Name: a versatile mounting method for long term imaging of zebrafish development
Uploaded: 2017-01-26T13:00:00
Description: Watch this Scientific Journal Video about A Versatile Mounting Method for Long Term Imaging of Zebrafish Development at JoVE.com

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-Te&#769;le&#769;thon fellowship (number 16829). He is now supported by a Wellcome Trust/Royal Society Sir Henry Dale Fellowship.

1. Preparation of Solutions and Pulled Glass Needle
<ol>
	<li>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 &#176;C. 
	NOTE: The anesthetic Tricaine acts preferentially on neural voltage-gated sodium channels thereby blocking muscle twitching and movement6.</li>
	<li>Make a working solution of Tricaine at a final concentration of ...

<ol>
	<li>Graeden, E., Sive, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+Imaging+of+the+Zebrafish+Embryonic+Brain+by+Confocal+Microscopy.">Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy.</a> J Vis Exp. (26), e1-e2 (2009).</li><li>Delaune, E. A., Fran&#231;ois, P., Shih, N. P., Amacher, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Cell-Resolution+Imaging+of+the+Impact+of+Notch+Signaling+and+Mitosis+on+Segmentation+Clock+Dynamics.">Single-Cell-Resolution Imaging of the Impact of Notch Signaling and Mitosis on Segmentation Clock Dynamics.</a> Dev Cell. 23 (5), 995-1005 (2012).</li><li>Lawton, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulated+tissue+fluidity+steers+zebrafish+body+elongation.">Regulated tissue fluidity steers zebrafish body elongation.</a> Development. 140 (3), 573-582 (2013).</li><li>Dray, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-Fibronectin+Interactions+Propel+Vertebrate+Trunk+Elongation+via+Tissue+Mechanics.">Cell-Fibronectin Interactions Propel Vertebrate Trunk Elongation via Tissue Mechanics.</a> Curr Biol. 23 (14), 1335-1341 (2013).</li><li>Steventon, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Species+tailoured+contribution+of+volumetric+growth+and+tissue+convergence+to+posterior+body+elongation+in+vertebrates.">Species tailoured contribution of volumetric growth and tissue convergence to posterior body elongation in vertebrates.</a> Development. 143, 1732-1741 (2016).</li><li>Attili, S., Hughes, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaesthetic+Tricaine+Acts+Preferentially+on+Neural+Voltage-Gated+Sodium+Channels+and+Fails+to+Block+Directly+Evoked+Muscle+Contraction.">Anaesthetic Tricaine Acts Preferentially on Neural Voltage-Gated Sodium Channels and Fails to Block Directly Evoked Muscle Contraction.</a> PLoS ONE. 9 (8), 103751-103756 (2014).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Zebrafish Book, 5th Edition; A guide for the laboratory use of zebrafish (Danio rerio). , (2007).</li><li>Schr&#246;ter, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+zebrafish+somitogenesis.">Dynamics of zebrafish somitogenesis.</a> Dev Dyn. 237 (3), 545-553 (2008).</li><li>Megason, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+toto+imaging+of+embryogenesis+with+confocal+time-lapse+microscopy.">In toto imaging of embryogenesis with confocal time-lapse microscopy.</a> Methods Mol Biol (Clifton, N.J.). 546, 317-332 (2009).</li><li>Sugiyama, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Illuminating+cell-cycle+progression+in+the+developing+zebrafish+embryo.">Illuminating cell-cycle progression in the developing zebrafish embryo.</a> PNAS. 106 (49), 20812-20817 (2009).</li></ol>

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...

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 to relate this directly to the morphogenesis of the entire structure.
Zebrafish embryos are ideal for imaging posterior body elongation as their optical transparency and small size allows for the application of minimally invasive light imaging approaches well suited to live imaging.1 This has been evidenced by a series of recent publications that have shed light on posterior body development at the level of molecules,2 single cells,3 and inter-tissue behaviors,4 as well as at the level of cell population and whole organ.5
Advanced imaging techniques such as confocal, multi-photon microscopy and selective plane illumination microscopy (SPIM) are enabling the long term imaging of developmental processes with decreased effect of light toxicity and photo-bleaching. Robust techniques for the mounting of live samples are required to achieve three goals: 1) sufficient support to maintain samples in the correct orientation during transfer to the microscope and during acquisition, 2) sufficient freedom of movement of the sample to allow for the outgrowth of the posterior body region without affecting its normal development, and finally 3) a certain degree in versatility of the mounting method to allow imaging on diverse imaging set-ups.
This protocol introduces a mounting technique for imaging the development of 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 the posterior body region to elongate and develop normally. As such, it is also an appropriate method for the long-term imaging of other regions of the developing body as the agarose enables imaging by standard light imaging techniques. This protocol demonstrates mounting of embryos in a lateral orientation, although it is also possible to mount embryos in alterative orientations. It will further show how to adapt the method for upright, inverted and vertical light-sheet microscopy setups.

<table border="0" cellpadding="0" cellspacing="0" width="727">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">CONSUMABLES</td>
			<td style="width:236px;"></td>
			<td style="width:135px;"></td>
			<td style="width:125px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass-bottomed dishes</td>
			<td>Mattek</td>
			<td>P35-1.5-10-C</td>
			<td>35mm petri dish, 10mm microwell. No. 1.5 cover glass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Capillaries for injection needles</td>
			<td>&#160;Sutter&#160;</td>
			<td>BF 120-94-10</td>
			<td>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.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro-scalpel</td>
			<td>Feather</td>
			<td>P-715</td>
			<td>Micro Feather disposable opthalmic scalpel with plastic handle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pasteur Pipettes</td>
			<td></td>
			<td></td>
			<td>230 mm long</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">REAGENTS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tricaine</td>
			<td>Sigma-Aldrich</td>
			<td>A5040</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Low-melting point agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A9414</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EQUIPMENT</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fine forceps&#160;</td>
			<td>FINE SCIENCE TOOLS GMBH&#160;</td>
			<td>11252-30&#160;</td>
			<td>Dumont #5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Needle puller&#160;</td>
			<td>Sutter&#160;</td>
			<td>P97</td>
			<td>Heating-filament needle puller</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Binocular dissecting microscope</td>
			<td>Leica</td>
			<td>S8 Apo</td>
			<td></td>
		</tr>
	</tbody>
</table>

a versatile mounting method for long term imaging of zebrafish 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.

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...

Watch this Scientific Journal Video about A Versatile Mounting Method for Long Term Imaging of Zebrafish Development at JoVE.com

A Versatile Mounting Method for Long Term Imaging of Zebrafish Development

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. ...

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