Name: generation of parabiotic zebrafish embryos by surgical fusion of developing blastulae
Uploaded: 2016-06-11T15:00:00
Description: Watch this Scientific Journal Video about Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae at JoVE.com

We thank Julie R. Perlin for helpful comments on the manuscript. D.I.S. is supported by grants from the American Society of Hematology, the Cooley's Anemia Foundation, and the NIH (K01DK085217 and R03DK100672). E.J.H. is a Howard Hughes Medical Institute Fellow of the Helen Hay Whitney Foundation. B.L. is a Howard Hughes Medical Institute Medical Research Fellow. B.W.B. is supported by an Irvington Fellowship from the Cancer Research Institute and a Young Investigator Award from the Conquer Cancer Foundation of ASCO. L.I.Z. is supported by grants from the NIH (R01CA103846, P01HL032262, and R01HL04880), Taub Foundation for MDS Research, Harvard Stem Cell Institute, and is an investigator of the Howard Hughes Medical Institute.

This protocol was approved by Boston Children&#39;s Hospital Animal Care and Use Committee. This protocol is modified from a previously published method 15.
1. Preparation of Reagents (Days or Weeks in Advance)
<ol>
	<li>Prepare 1.5% agarose-coated dishes. Add 1.5 g agarose to 100 ml Zebrafish E3 medium in an Erlenmeyer flask. Heat, dissolve the agarose, and then pour approximately 5 ml into 100 mm diameter x 20 mm deep petri dishes. 
	Note: These are used for dechor...

<ol>
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Parabiotic fusion has been a powerful tool to investigate cellular functions of candidate genes in adult murine models and chick embryos10-14. More recently, a blastula fusion method has been described for generating conjoined zebrafish embryos15. In the present protocol, video-based tutorials are used to demonstrate and better describe the methodology for creating parabiotic zebrafish embryos of different genetic backgrounds in order to study temporal, cell-intrinsic, and cell-extrinsic roles of ca...

Creation of genetic mosaics (chimeras) between wild-type and genetically modified animals&#160;is a well-established and classical strategy for investigating cell-intrinsic versus cell-extrinsic functions of candidate genes1-6. Blastula transplantation in zebrafish has been widely utilized to generate chimeric embryos for studies of cell-autonomy7-9. Depending on the tissue of interest, however, it can be challenging to predictably target donor cells to the desired tissue (e.g., blood) 1-3, 7-9. Mouse geneticists have long utilized parabiotic surgical methods to generate conjoined organisms with a shared circulation 10-14. Because the parabiotic animals share a common bloodstream, interactions between cells that originated from one animal with cells of the other animal of a different genetic background can be studied 10-16. Recently, Karima Kissa&#39;s group elegantly demonstrated the ability to create conjoined zebrafish embryos and then use this system to study hematopoietic stem and progenitor cell (HSPC) migration15. In addition, zebrafish parabiosis was recently used to investigate the role of endothelial cadherin 5 in hematopoietic stem cell (HSC) emergence and migration,16 and to study the role of stromal cells in the HSPC niche during zebrafish development 17.&#160;
Unlike mice, zebrafish embryos are transparent and allow for direct visualization of cells during parabiotic development, making the system uniquely powerful. The utility of parabiosis in zebrafish, however, is limited by a steep learning curve, and parabiotic surgery on the delicate embryos can be technically challenging without detailed instruction and visual demonstration. The goal of this protocol is to provide a set of clear, step-by-step instructions to accompany video-based tutorials on how to generate parabiotic zebrafish embryos for studying temporal, cell-intrinsic, or cell-extrinsic functions of a candidate gene(s) by surgical fusion of developing blastulae. Key modifications and additional recommendations for increasing parabiont survival and new experimental applications are included.

