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...

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I., 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=Mitochondrial+Atpif1+regulates+haem+synthesis+in+developing+erythroblasts.">Mitochondrial Atpif1 regulates haem synthesis in developing erythroblasts.</a> Nature. 491 (7425), 608-612 (2012).</li><li>Tamplin, O. J., 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=Hematopoietic+stem+cell+arrival+triggers+dynamic+remodeling+of+the+perivascular+niche.">Hematopoietic stem cell arrival triggers dynamic remodeling of the perivascular niche.</a> Cell. 160 (1-2), 241-252 (2015).</li><li>Ad&#225;m, A., B&#225;rtfai, R., Lele, Z., Krone, P. H., Orb&#225;n, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat-inducible+expression+of+a+reporter+gene+detected+by+transient+assay+in+zebrafish.">Heat-inducible expression of a reporter gene detected by transient assay in zebrafish.</a> Exp. Cell Res. 256 (1), 282-290 (2000).</li></ol>

The authors declare that they have no conflict of interest.

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 ...

The overall goal of this method is to generate parabiotic zebrafish embryos, which can be used for studying cell intrinsic versus cell extrinsic functions for candidate genes of interest. This method can help answer key questions of cell autonomy. For example, in the hematopoetic system, vital defects in HSC migration are intrinsic to the HSCs or the niche microenvironment.The main advantage of this protocol is an improved ability to successfully generate parabiotic zebrafish embryos. Generally, individuals new to this method will struggle, because the developing embryos are extremely fragile, and easily damaged. Visual demonstration of this method is critical, as the surgical stitching steps can be difficult to learn because they require a high degree of accuracy and precision.Here we provide step-by-step instructions to generate parabiotic zebrafish embryos by surgical fusion of developing blastulas. This method demonstrates how to increase efficiency of parabiotic fusions and enhance survival of conjoined embryos. After preparing reagents and tools according to the text protocol, prepare two to three modified Pasteur pipettes by briefly heating the end of the pipette over a Bunsen burner.Then, using large forceps, while it is still hot, bend the end of the pipette approximately 45 degrees. To ensure that there are no sharp edges that might damage the embryos, keep the end of the pipette over a Bunsen burner for one to two seconds. Using plasma DNA, mRNA or morpholino, micro-inject embryos within one hour of collection, and allow them to develop to the 256 cell stage.As the embryos approach the 256 cell stage, transfer each genetic background into separate agarose coated dishes, scratch-free glass beakers or petri dishes. After dechorionating the embryos according to the text protocol, thaw previously prepared methylcellulose, and use a tabletop centrifuge at maximum speed to pellet undissolved crystals, which will damage the embryos or rupture the yolks. In the meantime, using a marker, mark 12 to 15 pre-determined spots on the bottom-side of a 100 milliliter agarose coated petri dish.Then, after centrifugation, using the clear upper fraction of the methylcellulose, transfer one to 1.5 centimeter drops into the marked spots in the petri dish. Next, prepare one 40 milliliter aliquot of high-calcium ringer, or HCR solution per agarose coated dish just prepared. To each aliquot, add antibiotics as listed here.When ready to perform blastula fusions, use the aliquots of HCR with antibiotics to carefully fill each dish containing the methylcellulose droplets, taking care to not disrupt the droplets. Attach a modified glass Pasteur pipette prepared earlier to a 10 milliliter pipette pump. Then, under a stereoscope, collect one dechorionated embryo from each background.Before dispensing the two embryos, use the Pasteur pipette to make a small depression in the middle of the methylcellulose drop. Then, deposit the two embryos into the depression. Now, using a teasing needle with a gel loading tip on the end, use gentle sweeping motions without directly touching the embryos to carefully reposition the embryos within the methylcellulose, such that their animal poles are directly touching one another.Properly orienting the developing embryos dictates how the embryos will fuse and develop. For head-to-head fusions, ensure that each fusion is aligned at their animal poles. Allow the embryos to sit for five minutes for the methylcellulose to settle in around them.During this time, load the next pair into another drop. To perform the surgery, using the glass needle tool, carefully wound each embryo at their point of contact. Then, with a gentle sewing motion, pull the cells from the first embryo into the second embryo and back again.At the end of the motion, hold the needle in place for two to three seconds before very slowly drawing it away. Precision with the glass needle is critical to ensure the embryos are properly stitched together. Care must be