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 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 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.
Creation of genetic mosaics (chimeras) between wild-type and genetically modified animals 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'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.
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.
This protocol was approved by Boston Children'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)
2. Setting up Breeding Pairs of Zebrafish and Collection of Embryos (Day -1 to Day 0)
3. Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae
4. Microscope Setup, Image Acquisition, Processing and Analysis
Consistent with previously published studies15, successful parabiotic fusion of zebrafish embryos depends on the staging and orientation of the two embryos 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 either alone or with a gel-loading tip or glass microinjection needle fixed to the end by lab-film (Figure 1).
After placing the two blastulae in 4% methyl cellulose and using the gel-loading tip to orient them with their animal poles facing one another (Figure 2A), the embryos were carefully wounded at their point of contact with the glass microinjection needle (Figure 2B). A greater degree of wounding (compare Figure 2B a Figure 2C), and revisiting embryo pairs to bolster an initial connection with additional wounding, increased the likelihood of the embryos maintaining a connection that resulted in successful fusion (Figure 3A-C, Movie 1), and without additional morphological defects or delayed development. When done carefully, nearly 100% of the embryo pairs were fused, although a fraction of these embryos (around 25%) never established healthy circulation in both halves. By orienting the two blastulae 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 (Figure 3D).
To confirm that the embryos indeed shared their circulation, fluorescent dextran was injected into circulation, which could be seen subsequently circulating through both embryos (Movie 2). Additionally, transgenic embryos that had GFP+ erythrocytes (lcr:eGFP) were fused to embryos that had mCherry+ vascular endothelial cells (flk1:HRAS-mCherry). By 48 hpf, GFP+ erythrocytes were observed circulating through the flk1:HRAS-mCherry partner embryo (Figure 4A and Movie 3). To expand the utility of the parabiotic system, temporal control of gene expression was added. Prior to fusion, one of the two embryos was injected with an hsp70:eGFP DNA construct 20. While the fused embryos were growing at 28.5 °C, no fluorescence was observed. In contrast, after a brief 30 min heat shock at 37 °C, a clear GFP signal was visible in one of the two embryos (Figure 4B). In some instances GFP+ cells were observed circulating in the un-injected partner embryo. Thus, placing a gene of interest under the control of a heat shock promoter provides additional temporal control to studies investigating cell-intrinsic or cell-extrinsic functions of candidate genes.
Figure 1. Tools for Generating Parabiotic Zebrafish Embryos.
(A) Annotated image shows tools used for surgical fusion of developing blastulae. Tools include a modified Pasteur pipette (top), which is used in conjunction with a 10 ml pipette pump (green). Wood handled teasing needles are used either alone or with a plastic gel-loading pipette tip or pulled glass microinjection needled fixed to the end with lab-film. (B) Image shows the ends of three glass microinjection needles that have been broken with a forceps at different diameters. The needles are lying on top of a micrometer; the smallest lines are spaced 10 µm apart. Please click here to view a larger version of this figure.
Figure 2. Surgical Fusion of Developing Blastulae.
High-magnification images show two zebrafish embryos at the high stage of development, oriented with their animal poles facing one another, prior to surgical stitching (A), immediately after minimal wounding (B), and after more substantial wounding (C). 20 min later it is apparent that the embryos have been successfully fused based on the uninterrupted bridge of cells between the two blastulae (D). Scale bar represents 250 µm. Please click here to view a larger version of this figure.
Figure 3. Development of Parabiotic Zebrafish Embryos.
(A-C) Images show a whole-parabiont view of early development just after wounding (A) and as the embryos continue to develop and undergo epiboly (B and C) These images correspond to Movie 1. (D) Image shows a parabiotic embryo pair at 36 hpf. The embryos are connected at their yolk sacs, have two hearts and share a common circulation. Scale bars represent 250 µm. Please click here to view a larger version of this figure.
Figure 4. Visualizing Parabiosis with Genetically Encoded Fluorescent Proteins.
(A) Images (fluorescence alone, right; overlay with transmitted light, left) show the head region of conjoined embryos at 48 hpf. The bottom embryo of this pair is transgenic for lcr:eGFP (erythroctyes, green) while its partner (top) is transgenic for flk1:HRAS-mCherry, which labels vascular endothelial cells (red). Green erythrocytes can be seen circulating through the partner embryo. This image corresponds to Movie 3. (B) The left embryo in this pair was injected with an hsp70:eGFP construct at the one-cell stage and then fused to an un-injected partner (right). When the animals were heat shocked at 37 °C for 30 min at 36 hpf, the green fluorescence of GFP was observed in the left embryo at 60 hpf. Scale bars represent 500 µm. Please click here to view a larger version of this figure.
Movie 1. Early development of surgically fused zebrafish blastulae. (Right click to download).
Movie shows the early development of a pair of surgically fused zebrafish blastulae. Epiboly can be seen occurring simultaneously in each embryo. This movie is related to Figure 3A-C.
Movie 2. Fluorescent dextran injection illuminates a shared circulation. (Right click to download).
Movie shows fluorescent dextran being injected into the common cardinal vein of a parabiotic embryo pair at 60 hpf. The fluorescent dye can be seen subsequently circulating through both embryos.
