A Versatile Mounting Method for Long Term Imaging of Zebrafish Development

Summary

Here, we present a versatile mounting method that allows for the long-term time-lapse imaging of the posterior body development of live zebrafish embryos without perturbing normal development.

Abstract

Zebrafish embryos offer an ideal experimental system to study complex morphogenetic processes due to their ease of accessibility and optical transparency. In particular, posterior body elongation is an essential process in embryonic development by which multiple tissue deformations act together to direct the formation of a large part of the body axis. In order to observe this process by long-term time-lapse imaging it is necessary to utilize a mounting technique that allows sufficient support to maintain samples in the correct orientation during transfer to the microscope and acquisition. In addition, the mounting must also provide sufficient freedom of movement for the outgrowth of the posterior body region without affecting its normal development. Finally, there must be a certain degree in versatility of the mounting method to allow imaging on diverse imaging set-ups. Here, we present a mounting technique for imaging the development of posterior body elongation in the zebrafish D. rerio. This technique involves mounting embryos such that the head and yolk sac regions are almost entirely included in agarose, while leaving out the posterior body region to elongate and develop normally. We will show how this can be adapted for upright, inverted and vertical light-sheet microscopy set-ups. While this protocol focuses on mounting embryos for imaging for the posterior body, it could easily be adapted for the live imaging of multiple aspects of zebrafish development.

Introduction

Posterior body elongation is an essential process in embryonic development by which the embryo extends to form a large part of the body axis. It is an example of a complex morphogenetic process by which multiple cell behaviors act coordinately to generate the morphogenesis at the level of individual tissues. These differential tissue deformations then act together to generate the elongation of the posterior body at the whole structure level. To understand how these processes are controlled and coordinated during development, we must be able to follow these processes at multiple scales (i.e. at the level of molecules, cells, cell populations and tissues) and t....

Protocol

1. Preparation of Solutions and Pulled Glass Needle

1. Make a 25x stock solution of Tricaine (3-amino benzoic acid ethyl ester, also called ethyl 3-aminobenzoate) at 4 mg/mL in 20 mM Tris pH 8.8 and bring that solution is at pH 7. Aliquot by 4 mL and store at -20 °C.
   NOTE: The anesthetic Tricaine acts preferentially on neural voltage-gated sodium channels thereby blocking muscle twitching and movement⁶.
2. Make a working solution of Tricaine at a final concentration of 0.17 mg/ml in E3 embryo medium.⁷
   NOTE: Make the Tricaine working solution extemporaneously as the pH of this solution d....

Results

The protocol outlined above details a versatile technique for the mounting of zebrafish embryos for long-term time lapse imaging. An example of this is shown in Figure 2A and in animated/video Figure 1. Embryos were injected at the 1 cell stage with mRNA encoding the photoconvertible fluorescent protein kikumeGR. At the 15 somite stage they were mounted as described above and imaged for 12 hr on an inverted confocal microscope with a 10X objective. The re.......

Discussion

This mounting technique enables embryos to be kept still during transfer to the microscope and over long-term time-lapse imaging experiments aimed at following posterior body elongation at multiple length scales. Furthermore, it is versatile in that it allows for imaging on both upright and inverted microscopy set-ups, and a suggestion is made for how this can be further adapted to vertically orientated SPIM.

A critical step in this protocol is the careful removal of excess agarose surrounding.......

Materials

Name	Company	Catalog Number	Comments
CONSUMABLES
Glass-bottomed dishes	Mattek	P35-1.5-10-C	35mm petri dish, 10mm microwell. No. 1.5 cover glass
Capillaries for injection needles	Sutter	BF 120-94-10	We use orosilicate glass with filament, OD 1.20 mm, ID 0.94 mm, length 10 cm. However, filament needles are not necessary and most injection standard needles should work.
Micro-scalpel	Feather	P-715	Micro Feather disposable opthalmic scalpel with plastic handle
Pasteur Pipettes			230 mm long
REAGENTS
Tricaine	Sigma-Aldrich	A5040
Low-melting point agarose	Sigma-Aldrich	A9414
EQUIPMENT
Fine forceps	FINE SCIENCE TOOLS GMBH	11252-30	Dumont #5
Needle puller	Sutter	P97	Heating-filament needle puller
Binocular dissecting microscope	Leica	S8 Apo