used not to damage the yolk sacs while maintaining enough connection for successful fusion.Allow the embryos to sit for 10 to 15 minutes at room temperature. In the meantime, stitch additional pairs of embryos. After the incubation, assess whether the embryos have remained attached, and repeat the wounding step if needed.Incubate the embryos in the same dish and medium at 28.5 degrees Celsius overnight. By the following morning, the embryos will have moved out of the mostly dissolved methylcellulose. Remove any embryos that have not successfully fused.Then, decant the medium and use 25 milliliters of E3 to replace it. To acquire images of the fused embryos, prepare 0.8%low melting point agarose. While still hot, aliquot 1 milliliter of the agarose into 1.5 milliliter microfuge tubes set in a 37 degree Celsius heating block.Anesthetize the parabiotic embryos by adding one milliliter of four milligrams per milliliter pH 7.0 tricaine to the 25 milliliters of E3 medium containing the embryos. Gently swirl the dish so that the embryos pool in the middle. Then, using a wide-tipped plastic transfer pipette, draw up the embryos in as little liquid as possible.Next, turn the pipette upright and gently bounce the embryos so that they settle to the very bottom of the pipette. Then, transfer the embryos to a 1 milliliter aliquot of low melting point agarose by lightly touching the pipette tip on the surface of the agarose. Dispose of any excess liquid from the pipette, and use the pipette to gently mix the embryos in the agarose.Then, with the pipette, transfer the agarose and embryos to the well of a glass bottom six-well plate. Under a stereomicroscope, use a gel loading tip affixed to the end of a teasing needle to position the embryos close to the cover glass and in the desired orientation for imaging. After the agarose has set, and medium with tricaine has been added to the wells, use an inverted widefield epifluorescence laser scanning confocal, or spinning disk confocal microscope to acquire images.For a whole embryo field of view, use a 4X objective. Use a 20X objective to image a specific tissue. Process the images according to the text protocol.As shown here, after orienting two blastulas with their animal poles facing one another, the embryos were carefully wounded at their point of contact. A greater degree of wounding increased the likelihood of the embryos maintaining a connection that resulted in a successful fusion without additional morphological defects or delayed development. As demonstrated in this figure, by orienting the two blastula with their animal poles directly aligned, reliable head-to-head or yolk sac-to-yolk sac fusions were generated with shared circulation.In most instances, the embryos had two hearts pumping a shared common circulation. To confirm that the embryos indeed shared their circulation, fused transgenic embryos that had GFP-positive erythrocytes were fused to embryos that had mCherry-positive vascular endothelial cells. As seen in this movie, by 48 hours post-fertilization, GFP-positive erythrocytes were observed circulating through the Flik 1 HRAS mCherry partner embryo.Finally, to investigate temporal control of gene expression, one of the two embryos was injected with an hsp70 EGFP DNA construct. After a brief 30-minute heat shock at 37 degrees Celsius, a clear GFP signal was established in one of the embryos, and in some cases, GFP-positive cells were seen circulating in the un-injected partner embryo. Once mastered, this technique can be done in five hours if it is performed properly.While attempting this procedure, it's important to remember to be careful when stitching the two blastulas together, and to revisit fusions if the surgery was not successful on the first attempt. Following this procedure, additional methods like live cell imaging can be used to answer additional questions, like whether wild-type HSCs can function normally in a mutant niche or conversely, whether mutant HSCs can function in a wild-type niche. This technique provides a powerful method for researchers in the field of hematology to study the regulation of blood development in zebrafish.Don't forget that tricaince can be hazardous and precautions such as wearing appropriate safety attire should always be taken while performing this procedure. After watching this video and following our recommended techniques, we hope that you will be able to generate better biotic zebrafish embryos with greater success.

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Research

JoVE Journal

Developmental Biology

11.3K Görüntüleme.  Boston Children’s Hospital. The overall goal of this method is to generate parabiotic zebrafish embryos, which can be used for studying cell intrinsic versus cell extrinsic functions for candidate genes of interest. This method can help answer key questions of cell autonomy. For example, in the hematopoetic system, vital defects in HSC migration are intrinsic to the HSCs or the niche microenvironment.The main advantage of this protocol is an improved ability to successfully generate parabiotic zebrafish embryos. ...