Abstract
The larval zebrafish (Danio rerio) is an excellent vertebrate model for in vivo imaging of biological phenomena at subcellular, cellular and systems levels. However, the optical accessibility of highly pigmented tissues, like the eyes, is limited even in this animal model. Typical strategies to improve the transparency of zebrafish larvae require the use of either highly toxic chemical compounds (e.g. 1-phenyl-2-thiourea, PTU) or pigmentation mutant strains (e.g. casper mutant). To date none of these strategies produce normally behaving larvae that are transparent in both the body and the eyes. Here we present crystal, an optically clear zebrafish mutant obtained by combining different viable mutations affecting skin pigmentation. Compared to the previously described combinatorial mutant casper, the crystal mutant lacks pigmentation also in the retinal pigment epithelium, therefore enabling optical access to the eyes. Unlike PTU-treated animals, crystal larvae are able to perform visually guided behaviours, such as the optomotor response, as efficiently as wild type larvae. To validate the in vivo application of crystal larvae, we performed whole-brain light-sheet imaging and two-photon calcium imaging of neural activity in the retina. In conclusion, this novel combinatorial pigmentation mutant represents an ideal vertebrate tool for completely unobstructed structural and functional in vivo investigations of biological processes, particularly when imaging tissues inside or between the eyes.
Highlights
IntroductionSurvival[8] (see Results and Discussion). In contrast, the second strategy is considerably less disruptive since it takes advantage of viable mutations affecting the function of genes expressed in specific subsets of cells where they are involved in defined processes of pigment production[5]
We validate the in vivo application of this novel mutant by performing whole-brain light-sheet imaging and two-photon functional recordings of neural activity in the retinae of crystal larvae
The optical clarity of crystal larvae is even superior to that of larvae treated with 200 μM PTU (Fig. 1c,d), since PTU inhibits melanin production but does not interfere with iridophore function[8]
Summary
Survival[8] (see Results and Discussion). In contrast, the second strategy is considerably less disruptive since it takes advantage of viable mutations affecting the function of genes expressed in specific subsets of cells where they are involved in defined processes of pigment production[5]. Since the formation of each pigment type is controlled independently of the others, the combination of different mutations is required to produce fully transparent zebrafish This strategy has been previously implemented to generate the double mutant casper, which lacks all melanophores and iridophores[4]. We further develop this strategy to generate a fully optically clear combinatorial mutant (crystal) that lacks all melanophores and iridophores, and has a non-pigmented RPE. This particular feature makes crystal larvae especially suited for imaging tissues inside or between the eyes while avoiding the use of chemical pigmentation blockers. We validate the in vivo application of this novel mutant by performing whole-brain light-sheet imaging and two-photon functional recordings of neural activity in the retinae of crystal larvae
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