scholarly journals Ultraspecific live imaging of the dynamics of zebrafish neutrophil granules by a histopermeable fluorogenic benzochalcone probe

2019 ◽  
Vol 10 (12) ◽  
pp. 3654-3670 ◽  
Author(s):  
Emma Colucci-Guyon ◽  
Ariane S. Batista ◽  
Suellen D. S. Oliveira ◽  
Magali Blaud ◽  
Ismael C. Bellettini ◽  
...  
Keyword(s):  
Live Imaging ◽  
Zebrafish Embryos ◽  
Wild Type

A fluorogenic benzochalcone specifically labels live neutrophil granules in whole wild-type, GFP- or RFP-expressing zebrafish embryos and larvae.

scholarly journals Anti-angiogenic Activity and Mechanism of Sesquiterpene Lactones from Centipeda minima

2016 ◽  
Vol 11 (4) ◽  
pp. 1934578X1601100 ◽  
Author(s):  
Weihuan Huang ◽  
Xiaobin Yu ◽  
Ning Liang ◽  
Wei Ge ◽  
Hin Fai Kwok ◽  
...  
Keyword(s):  
Sesquiterpene Lactones ◽  
Ethanol Extract ◽  
Zebrafish Embryos ◽  
Vegf Receptor ◽  
Wild Type ◽  
Zebrafish Model ◽  
Angiogenic Activity ◽  
Vessel Formation ◽  
Control Value ◽  
Centipeda Minima

Centipeda minima is a Chinese herbal medicine used in the treatment of various diseases including cancer. An ethanol extract of the herb, its four fractions with different polarities, and two volatile oils prepared by steam distillation (SD) and supercritical fluid extraction (SFE) were investigated for their anti-angiogenic activity in a wild-type zebrafish model using a quantitative endogenous alkaline phosphatase (EAP) assay. The SFE oil displayed potent anti-angiogenic activity. Fifteen sesquiterpene lactones (SLs; compounds 1–15) isolated from the SFE oil were evaluated for their anti-angiogenic effect. Results revealed that pseudoguaianolide type SLs (1–8) inhibited vessel formation in the zebrafish embryos while guaianolide type SLs (9–15) showed little effect. Among the active ones, 6- O-angeloylenolin (1), a major component of SFE oil, possessed the strongest effect by reducing vessel formation in zebrafish embryos to 40% of the control value at 29.7 μM. Further study using the Tg ( fli1a:EGFP) y1-type zebrafish model revealed that it blocked both intersegmental blood vessels (ISVs) and subintestinal vessels plexus (SIVs) formation in zebrafish embryos. Real-time polymerase chain reaction assay on the wild-type zebrafish embryos suggested that 6- O-angeloylenolin affected multiple molecular targets related to angiogenesis including VEGF receptor, angiopoietin, and its receptors. Taken together, our findings demonstrate that C. minima possesses anti-angiogenic activity, and 6- O-angeloylenolin is a promising candidate for the development of an anti-angiogenic agent.

scholarly journals Fully Automated Pipetting Sorting System for Different Morphological Phenotypes of Zebrafish Embryos

2017 ◽  
Vol 23 (2) ◽  
pp. 128-133 ◽  
Author(s):  
Helmut Breitwieser ◽  
Thomas Dickmeis ◽  
Marcel Vogt ◽  
Marco Ferg ◽  
Christian Pylatiuk
Keyword(s):  
Template Matching ◽  
Zebrafish Embryo ◽  
Petri Dish ◽  
Microtiter Plate ◽  
Model Organisms ◽  
Zebrafish Embryos ◽  
Wild Type ◽  
Patterning Defect ◽  
Glucocorticoid Deficiency ◽  
Sorting System

Systems biology methods, such as transcriptomics and metabolomics, require large numbers of small model organisms, such as zebrafish embryos. Manual separation of mutant embryos from wild-type embryos is a tedious and time-consuming task that is prone to errors, especially if there are variable phenotypes of a mutant. Here we describe a zebrafish embryo sorting system with two cameras and image processing based on template-matching algorithms. In order to evaluate the system, zebrafish rx3 mutants that lack eyes due to a patterning defect in brain development were separated from their wild-type siblings. These mutants show glucocorticoid deficiency due to pituitary defects and serve as a model for human secondary adrenal insufficiencies. We show that the variable phenotypes of the mutant embryos can be safely distinguished from phenotypic wild-type zebrafish embryos and sorted from one petri dish into another petri dish or into a 96-well microtiter plate. On average, classification of a zebrafish embryo takes approximately 1 s, with a sensitivity and specificity of 87% to 95%, respectively. Other morphological phenotypes may be classified and sorted using similar techniques.

