Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae

Summary

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.

Abstract

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.

Introduction

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 genes^1-6. Blastula transplantation in zebrafish has been widely utilized to generate chimeric embryos for studies of cell-autonomy^7-9. 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....

Protocol

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)

1. 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 dechorionation of embryos (Step 3.1) and for the parabiotic surgery (Step 3.3). Store agarose-coated dishes at 4 °C for a few weeks; take them out of t....

Results

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 eith.......

Discussion

Parabiotic fusion has been a powerful tool to investigate cellular functions of candidate genes in adult murine models and chick embryos^10-14. 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.......

Materials

Name	Company	Catalog Number	Comments
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