Intravital imaging of hair-cell development and regeneration in the zebrafish

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.

Keywords: auditory; development; hair cells; intravital fluorescence microscopy; lateral line system; regeneration; zebrafish.

Figure 1

The lateral line and neuromasts in the zebrafish larva. (A) Low magnification view of a zebrafish larva under transmitted light. (B) Hair cells in the lateral line were labeled with the fluorescent vital dye DiAsp (bright orange). It shows the superficial distribution of neuromasts along the anterior (head) and posterior (trunk and tail) lateral-line systems. (C) Schematic representation of the distribution of the neuromasts in the lateral line of a zebrafish larva. Orange highlights the anterior neuromasts, green the posterior neuromasts, and blue the dorsal neuromasts. (D,E) High magnification confocal image of a frontal view of a posterior neuromast revealing the hair cells (blue), supporting cells (red and green). (F) Schematic representation of a neuromast viewed from the side, depicting every known cell, including the neurons. (G) High magnification side-view image of a mature neuromast labeled with the vital dye DiAsp. Scale bars are 10μm.

Figure 2

UHCPs division and planar cell inversion. Frames from Supplementary Movie 1 at the time-points indicated A 916′ min series of confocal images of a double-transgenic Tg[Cldnb:mGFP;SqET4] regenerating neuromast. Two prospective UHCPs were identified retrospectively by playing the time series backwards and labeled with a yellow and green dot. Each UHCP eventually divides into a pair of sister hair cells, labeled in Cyan/Red and Green/Orange, respectively. On minute 624′ the sibling hair cells in the lower part of the neuromast begin to rotate around their contact point, eventually breaking the line of mirror symmetry. This complete inversion of the sister hair cells restores the line of mirror symmetry realigning the cells along the neuromast axis of planar polarity (double-headed arrow in the first panel). All images correspond to a single focal plane with the cells of interest in focus. Scale bar is 10 μm.

Figure 3

Sample mounting for confocal imagin. One to four larvae are, laterally and equally oriented, placed in the center of a cover glass bottom dish. One drop or two of 1% LMP (low–melting point) agarose in E3 are added covering the larvae. If needed readjust the position of the larvae using an embryo loop. Once the agarose solidifies, the embryos are covered with methylene-blue-free E3 medium containing tricaine and the lid is placed on top of the dish.

Figure 4

Sample mounting for SPIM imaging. (A) Glass capillary (C) with plunger (P). (B) The plunger is pulled back, drawing liquid agarose (A, cyan) into the capillary. (C) Before the agarose solidifies, the sample (S) is carefully injected into the liquid agarose in the capillary by using a p20-pipette and a yellow tip cut in the end. (D) After the agarose solidifies, the plunger is pushed down, extruding the agarose plug containing the sample from the capillary. (E) The sample is mounted on a xyzθ positioning stage in the SPIM. The laser light sheet (red) illuminates the sample along the x-axis, and the detected light (green) is collected orthogonally (along the y-axis) by the microscope optics to form an image on the camera.

Figure 5

Neuromast identification (A) Tg[SeqEt4] upon neomycin treatment, typically 1–4 hair cells can be found in the neuromasts. (B,C) Top (B) and lateral (C) brightfield view of a neuromast. (D) Scheme depicting primordium migration in a 20 hpf larvae and top brightfield view of a migrating primordium, identifiable as a group of ~100 cells just under the epidermis. Scale bars are 10 μm.

Figure 6

Three-dimensional imaging by SPIM. Frames from Supplementary Movie 2 at the time-points indicated. Top, full field of view. Bottom, selected regions of interest containing neuromasts 1 and 2. A 2-channel 68 h time-lapse recorded using SPIM, showing the deposition of 2 neuromasts and their subsequent development. Ath1 (showed in red) is expressed in hair-cells and can be detected in hair-cell progenitors a few hours before the neuromasts are deposited. ath1:RFP is observed later in dividing, young and mature hair-cells. All the cell nuclei are stained in green (H2B:GFP RNA) and the lateral line system is highlighted in green with the Cldnb:mEGFP reporter. All images are maximum-value projections along the detection axis. The brightness of each image has been normalized independently, so that the tdTomato fluorescence at early time-points is visible. Scale bars are 25 μm.