Abstract
Direct videomicroscopic visualization of organ formation and regeneration in toto is a powerful strategy to study cellular processes that often cannot be replicated in vitro. Intravital imaging aims at quantifying changes in tissue architecture or subcellular organization over time during organ development, regeneration or degeneration. A general feature of this approach is its reliance on the optical isolation of defined cell types in the whole animals by transgenic expression of fluorescent markers. Here we describe a simple and robust method to analyze sensory hair-cell development and regeneration in the zebrafish lateral line by high-resolution intravital imaging using laser-scanning confocal microscopy (LSCM) and selective plane illumination microscopy (SPIM). The main advantage of studying hair-cell regeneration in the lateral line is that it occurs throughout the life of the animal, which allows its study in the most natural context. We detail protocols to achieve continuous videomicroscopy for up to 68 hours, enabling direct observation of cellular behavior, which can provide a sensitive assay for the quantitative classification of cellular phenotypes and cell-lineage reconstruction. Modifications to this protocol should facilitate pharmacogenetic assays to identify or validate otoprotective or reparative drugs for future clinical strategies aimed at preserving aural function in humans.
Highlights
Direct in toto visualization of organ formation and repair is a powerful strategy to study cellular processes that often cannot be replicated in vitro (Megason and Fraser, 2007; Pittet and Weissleder, 2011; Rompolas et al, 2012)
Intravital imaging aims at quantifying changes in tissue architecture or subcellular organization over time during organ development, regeneration or degeneration
A general feature of high-resolution intravital imaging is its reliance on the optical isolation of cells or sub-cellular structures in the whole animal by the transgenic expression of fluorescent markers (Faucherre et al, 2010; Dempsey et al, 2012)
Summary
HANDLING OF THE ZEBRAFISH The success of these procedures crucially depends on the proper handling and mounting of the zebrafish embryo or larva. The larvae should be mounted all in the same orientation and in the center of the dish (Figure 3) This step is essential if the imaging needs to be done at the early stages of neuromast development. If the researcher wishes to image neuromasts where no hair cells are present, the screening has to be done using a 40× objective In this case favor the use of Tg[Cldnb:mGFP; SqET4] larvae where neuromast identification can be done by the expression of Tg[Cldnb:mGFP]. If imaging a Tg[SeqEt4] larvae where no hair cells are present use the skin of the animal as a reference to set the beginning boundary and from there set a ∼20 μm stack.
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