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

eLife, Год журнала: 2021, Номер 10

Опубликована: Янв. 8, 2021

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

Язык: Английский

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High-throughput functional analysis of autism genes in zebrafish identifies convergence in dopaminergic and neuroimmune pathways

Cell Reports, Год журнала: 2023, Номер 42(3), С. 112243 - 112243

Опубликована: Март 1, 2023

Advancing from gene discovery in autism spectrum disorders (ASDs) to the identification of biologically relevant mechanisms remains a central challenge. Here, we perform parallel vivo functional analysis 10 ASD genes at behavioral, structural, and circuit levels zebrafish mutants, revealing both unique overlapping effects loss function. Whole-brain mapping identifies forebrain cerebellum as most significant contributors brain size differences, while regions involved sensory-motor control, particularly dopaminergic regions, are associated with altered baseline activity. Finally, show global increase microglia resulting function select implicating neuroimmune dysfunction key pathway biology.

Язык: Английский

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Convergence of coronary artery disease genes onto endothelial cell programs

Nature, Год журнала: 2024, Номер 626(8000), С. 799 - 807

Опубликована: Фев. 7, 2024

Язык: Английский

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Copper regulates the host innate immune response against bacterial infection via activation of ALPK1 kinase

Proceedings of the National Academy of Sciences, Год журнала: 2024, Номер 121(4)

Опубликована: Янв. 17, 2024

Copper is an essential trace element for the human body, and its requirement optimistic immune functions has been recognized decades. How copper involved in innate pathway, however, remains to be clarified. Here, we report that serves as a signal molecule regulate kinase activity of alpha-kinase 1 (ALPK1), cytosolic pattern-recognition receptor (PRR), therefore promotes host cell defense against bacterial infection. We show response infection, cells actively accumulate cytosol, accumulated enhances evading pathogens, including intracellular and, unexpectedly, extracellular bacteria. Subsequently, demonstrate activates pathway ALPK1-dependent manner. Further mechanistic studies reveal binds ALPK1 directly this PRR. Moreover, binding sensitivity metabolite ADP-heptose eventually prompts elicit enhanced during Finally, using zebrafish vivo model, copper-treated shows increased production proinflammatory cytokines, recruitment phagosome cells, promoted clearance. Our findings uncover previously unrecognized role modulation pathogens advance our knowledge on cross talk between homeostasis system.

Язык: Английский

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Parp1 promotes sleep, which enhances DNA repair in neurons

Molecular Cell, Год журнала: 2021, Номер 81(24), С. 4979 - 4993.e7

Опубликована: Ноя. 18, 2021

Язык: Английский

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Zebrafish Models of Cardiac Disease: From Fortuitous Mutants to Precision Medicine

Circulation Research, Год журнала: 2022, Номер 130(12), С. 1803 - 1826

Опубликована: Июнь 9, 2022

Heart disease is the leading cause of death worldwide. Despite decades research, most heart pathologies have limited treatments, and often only curative approach transplantation. Thus, there an urgent need to develop new therapeutic approaches for treating cardiac diseases. Animal models that reproduce human pathophysiology are essential uncovering biology diseases discovering therapies. Traditionally, mammals been used as disease, but cost generating maintaining exorbitant, studies very low throughput. In last decade, zebrafish has emerged a tractable model diseases, owing several characteristics made this animal popular among developmental biologists. Zebrafish fertilization development external; embryos can be obtained in high numbers, cheap easy maintain, manipulated create genetic models. Moreover, exhibit exceptional ability regenerate their after injury. This review summarizes 25 years research using study heart, from classical forward screenings contemporary methods mutations found patients with disease. We discuss advantages limitations organism introduce experimental exploited zebrafish, including reverse genetics chemical screenings. Last, we induce injury ideas derived studying natural regeneration. Studies potential accelerate discovery strategies treat

Язык: Английский

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CRISPR gRNA phenotypic screening in zebrafish reveals pro-regenerative genes in spinal cord injury

