scholarly journals MicroCT-based phenomics in the zebrafish skeleton reveals virtues of deep phenotyping in a distributed organ system

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Matthew Hur ◽  
Charlotte A Gistelinck ◽  
Philippe Huber ◽  
Jane Lee ◽  
Marjorie H Thompson ◽  
...  
Keyword(s):  
Axial Skeleton ◽  
Thyroid Stimulating Hormone ◽  
Organ System ◽  
Genetic Screens ◽  
Spatially Distributed ◽  
Functional Understanding ◽  
Increase Productivity ◽  
Brittle Bone Disease ◽  
Mutant Phenotypes ◽  
Deep Phenotyping

Phenomics, which ideally involves in-depth phenotyping at the whole-organism scale, may enhance our functional understanding of genetic variation. Here, we demonstrate methods to profile hundreds of phenotypic measures comprised of morphological and densitometric traits at a large number of sites within the axial skeleton of adult zebrafish. We show the potential for vertebral patterns to confer heightened sensitivity, with similar specificity, in discriminating mutant populations compared to analyzing individual vertebrae in isolation. We identify phenotypes associated with human brittle bone disease and thyroid stimulating hormone receptor hyperactivity. Finally, we develop allometric models and show their potential to aid in the discrimination of mutant phenotypes masked by alterations in growth. Our studies demonstrate virtues of deep phenotyping in a spatially distributed organ system. Analyzing phenotypic patterns may increase productivity in genetic screens, and facilitate the study of genetic variants associated with smaller effect sizes, such as those that underlie complex diseases.

scholarly journals microCT-Based Phenomics in the Zebrafish Skeleton Reveals Virtues of Deep Phenotyping in a Distributed Organ System

2017 ◽  
Author(s):  
Matthew Hur ◽  
Charlotte A. Gistelinck ◽  
Philippe Huber ◽  
Jane Lee ◽  
Marjorie H. Thompson ◽  
...  
Keyword(s):  
Axial Skeleton ◽  
Thyroid Stimulating Hormone ◽  
Effect Sizes ◽  
Genetic Screens ◽  
Spatially Distributed ◽  
Functional Understanding ◽  
Increase Productivity ◽  
Brittle Bone Disease ◽  
Mutant Phenotypes ◽  
Deep Phenotyping

ABSTRACTPhenomics, which ideally involves in-depth phenotyping at the whole-organism scale, may enhance our functional understanding of genetic variation. Here, we demonstrate methods to profile hundreds of measures comprised of morphological and densitometric traits from a large number sites in the axial skeleton of adult zebrafish. We show the potential for vertebral patterns to confer heightened sensitivity, with similar specificity, in discriminating mutant populations compared to analyzing individual vertebrae in isolation. We identify phenotypes associated with human brittle bone disease and thyroid stimulating hormone receptor hyperactivity. Finally, we develop allometric models and show their potential to aid in the discrimination of mutant phenotypes masked by alterations in growth. Our studies demonstrate virtues of deep phenotyping in a spatially distributed organ. Analyzing phenotypic patterns may increase productivity in genetic screens, and could facilitate the study of genetic variants associated with smaller effect sizes, such as those that underlie complex diseases.

scholarly journals A simple and effective F0 knockout method for rapid screening of behaviour and other complex phenotypes

2020 ◽  
Author(s):  
François Kroll ◽  
Gareth T Powell ◽  
Marcus Ghosh ◽  
Paride Antinucci ◽  
Timothy J Hearn ◽  
...  
Keyword(s):  
Neurological Diseases ◽  
Rapid Screening ◽  
Genetic Screens ◽  
Behavioural Phenotype ◽  
Larval Zebrafish ◽  
Complex Phenotypes ◽  
Human Genes ◽  
Behavioural Phenotypes ◽  
Mutant Phenotypes ◽  
Genetic Contributions

