Application of Infrared Laser to the Zebrafish Vascular System

Kimura, Eiji; Deguchi, Tomonori; Kamei, Yasuhiro; Watanabe, Shôji; Yuba, Shunsuke; Hitomi, Jiro

doi:10.1161/atvbaha.112.300602

Cited by 20 publications

(9 citation statements)

References 14 publications

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“…We also try to establish the knockout line of the some genes which may be associate with the ocular vasculature formation using this system. Furthermore we succeeded in regulating spatio-temporal gene expression in targeted single cells using infrared laser, and this method will be useful for rescue of the deficient gene in the targeted cell [42]. By combining these new methods with the morphological and genetic information, we will elucidate both the morphogenetic and pathogenic mechanisms of ocular vasculature formation in future.…”

Section: Discussionmentioning

confidence: 99%

Live imaging of primary ocular vasculature formation in zebrafish

et al. 2017

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Ocular vasculature consists of the central retinal and ciliary vascular systems, which are essential to maintaining visual function. Many researchers have attempted to determine their origins and development; however, the detailed, stepwise process of ocular vasculature formation has not been established. In zebrafish, two angioblast clusters, the rostral and midbrain organizing centers, form almost all of the cranial vasculature, including the ocular vasculature, and these are from where the cerebral arterial and venous angioblast clusters, respectively, differentiate. In this study, we first determined the anatomical architecture of the primary ocular vasculature and then followed its path from the two cerebral angioblast clusters using a time-lapse analysis of living Tg(flk1:EGFP)k7 zebrafish embryos, in which the endothelial cells specifically expressed enhanced green fluorescent protein. We succeeded in capturing images of the primary ocular vasculature formation and were able to determine the origin of each ocular vessel. In zebrafish, the hyaloid and ciliary arterial systems first organized independently, and then anastomosed via the inner optic circle on the surface of the lens by the lateral transfer of the optic vein. Finally, the choroidal vascular plexus formed around the eyeball to complete the primary ocular vasculature formation. To our knowledge, this study is the first to report successful capture of circular integration of the optic artery and vein, lateral transfer of the optic vein to integrate the hyaloidal and superficial ocular vasculatures, and formation of the choroidal vascular plexus. Furthermore, this new morphological information enables us to assess the entire process of the primary ocular vasculature formation, which will be useful for its precise understanding.

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Section: Discussionmentioning

confidence: 99%

Live imaging of primary ocular vasculature formation in zebrafish

et al. 2017

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“…Although it is technically impossible to hit the same neuronal progenitor cells reproducibly, lineage analysis can be achieved by heat-induction in the same micro areas based on embryo morphology or the detection of fluorescent proteins. The use of a low laser intensity to theoretically target a single cell would allow us to induce recombination in a single or a few adjacent cells [15], [42]. Determination of the distribution of GFP-expressing cells by repeated analysis would enable us to identify clonal units derived from the target micro area.…”

Section: Discussionmentioning

confidence: 99%

Controlled Cre/loxP Site-Specific Recombination in the Developing Brain in Medaka Fish, Oryzias latipes

et al. 2013

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BackgroundGenetic mosaic techniques have been used to visualize and/or genetically modify a neuronal subpopulation within complex neural circuits in various animals. Neural populations available for mosaic analysis, however, are limited in the vertebrate brain.Methodology/Principal FindingsTo establish methodology to genetically manipulate neural circuits in medaka, we first created two transgenic (Tg) medaka lines, Tg (HSP:Cre) and Tg (HuC:loxP-DsRed-loxP-GFP). We confirmed medaka HuC promoter-derived expression of the reporter gene in juvenile medaka whole brain, and in neuronal precursor cells in the adult brain. We then demonstrated that stochastic recombination can be induced by micro-injection of Cre mRNA into Tg (HuC:loxP-DsRed-loxP-GFP) embryos at the 1-cell stage, which allowed us to visualize some subpopulations of GFP-positive cells in compartmentalized regions of the telencephalon in the adult medaka brain. This finding suggested that the distribution of clonally-related cells derived from single or a few progenitor cells was restricted to a compartmentalized region. Heat treatment of Tg(HSP:Cre x HuC:loxP-DsRed-loxP-GFP) embryos (0–1 day post fertilization [dpf]) in a thermalcycler (39°C) led to Cre/loxP recombination in the whole brain. The recombination efficiency was notably low when using 2–3 dpf embyos compared with 0–1 dpf embryos, indicating the possibility of stage-dependent sensitivity of heat-inducible recombination. Finally, using an infrared laser-evoked gene operator (IR-LEGO) system, heat shock induced in a micro area in the developing brains led to visualization of clonally-related cells in both juvenile and adult medaka fish.Conclusions/SignificanceWe established a noninvasive method to control Cre/loxP site-specific recombination in the developing nervous system in medaka fish. This method will broaden the neural population available for mosaic analyses and allow for lineage tracing of the vertebrate nervous system in both juvenile and adult stages.

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“…Although we have focused only on the generation of pulses, irradiation can be further tuned by modifying the pulse frequency and duration. Recently, IR-LEGO was successfully used for inducing local gene expression in zebrafish, medaka fish, and Arabidopsis thaliana [8], [25]. The innovations reported here would be useful in achieving more efficient, target-specific, and elaborated gene induction in other organisms as well.…”

Section: Resultsmentioning

confidence: 98%

Pulsed Irradiation Improves Target Selectivity of Infrared Laser-Evoked Gene Operator for Single-Cell Gene Induction in the Nematode C. elegans

2014

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Methods for turning on/off gene expression at the experimenter’s discretion would be useful for various biological studies. Recently, we reported on a novel microscope system utilizing an infrared laser-evoked gene operator (IR-LEGO) designed for inducing heat shock response efficiently in targeted single cells in living organisms without cell damage, thereby driving expression of a transgene under the control of a heat shock promoter. Although the original IR-LEGO can be successfully used for gene induction, several limitations hinder its wider application. Here, using the nematode Caenorhabditis elegans (C. elegans) as a subject, we have made improvements in IR-LEGO. For better spatial control of heating, a pulsed irradiation method using an optical chopper was introduced. As a result, single cells of C. elegans embryos as early as the 2-cell stage and single neurons in ganglia can be induced to express genes selectively. In addition, the introduction of site-specific recombination systems to IR-LEGO enables the induction of gene expression controlled by constitutive and cell type-specific promoters. The strategies adopted here will be useful for future applications of IR-LEGO to other organisms.

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Application of Infrared Laser to the Zebrafish Vascular System

Cited by 20 publications

References 14 publications

Live imaging of primary ocular vasculature formation in zebrafish

Live imaging of primary ocular vasculature formation in zebrafish

Controlled Cre/loxP Site-Specific Recombination in the Developing Brain in Medaka Fish, Oryzias latipes

Pulsed Irradiation Improves Target Selectivity of Infrared Laser-Evoked Gene Operator for Single-Cell Gene Induction in the Nematode C. elegans

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