Movie 3. Green blood flowing through red vessels. (Right click to download).
Movie shows the head region of conjoined embryos at 48 hpf. The bottom embryo of this pair is transgenic for lcr:eGFP (erythroctyes, green) while its partner (top) is transgenic for flk1:HRAS-mCherry, which labels vascular endothelial cells (red). Green erythrocytes are seen circulating through the partner embryo. This movie is related to Figure 4A.
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 candidate genes of interest in hematopoietic development and beyond.
The method described in this paper was recently used to track HSC emergence in one embryo, migration via blood-circulation to an embryo of different genetic background, and subsequent engraftment and differentiation 16. In agreement with the Kissa group’s findings, staging and embryo positioning were found to determine the success rate and anatomical nature of the parabiotic fusion. With a few key modifications to the protocol, parabionts were generated with a significantly higher survival rate. These include wounding the blastulae to a greater degree, holding the needle in place for 2 – 3 sec after wounding, and then revisiting blastula pairs after 15 min to reinforce an initial connection with additional wounding, if necessary. With these additional recommendations, nearly 100% of the parabionts survived and remained attached, and 75% of them shared a common circulation.
To incorporate an additional element of experimental control to the method, parabiotic embryos are demonstrated here to be tolerant to heat shock, allowing for temporal control of gene expression within the parabiotic context 20. A limitation of this technique is that in some instances the parabiotic partner embryos are not in the same plane after parabiotic fusion. This can make it difficult to mount embryos in LMP agarose to track cells in both embryos simultaneously using time-lapse microscopy. To circumvent this potential issue, one should plan to generate multiple parabiotic embryos to ensure the desired orientation is achieved. Alternatively, embryos can be recovered from the LMP using a fine forceps and Pasteur pipette and re-mounted for imaging from a different perspective. The goal of this protocol was to provide a set of clear, concise instructions to accompany video-based tutorials on how to surgically fuse developing blastulae to generate parabiotic zebrafish embryos.
With the key modifications and additional recommendations for increasing parabiont survival, this protocol will hopefully empower investigators who wish to utilize conjoined embryos to investigate factors regulating cell migration, fate specification, and differentiation in a range of tissues and cellular contexts.
The authors have nothing to disclose.
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.
Methyl cellulose | Sigma | M0387 |
Individual components for E3/HCR: | For 1L: 14,61g NaCl, 0,63g KCl, 2,43g CaCl2-2H2O, 1,99g MgSO4 | |
NaCl (Sodium chloride) | Sigma-Aldrich | S9888 |
KCl (Potassium chloride) | Sigma-Aldrich | P9541 |
CaCl2 (Calcium chloride dihydrate) | Sigma-Aldrich | 223506 |
MgSO4 (Magnessium sulfate heptahydrate) | Sigma-Aldrich | 230391 |
Hepes (1M ) buffer solution | ThermoFisher | 15630-080 |
Name | Company | Catalog Number |
Antibiotics: | ||
Pen/Strep | gibco by Life technologies | 15140-120 |
Ampicilin sodium salt | Sigma Life Science | A0166 |
Kanamycin sulfate from Streptomyces kanamyceticus | Sigma Life Science | K1377 |
Name of Reagent/ Equipment | Company | Catalog Number |
50 mg/ml Pronase from Streptomyces griseus | Roche | 11459643001 |
capillary glass used for needles (Capillary Glass & Filaments) | Sutter Instrument | ITEM#: BF 100-50-10 |
Teasing needles with wooden handles | Fisher Scientific | S07894 |
Glass Pasteur pipettes | Fisherbrand | 13-678-20A |
10 mL pipette pump (green) (Pipette Pump Pipettor) | Novatech International | F37898-0000 |
100 mm diameter/ 20 mm deep plastic petri dishes (PETRI DISH, 100/20 MM, PS, CLEAR, WITH VENTS, HEAVY DESIGN, 15 PCS./BAG ) |
Greiner Bio-one | 664102 |
Dextran, Cascade Blue, 10,000 MW, Anionic, Lysine Fixable | ThermoFisher | D-1976 |
PTU. working stock is 0.003% (50X is 0.15%). for 500ml, 0.75 g N-Phenylthiourea | Sigma-Aldrich | P7629 |
Tricaine (powder) (Tricaine Methanesulfonato, Tricaine-S) | Western Chemical Inc | MS 222 |
LMP agarose (Ultrapure LMP agarose) | Invitrogen | 16520100 |
plastic transfer pipette (just the wide ended one I think) | Fisherbrand | 137115AM |
Glass-bottom 6-well plates used for imaging | MatTek | P06G1.5-20-F |
plastic western gel loading tip fixed on the end of a wood-handled dissecting needle (GELoader tips) | Eppendorf | 22351656 |
glass cover slips, slides and vacuum grease if mounting for an upright microscope: | ||
Vaccum grease ( Dow Corning® high-vacuum silicone grease colorless, weight 5.3 oz (tube) ) |
Dow Corning | Z273554 |
Glass cover slips | Corning Life Sciences | 2960-244 |