ABSTRACTHundreds of human genes are associated with neurological diseases, but translation into tractable biological mechanisms is lagging. Larval zebrafish are an attractive model to investigate genetic contributions to neurological diseases. However, current CRISPR-Cas9 methods are difficult to apply to large genetic screens studying behavioural phenotypes. To facilitate rapid genetic screening, we developed a simple sequencing-free tool to validate gRNAs and a highly effective CRISPR-Cas9 method capable of converting >90% of injected embryos directly into F0 biallelic knockouts. We demonstrate that F0 knockouts reliably recapitulate complex mutant phenotypes, such as altered molecular rhythms of the circadian clock, escape responses to irritants, and multi-parameter day-night locomotor behaviours. The technique is sufficiently robust to knockout multiple genes in the same animal, for example to create the transparent triple knockout crystal fish for imaging. Our F0 knockout method cuts the experimental time from gene to behavioural phenotype in zebrafish from months to one week.

scholarly journals A simple and effective F0 knockout method for rapid screening of behaviour and other complex phenotypes

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
François Kroll ◽  
Gareth T Powell ◽  
Marcus Ghosh ◽  
Gaia Gestri ◽  
Paride Antinucci ◽  
...  
Keyword(s):  
Neurological Diseases ◽  
Rapid Screening ◽  
Genetic Screens ◽  
Behavioural Phenotype ◽  
Larval Zebrafish ◽  
Complex Phenotypes ◽  
Human Genes ◽  
Behavioural Phenotypes ◽  
Mutant Phenotypes ◽  
Genetic Contributions

Hundreds of human genes are associated with neurological diseases, but translation into tractable biological mechanisms is lagging. Larval zebrafish are an attractive model to investigate genetic contributions to neurological diseases. However, current CRISPR-Cas9 methods are difficult to apply to large genetic screens studying behavioural phenotypes. To facilitate rapid genetic screening, we developed a simple sequencing-free tool to validate gRNAs and a highly effective CRISPR-Cas9 method capable of converting >90% of injected embryos directly into F0 biallelic knockouts. We demonstrate that F0 knockouts reliably recapitulate complex mutant phenotypes, such as altered molecular rhythms of the circadian clock, escape responses to irritants, and multi-parameter day-night locomotor behaviours. The technique is sufficiently robust to knockout multiple genes in the same animal, for example to create the transparent triple knockout crystal fish for imaging. Our F0 knockout method cuts the experimental time from gene to behavioural phenotype in zebrafish from months to one week.

scholarly journals MicroCT-Based Phenomics in the Zebrafish Skeleton Reveals Virtues of Deep Phenotyping in a Distributed Organ System

Zebrafish ◽  
2018 ◽  
Vol 15 (1) ◽  
pp. 77-78 ◽  
Author(s):  
Matthew Hur ◽  
Charlotte A. Gistelinck ◽  
Philippe Huber ◽  
Jane Lee ◽  
Marjorie H. Thompson ◽  
...  
Keyword(s):  
Organ System ◽  
Deep Phenotyping

scholarly journals Author response: MicroCT-based phenomics in the zebrafish skeleton reveals virtues of deep phenotyping in a distributed organ system

2017 ◽  
Author(s):  
Matthew Hur ◽  
Charlotte A Gistelinck ◽  
Philippe Huber ◽  
Jane Lee ◽  
Marjorie H Thompson ◽  
...  
Keyword(s):  
Organ System ◽  
Author Response ◽  
Deep Phenotyping

scholarly journals Integration of Odor-Induced Activity of Kenyon Cells in an Electrotonically Compact Drosophila Mushroom Body Output Neuron (MBON)

2019 ◽  
Author(s):  
Omar Hafez ◽  
Benjamin Escribano ◽  
Jan Pielage ◽  
Ernst Niebur
Keyword(s):  
Mushroom Body ◽  
Behavioral Outcomes ◽  
Dendritic Structure ◽  
Internal Representation ◽  
Kenyon Cell ◽  
Sensory Stimuli ◽  
Output Neuron ◽  
Spatially Distributed ◽  
Functional Understanding ◽  
Distributed Cluster