scholarly journals The microtubule organization in the zebrafish yolk adapts to transgene-mediated phenotypic variations

2020 ◽  
Author(s):  
Maria Marsal ◽  
Matteo Bernardello ◽  
Emilio J. Gualda ◽  
Pablo Loza-Alvarez
Keyword(s):  
Light Sheet ◽  
Variable Number ◽  
Zebrafish Embryos ◽  
Wild Type ◽  
Microtubule Organization ◽  
Molecular Machinery ◽  
Phenotypic Variations ◽  
Microtubule Networks ◽  
Vast Network ◽  
Light Sheet Fluorescence Microscopy

SUMMARYThe organization of microtubule networks in the cells is orchestrated by subcellular structures named microtubule organizing centers (MTOCs). In zebrafish embryos, the yolk is surrounded by a cytoplasmic layer containing a vast network of microtubules. In order to understand how this complex network is organized, we use dclk2-GFP zebrafish embryos, as a microtubule reporter line, and Light Sheet Fluorescence Microscopy. We find that this organization is mediated by a variable number of aster-like MTOCs during epiboly, and that it does not follow a rigid scheme, exemplifying developmental robustness. We characterize asters morphology, dynamics, and their uniform distribution in the yolk sphere. Consistent with their role as MTOCs we find that they contain key molecular machinery for MTs dynamics, amongst which centrin marks the assignation of MTOCs over time. Finally, we demonstrate that merely the overexpression of dclk2-GFP in wild type embryos can induce the formation of asters. We propose dclk2-GFP embryos as a model for the study of the collective behaviour of microtubules in complex systems.

scholarly journals In vivo dynamics of skeletal muscle Dystrophin in zebrafish embryos revealed by improved FRAP analysis

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Fernanda Bajanca ◽  
Vinicio Gonzalez-Perez ◽  
Sean J Gillespie ◽  
Cyriaque Beley ◽  
Luis Garcia ◽  
...  
Keyword(s):  
Turnover Rate ◽  
Zebrafish Embryos ◽  
Wild Type ◽  
High Turnover ◽  
Complex Protein ◽  
In Vivo Analysis ◽  
Membrane Bound ◽  
First Time ◽  
Developing Muscle

Dystrophin forms an essential link between sarcolemma and cytoskeleton, perturbation of which causes muscular dystrophy. We analysed Dystrophin binding dynamics in vivo for the first time. Within maturing fibres of host zebrafish embryos, our analysis reveals a pool of diffusible Dystrophin and complexes bound at the fibre membrane. Combining modelling, an improved FRAP methodology and direct semi-quantitative analysis of bleaching suggests the existence of two membrane-bound Dystrophin populations with widely differing bound lifetimes: a stable, tightly bound pool, and a dynamic bound pool with high turnover rate that exchanges with the cytoplasmic pool. The three populations were found consistently in human and zebrafish Dystrophins overexpressed in wild-type or dmdta222a/ta222a zebrafish embryos, which lack Dystrophin, and in Gt(dmd-Citrine)ct90a that express endogenously-driven tagged zebrafish Dystrophin. These results lead to a new model for Dystrophin membrane association in developing muscle, and highlight our methodology as a valuable strategy for in vivo analysis of complex protein dynamics.

Retinoic Acid Blockade Increases Primitive Blood Cell Formation in cdx4 Mutant Zebrafish Embryos, Murine Yolk Sac Explants and Differentiated Embryonic Stem Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 201-201
Author(s):  
Jill L. de Jong ◽  
Alan J. Davidson ◽  
Yuan Wang ◽  
James Palis ◽  
George Q. Daley ◽  
...  
Keyword(s):  
Stem Cells ◽  
Retinoic Acid ◽  
Embryonic Stem Cells ◽  
Embryoid Body ◽  
Embryonic Stem ◽  
Yolk Sac ◽  
Zebrafish Embryos ◽  
Retinoic Acid Signaling ◽  
Wild Type ◽  
Hematopoietic Stem