<table border="0" cellpadding="0" cellspacing="0" width="686">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Methyl cellulose</td>
			<td style="width:87px;">Sigma</td>
			<td style="width:167px;">M0387</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Individual components for E3/HCR:</td>
			<td></td>
			<td style="width:167px;">For 1L: 14,61g NaCl, 0,63g KCl, 2,43g CaCl2-2H2O, 1,99g MgSO4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl (Sodium chloride)</td>
			<td style="width:87px;">Sigma-Aldrich</td>
			<td style="width:167px;">S9888</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl (Potassium chloride)</td>
			<td>Sigma-Aldrich</td>
			<td style="width:167px;">P9541</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CaCl2 (Calcium chloride dihydrate)</td>
			<td>Sigma-Aldrich</td>
			<td style="width:167px;">223506</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgSO4 (Magnessium sulfate heptahydrate)</td>
			<td>Sigma-Aldrich</td>
			<td style="width:167px;">230391</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hepes (1M ) buffer solution</td>
			<td style="width:87px;">ThermoFisher</td>
			<td>15630-080</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Name</td>
			<td style="width:87px;">Company</td>
			<td style="width:167px;">Catalog Number</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antibiotics:</td>
			<td style="width:87px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pen/Strep</td>
			<td style="width:87px;">gibco by Life technologies</td>
			<td>15140-120</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ampicilin sodium salt</td>
			<td style="width:87px;">Sigma Life Science</td>
			<td style="width:167px;">A0166</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kanamycin sulfate from Streptomyces kanamyceticus</td>
			<td style="width:87px;">Sigma Life Science</td>
			<td style="width:167px;">K1377</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Name of Reagent/ Equipment</td>
			<td style="width:87px;">Company</td>
			<td style="width:167px;">Catalog Number</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 mg/ml Pronase from Streptomyces griseus</td>
			<td style="width:87px;">Roche</td>
			<td style="width:167px;">11459643001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">capillary glass used for needles (Capillary Glass &#38; Filaments)</td>
			<td style="width:87px;">Sutter Instrument</td>
			<td style="width:167px;">ITEM#: BF 100-50-10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Teasing needles with wooden handles</td>
			<td style="width:87px;">Fisher Scientific</td>
			<td>S07894</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass Pasteur pipettes</td>
			<td style="width:87px;">Fisherbrand</td>
			<td style="width:167px;">13-678-20A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10 mL pipette pump (green) (Pipette Pump Pipettor)</td>
			<td style="width:87px;">Novatech International</td>
			<td style="width:167px;">F37898-0000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 mm diameter/ 20 mm deep plastic petri dishes (PETRI DISH, 100/20 MM, PS, CLEAR, WITH VENTS, 
			HEAVY DESIGN, 15 PCS./BAG )</td>
			<td>Greiner Bio-one</td>
			<td>664102</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dextran, Cascade Blue, 10,000 MW, Anionic, Lysine Fixable</td>
			<td>ThermoFisher</td>
			<td>D-1976</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PTU. working stock is 0.003% (50X is 0.15%). for 500ml, 0.75 g N-Phenylthiourea</td>
			<td style="width:87px;">Sigma-Aldrich</td>
			<td style="width:167px;">P7629</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tricaine (powder) (Tricaine Methanesulfonato, Tricaine-S)</td>
			<td style="width:87px;">Western Chemical Inc</td>
			<td style="width:167px;">MS 222</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LMP agarose (Ultrapure LMP agarose)</td>
			<td style="width:87px;">Invitrogen</td>
			<td style="width:167px;">16520100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">plastic transfer pipette (just the wide ended one I think)</td>
			<td style="width:87px;">Fisherbrand</td>
			<td style="width:167px;">137115AM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass-bottom 6-well plates used for imaging</td>
			<td style="width:87px;">MatTek</td>
			<td>P06G1.5-20-F</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">plastic western gel loading tip fixed on the end of a wood-handled dissecting needle (GELoader tips)</td>
			<td style="width:87px;">Eppendorf</td>
			<td style="width:167px;">22351656</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">glass cover slips, slides and vacuum grease if mounting for an upright microscope:</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Vaccum grease ( Dow Corning&#174; high-vacuum silicone grease 
			colorless, weight 5.3 oz (tube) )</td>
			<td style="width:87px;">Dow Corning</td>
			<td>Z273554</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">Glass cover slips</td>
			<td style="width:87px;">Corning Life Sciences</td>
			<td style="width:167px;">2960-244</td>
		</tr>
	</tbody>
</table>

generation of parabiotic zebrafish embryos by surgical fusion of developing blastulae

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for candidate genes of interest, migratory behaviors of cells, and secreted signals in distinct genetic settings. Because parabiotic animals share a common circulation, any blood or blood-borne factor from one animal will be exchanged with its partner and vice versa. Thus, cells and molecular factors derived from one genetic background can be studied in the context of a second genetic background. Parabiosis of adult mice has been &#160;used extensively to research aging, cancer, diabetes, obesity, and brain development. More recently, parabiosis of zebrafish embryos has been used to study the developmental biology of hematopoiesis. In contrast to mice, the transparent nature of zebrafish embryos permits the direct visualization of cells in the parabiotic context, making it a uniquely powerful method for investigating fundamental cellular and molecular mechanisms. The utility of this technique, however, is limited by a steep learning curve for generating the parabiotic zebrafish embryos. This protocol provides a step-by-step method on how to surgically fuse the blastulae of two zebrafish embryos of different genetic backgrounds to investigate the role of candidate genes of interest. In addition, the parabiotic zebrafish embryos are tolerant to heat shock, making temporal control of gene expression possible. This method does not require a sophisticated set-up and has broad applications for studying cell migration, fate specification, and differentiation in vivo during embryonic development.

Consistent with previously published studies15, successful parabiotic fusion of zebrafish embryos depends on the staging and orientation of the two embryos&#160;and the concentration of methylcellulose. With just a few simple, inexpensive tools, surgically fused developing blastulae were generated that grew into parabiotic embryos with shared circulation. These tools included a modified Pasteur pipette, a 10 ml pipette pump, and wood handled teasing needles which were used eith...

Watch this Scientific Journal Video about Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae at JoVE.com

Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae

This protocol provides step-by-step instruction on how to generate parabiotic zebrafish embryos of different genetic backgrounds. When combined with the unparalleled imaging capabilities of the zebrafish embryo, this method provides a uniquely powerful means to investigate cell-autonomous versus non-cell-autonomous functions for candidate genes of interest.

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for ...

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