PLoS Genetics, Год журнала: 2021, Номер 17(4), С. e1009515 - e1009515

Опубликована: Апрель 29, 2021

Zebrafish exhibit robust regeneration following spinal cord injury, promoted by macrophages that control post-injury inflammation. However, the mechanistic basis of how regulate is poorly understood. To address this gap in understanding, we conducted a rapid vivo phenotypic screen for macrophage-related genes promote after injury. We used acute injection synthetic RNA Oligo CRISPR guide RNAs (sCrRNAs) were pre-screened high activity . Pre-screening over 350 sCrRNAs allowed us to rapidly identify highly active (up half, abbreviated as haCRs) and effectively target 30 potentially genes. Disruption 10 these impaired axonal selected 5 further analysis generated stable mutants using haCRs. Four ( tgfb1a , tgfb3 tnfa sparc ) retained haCR phenotype, validating approach. Mechanistically, haCR-injected mutant zebrafish fail resolve inflammation, indicated prolonged presence neutrophils increased levels il1b expression. Inhibition Il-1β rescues axon mutant. Hence, our scalable screening approach has identified functional regulators regeneration, but can be applied any biological function interest.

Язык: Английский

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Myelination generates aberrant ultrastructure that is resolved by microglia

The Journal of Cell Biology, Год журнала: 2022, Номер 222(3)

Опубликована: Дек. 22, 2022

To enable rapid propagation of action potentials, axons are ensheathed by myelin, a multilayered insulating membrane formed oligodendrocytes. Most the myelin is generated early in development, resulting generation long-lasting stable structures. Here, we explored structural and dynamic changes central nervous system during development. achieve this, performed an ultrastructural analysis mouse optic nerves serial block face scanning electron microscopy (SBF-SEM) confocal time-lapse imaging zebrafish spinal cord. We found that undergoes extensive postnatal Myelin degeneration profiles were engulfed phagocytosed microglia using exposed phosphatidylserine as one “eat me” signal. In contrast, retractions entire sheaths occurred independently involved uptake oligodendrocyte itself. Our findings show development inaccurate process associated with aberrant features require substantial refinement.

Язык: Английский

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Variant-to-gene mapping followed by cross-species genetic screening identifies GPI-anchor biosynthesis as a regulator of sleep

Science Advances, Год журнала: 2023, Номер 9(1)

Опубликована: Янв. 6, 2023

Genome-wide association studies (GWAS) in humans have identified loci robustly associated with several heritable diseases or traits, yet little is known about the functional roles of underlying causal variants regulating sleep duration quality. We applied an ATAC-seq/promoter focused Capture C strategy human iPSC-derived neural progenitors to carry out a "variant-to-gene" mapping campaign that 88 candidate effector genes connected relevant GWAS signals. To functionally validate role implicated regulation, we performed neuron-specific RNA interference screen fruit fly,

Язык: Английский

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DOPAL initiates αSynuclein-dependent impaired proteostasis and degeneration of neuronal projections in Parkinson’s disease

npj Parkinson s Disease, Год журнала: 2023, Номер 9(1)

Опубликована: Март 25, 2023

Abstract Dopamine dyshomeostasis has been acknowledged among the determinants of nigrostriatal neuron degeneration in Parkinson’s disease (PD). Several studies experimental models and postmortem PD patients underlined increasing levels dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL), which is highly reactive towards proteins. DOPAL shown to covalently modify presynaptic protein αSynuclein (αSyn), whose misfolding aggregation represent a major trait pathology, triggering αSyn oligomerization dopaminergic neurons. Here, we demonstrated that elicits accumulation hampers clearance primary DOPAL-induced buildup lessens neuronal resilience, compromises synaptic integrity, overwhelms quality control pathways neurites. The progressive decline homeostasis further leads loss motor impairment, as showed vivo models. Finally, developed specific antibody detected increased DOPAL-modified human striatal tissues from idiopathic patients, corroborating translational relevance αSyn-DOPAL interplay neurodegeneration.

Язык: Английский

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