaired. A homology model of the COPI trunk domain illustrates the recognition of R-based signals by COPI

A high-throughput chemically induced inflammation assay in zebrafish

Artículo de publicación ISIBackground: Studies on innate immunity have benefited from the introduction of zebrafish as a model system. Transgenic fish expressing fluorescent proteins in leukocyte populations allow direct, quantitative visualization of an inflammatory response in vivo. It has been proposed that this animal model can be used for high-throughput screens aimed at the identification of novel immunomodulatory lead compounds. However, current assays require invasive manipulation of fish individually, thus preventing high-content screening. Results: Here we show that specific, noninvasive damage to lateral line neuromast cells can induce a robust acute inflammatory response. Exposure of fish larvae to sublethal concentrations of copper sulfate selectively damages the sensory hair cell population inducing infiltration of leukocytes to neuromasts within 20 minutes. Inflammation can be assayed in real time using transgenic fish expressing fluorescent proteins in leukocytes or by histochemical assays in fixed larvae. We demonstrate the usefulness of this method for chemical and genetic screens to detect the effect of immunomodulatory compounds and mutations affecting the leukocyte response. Moreover, we transformed the assay into a high-throughput screening method by using a customized automated imaging and processing system that quantifies the magnitude of the inflammatory reaction. Conclusions: This approach allows rapid screening of thousands of compounds or mutagenized zebrafish for effects on inflammation and enables the identification of novel players in the regulation of innate immunity and potential lead compounds toward new immunomodulatory therapies. We have called this method the chemically induced inflammation assay, or ChIn assay.This work was supported by grants to MA from Fondecyt (1070867), FONDAP (15090007), ICM (P06-039F), CORFO-Innova (09MCSS-6705), DFG-Conicyt 075-2009; to CD from UNAB (DI- 01-09/1) and Fondecyt (24090004); to UL from Dopaminet (EU FP7 223744); and to CG by a Marie Curie International Reintegration Grant (EU FP7; PIRG07-GA-2010-267552)

Localization of orphanin FQ (nociceptin) peptide and messenger RNA in the central nervous system of the rat

Orphanin FQ (OFQ) is the endogenous agonist of the opioid receptor-like receptor (ORL-1). It and its precursor, prepro-OFQ, exhibit structural features suggestive of the opioid peptides. A cDNA encoding the OFQ precursor sequence in the rat recently has been cloned, and the authors recently generated a polyclonal antibody directed against the OFQ peptide. In the present study, the authors used in situ hybridization and immunohistochemistry to examine the distribution of OFQ peptide and mRNA in the central nervous system of the adult rat. OFQ immunoreactivity and prepro-OFQ mRNA expression correlated virtually in all brain areas studied. In the forebrain, OFQ peptide and mRNA were prominent in the neocortex endopiriform nucleus, claustrum, lateral septum, ventral forebrain, hypothalamus, mammillary bodies, central and medial nuclei of the amygdala, hippocampal formation, paratenial and reticular nuclei of the thalamus, medial habenula, and zona incerta. No OFQ was observed in the pineal or pituitary glands. In the brainstem, OFQ was prominent in the ventral tegmental area, substantia nigra, nucleus of the posterior commissure, central gray, nucleus of Darkschewitsch, peripeduncular nucleus, interpeduncular nucleus, tegmental nuclei, locus coeruleus, raphe complex, lateral parabrachial nucleus, inferior olivary complex, vestibular nuclear complex, prepositus hypoglossus, solitary nucleus, nucleus ambiguous, caudal spinal trigeminal nucleus, and reticular formation. In the spinal cord, OFQ was observed throughout the dorsal and ventral horns. The wide distribution of this peptide provides support for its role in a multitude of functions, including not only nociception but also motor and balance control, special sensory processing, and various autonomic and physiologic processes. J. Comp. Neurol. 406:503–547, 1999. © 1999 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/34452/1/7_ftp.pd

ZebrafishMiner: an open source software for interactive evaluation of domain-specific fluorescence in zebrafish

High-throughput microscopy makes it possible to observe the morphology of zebrafish on large scale to quantify genetic, toxic or drug effects. The image acquisition is done by automated microscopy, images are evaluated automatically by image processing pipelines, tailored specifically to the requirements of the scientific question. The transfer of such algorithms to other projects, however, is complex due to missing guidelines and lack of mathematical or programming knowledge. In this work, we implement an image processing pipeline for automatic fluorescence quantification in user-defined domains of zebrafish embryos and larvae of different age. The pipeline is capable of detecting embryos and larvae in image stacks and quantifying domain activity. To make this protocol available to the community, we developed an open source software package called „ZebrafishMiner“ which guides the user through all steps of the processing pipeline and makes the algorithms available and easy to handle. We implemented all routines in an MATLAB-based graphical user interface (GUI) that gives the user control over all image processing parameters. The software is shipped with a manual of 30 pages and three tutorial datasets, which guide the user through the manual step by step. It can be downloaded at https://sourceforge.net/projects/scixminer/

Protocol for intelligent high-content screening of zebrafish embryos on a standard widefield screening microscope

Here we demonstrate a toolset for automated intelligent high-content screening of whole zebrafish embryos at cellular resolution on a standard wide-field screening microscope. Using custom-developed algorithms, predefined regions of interest, such as the brain, are automatically detected. The regions of interest are subsequently imaged automatically at high magnification, enabling rapid capture of cellular resolution data. standardized imaging of internal organs and cellular structures.</p