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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.
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.
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....
1. Preparation of Solutions and Pulled Glass Needle
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.......
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.......
Estelle Hirsinger: Core funding from the Institut Pasteur and Agence Nationale de la Recherche (ANR-10-BLAN-121801 DEVPROCESS). Estelle Hirsinger is from the Centre National de la Recherche Scientifique (CNRS). Benjamin Steventon was funded by the Agence Nationale de la Recherche (ANR- 10-BLAN-121801 DEVPROCESS), then a Roux fellowship (Institut Pasteur) then an AFM-Téléthon fellowship (number 16829). He is now supported by a Wellcome Trust/Royal Society Sir Henry Dale Fellowship.
....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 |
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