AbstractThe formation of an ecologically useful lasting memory requires that the brain has an accurate internal representation of the surrounding environment. In addition, it must have the ability to integrate a variety of different sensory stimuli and associate them with rewarding and aversive behavioral outcomes. Over the previous years, a number of studies have dissected the anatomy and elucidated some of the working principles of the Drosophila mushroom body (MB), the fly’s center for learning and memory. As a consequence, we now have a functional understanding of where and how in the MB sensory stimuli converge and are associated. However, the molecular and cellular dynamics at the critical synaptic intersection for this process, the Kenyon cell-mushroom body output neuron (KC-MBON) synapse, are largely unknown. Here, we introduce a first approach to understand this integration process and the physiological changes occurring at the KC-MBON synapse during Kenyon cell (KC) activation. We use the published connectome of the Drosophila MB to construct a functional computational model of the MBON-α3-A dendritic structure. We simulate synaptic input by individual KC-MBON synapses by current injections into precisely (μm) identified local dendritic sections, and the input from a model population of KCs representing an odor by a spatially distributed cluster of current injections. By recording the effect of the simulated current injections on the membrane potential of the neuron, we show that the MBON-α3-A is electrotonically compact. This suggests that odor-induced MBON activity is likely governed by input strength while the positions of KC input synapses are largely irrelevant.

scholarly journals Eukaryotic MCM Proteins: Beyond Replication Initiation

2004 ◽  
Vol 68 (1) ◽  
pp. 109-131 ◽  
Author(s):  
Susan L. Forsburg
Keyword(s):  
Genome Stability ◽  
Dna Helicase ◽  
Replication Initiation ◽  
Genetic Screens ◽  
Minichromosome Maintenance ◽  
Experimental Systems ◽  
Damage Response ◽  
Mcm Proteins ◽  
Mutant Phenotypes ◽  
Related Proteins

SUMMARY The minichromosome maintenance (or MCM) protein family is composed of six related proteins that are conserved in all eukaryotes. They were first identified by genetic screens in yeast and subsequently analyzed in other experimental systems using molecular and biochemical methods. Early data led to the identification of MCMs as central players in the initiation of DNA replication. More recent studies have shown that MCM proteins also function in replication elongation, probably as a DNA helicase. This is consistent with structural analysis showing that the proteins interact together in a heterohexameric ring. However, MCMs are strikingly abundant and far exceed the stoichiometry of replication origins; they are widely distributed on unreplicated chromatin. Analysis of mcm mutant phenotypes and interactions with other factors have now implicated the MCM proteins in other chromosome transactions including damage response, transcription, and chromatin structure. These experiments indicate that the MCMs are central players in many aspects of genome stability.

Tissue section lifting for electron microscopy

1978 ◽  
Vol 36 (2) ◽  
pp. 86-87
Author(s):  
Adrian F. van Dellen
Keyword(s):  
Infectious Disease ◽  
Electron Microscopy ◽  
Tissue Culture ◽  
Organ System ◽  
Surrounding Tissue ◽  
Paraffin Sections ◽  
Surface Areas ◽  
Specific Determination ◽  
High Degree ◽  
Accuracy And Repeatability

The morphologic pathologist may require information on the ultrastructure of a non-specific lesion seen under the light microscope before he can make a specific determination. Such lesions, when caused by infectious disease agents, may be sparsely distributed in any organ system. Tissue culture systems, too, may only have widely dispersed foci suitable for ultrastructural study. In these situations, when only a few, small foci in large tissue areas are useful for electron microscopy, it is advantageous to employ a methodology which rapidly selects a single tissue focus that is expected to yield beneficial ultrastructural data from amongst the surrounding tissue. This is in essence what "LIFTING" accomplishes. We have developed LIFTING to a high degree of accuracy and repeatability utilizing the Microlift (Fig 1), and have successfully applied it to tissue culture monolayers, histologic paraffin sections, and tissue blocks with large surface areas that had been initially fixed for either light or electron microscopy.

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