9.3.1 Labeling of embryo and transplantation of the embryonic shield

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9.3.1 Labeling of embryo and transplantation of the embryonic shield

Keiji Inohaya
Tokyo Institute of Technology

9.3.1.1 Equipment and reagents

• Glass tube (Model G-1, Narishige Co.)
• Pipette puller (PB-7, Narishige Co.)
• Grinder (EG-4, Narishige Co.)
• Microforge (MF-90, Narishige Co.)
• Biotin dextran (molecular weight 10,000; Molecular Probes): Biotin dextran is dissolved in 0.2M KCl to give 3% solution and the solution are centrifuged through a 20 µm pore filter.
• Partially purified hatching enzyme (Step1)
• 1.6% methylcellulose (4,000 cP) dissolved in the medaka Ringer
• ABC Vectastain Kit (Vector Laboratories, Inc.)
• ABC solution (avidin-biotinylated peroxidase complex in 1% DMSO, 0.1% Tween-20 in phosphate-buffered saline)
• AEC (3-Amino-9-ethylcarbazole; Vector Laboratories, Inc.)
• Avidin-fluorescein conjugate (NeutraLite, Molecular Probes)

9.3.1.2 Preparation of micropipettes

1. Pull micropipette from glass tube with no capillary with a vertical microelectrode puller.
2. Break the tips of the capillaries to the inner diameter 40 µm using a knife.
3. Polish the tips of the capillaries with a grinder.
4. Fashion a sharp spear-tip with a microforge.

9.3.1.3 Labeling of donor embryos

1. Pull micropipette from capillary glass tubes with a vertical microelectrode puller.
2. Break the tips of the capillaries slightly, and fill with the labeling solution (0.3% biotin dextran / 0.1% neutral red in 0.2M KCl)
3. Inject the labeling solution into one of blastomeres at 1- or 4-cell stage by pressure. (The injected dye spread through intercellular cytoplasmic connections to all cells of blastoderm. Intravitelline injection of the dye does not label the medaka blastomeres.)
4. Incubate the injected embryos until the early gastrula stage. (The embryos at an early blastula stage are kept at 4 ºC overnight. The exposure to 4 ºC had no adverse effect on development of medaka embryos).

9.3.1.4 Transplantation of the embryonic shield

1. Dechorionate donor and host embryos with partially purified hatching enzyme before the early gastrula stage.
2. Transfer the donor and host embryos on a depression slide in 1.6% methylcellulose.
3. Under a stereomicroscope, draw cells of the embryonic shield directly into the pipette.
4. Insert the transplanting pipette and transplant the labeled donor cells into the ventral margin of an unlabeled host embryo.
5. Incubate the host and donor embryos in the medaka Ringer until appropriate stages.

9.3.1.5 Detection of the progeny of the transplanted cells

Whole-mount staining of labeled cells

1. Fix the host embryos in 4% paraformaldehyde in phosphate-buffered saline (PBS) overnight at 4 ºC.
2. Wash the specimens 3 times in PBS (10 min each).
3. Incubated the specimens with ABC solution. avidin-biotinylated peroxidase complex in 1% DMSO, 0.1% Tween-20 in PBS for 2 hours at room temperature.
4. Wash the specimens 3 times with 1%DMSO, 0.1% Tween-20 in PBS (15, 30 and 45 min each).
5. Stain the donor-derived cells with AEC solution.

Detection of labeled cells in sections with fluorescence

1. Fix the host embryos in 4% paraformaldehyde in PBS overnight at 4 ºC, and prepare the frozen sections.
2. Incubate with the avidin-fluorescein conjugate in PBS containing 0.1% Tween-20 (PBST) at room temperature for 1 hour.
3. Wash the sections in PBST
4. Observe with a confocal microscope.

9.3.1.6 References

Inohaya K, Yasumasu S, Yasumasu I, Iuchi I and Yamagami K (1999). Analysis of the origin and development of hatching gland cells by transplantation of the embryonic shield in the fish, Oryzias latipes. Develop. Growth. Differ. 41, 557-566

Westerfield M (1995). The Zebrafish Book (Ed.3). A guide for the laboratory use of zebrafish (Brachydanio rerio). Oregon: University of Oregon Press.

Kane DA and Kishimoto Y (2002). Cell labeling and transplantation techniques. In Zebrafish. Oxford University Press.