Abstract Retinoic acid signaling is critical for proper development, as well as the regulation of hematopoiesis in murine and human cells. It has been reported that retinoic acid expands definitive hematopoietic stem cells and confers an advantage in transplantation of murine HSCs. We demonstrate here that all trans retinoic acid (ATRA) blocks early hematopoiesis in zebrafish embryos as measured by decreased expression of scl and gata1. This loss of gata1 expression after exposure to ATRA is reminiscent of the lack of gata1 expression in cdx4 mutant zebrafish embryos, prompting the hypothesis that retinoic acid and cdx4 share a common signaling pathway regulating the emergence of the hematopoietic system, and that inhibition of ATRA might rescue blood development in cdx4 mutants. We tested 4-diethylamino benzaldehyde (DEAB), an inhibitor of retinoic acid biosynthesis, and AGN193109, a pan-RAR antagonist, for their ability to rescue gata1 expression in cdx4 mutant embryos. We found that treatment with either DEAB or AGN193109 results in increased gata1 expression in the posterior mesoderm of both cdx4 mutants and wild type zebrafish embryos. This rescue of gata1 expression in the cdx4 mutants is observed only when the DEAB treatment occurs during late gastrulation. Mutant embryos exposed to DEAB only during early gastrulation or somitogenesis did not have increased gata1 expression. While this observation may reflect the complex activity of ATRA in both hematopoiesis and in mesodermal patterning, we conclude that it is also consistent with a switch to the stimulatory effect of retinoic acid on hematopoiesis observed by others in adult mice. Similarly, DEAB treatment increased primitive erythroid cells in murine hematopoietic cultures. We show that E7.5 murine yolk sac explant cultures exposed to DEAB have increased primitive erythroid colony forming cells (EryP-CFC) and definitive erythroid burst forming units (BFU-E). In contrast, ATRA markedly inhibits all erythroid colony formation in murine yolk sac explants. Likewise, murine embryonic stem cells treated with DEAB during embryoid body development have an 8-fold expansion of EryP-CFC, and a 2-fold expansion of multipotent GEMM colonies. Previously we published that overexpression of hoxA9 mRNA rescues gata1 expression in cdx4 mutant embryos. However, the hematopoietic defect in cdx4 mutant embryos is not rescued by overexpression of scl. To test the hypothesis that hoxA9 and scl are both signaling downstream of retinoic acid in the stimulation of primitive erythroid development, we overexpressed hoxA9 or scl by microinjecting mRNA into single-cell wild type zebrafish embryos followed by treatment with ATRA. We find that overexpression of either hoxA9 or scl partially rescues the block of hematopoiesis induced by ATRA. Taken together, these data indicate that the commitment of mesodermal cells to hematopoietic fates is inhibited by retinoic acid, and that the retinoic acid signal is acting downstream of cdx4 in the zebrafish embryo, while scl and hoxA9 are acting downstream of retinoic acid signaling.

scholarly journals Abrogating ALIX Interactions Results in Stuttering of the ESCRT Machinery

Viruses ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 1032 ◽  
Author(s):  
Shilpa Gupta ◽  
Mourad Bendjennat ◽  
Saveez Saffarian
Keyword(s):  
Binding Site ◽  
Linear Model ◽  
Residence Time ◽  
Live Imaging ◽  
Assembly Model ◽  
Wild Type ◽  
Cellular Membranes ◽  
Virion Assembly ◽  
Endosomal Sorting ◽  
Escrt Machinery

Endosomal sorting complexes required for transport (ESCRT) proteins assemble on budding cellular membranes and catalyze their fission. Using live imaging of HIV virions budding from cells, we followed recruitment of ESCRT proteins ALIX, CHMP4B and VPS4. We report that the ESCRT proteins transiently co-localize with virions after completion of virion assembly for durations of 45 ± 30 s. We show that mutagenizing the YP domain of Gag which is the primary ALIX binding site or depleting ALIX from cells results in multiple recruitments of the full ESCRT machinery on the same virion (referred to as stuttering where the number of recruitments to the same virion >3). The stuttering recruitments are approximately 4 ± 3 min apart and have the same stoichiometry of ESCRTs and same residence time (45 ± 30 s) as the single recruitments in wild type interactions. Our observations suggest a role for ALIX during fission and question the linear model of ESCRT recruitment, suggesting instead a more complex co-assembly model.

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