Cell transplantation in glycosyltransferase-depleted medaka embryo
To investigate glycan functions in vertebrate development, we employed a loss-of-function approach for studying several glycosyltransferases using morpholino antisense oligonucleotides in the medaka, Oryzias latipes. Cell transplantation was used to distinguish the cell-autonomous and non-cell-autonomous functions of glycosyltransferases in our previous report. Here we describe the method of cell transplantation at the early developmental stage in medaka.
Yamamoto’s Ringer’s solution (0.75% NaCl, 0.02% KCl, 0.02% CaCl2, 0.002% NaHCO3; adjust to pH 7.3 using NaHCO3)
Morpholino antisense oligonucleotides against beta-1,4-galactosyltransferase 2 mRNA (β4GalT2 MO) (Gene Tools, LLC)
FITC-dextran, MW10, 000 (Molecular Probes)
Rhodamine-dextran, MW10, 000 (Molecular Probes)
Hatching enzyme (supplied by National BioResource Project (NBRP) Medaka)
Penicillin and streptomycin (GIBCO)
FluorinertTM FC-77 (Sumitomo 3M)
Stereomicroscope (Olympus, SZX12)
Watchmaker’s forceps (INOX No.5)
Fluorescent stereomicroscope (Leica, MZ16FA)
Micropipette puller PC-10 (Narishige)
Glass capillary G-1 (1 × 90 mm, Narishige)
Incubator HCRCS2V150W-A1202 (Ikuta Industries)
Fine probe (The tip of a micropipette which is fire-polished and attached to a stick)
50-μl syringe (Hamilton, 1705TLL)
Microinjector IM-5B (Narishige)
Acrylic mold (used as a male component; the design of the mold was according to that specified in the EMBO practical course, 1998)
50-ml syringe (Terumo)
100-mm plastic Petri dish (Becton Dickinson)
Three-way cock, Teflon tube and Fittings (Diba industries Inc.)
Setting up transplant apparatus
Place each equipment, as well as the stereomicroscope, the microinjector, and the micromanipulator, as shown in Fig.1A.
Connect the three-way cock and 50-ml syringe filled with the Fluorinert (a), the three-way cock and the micropipette holder (b), and the three-way cock and the microinjector (c) using the Teflon tubes and fittings as shown in Fig.1B.
Preparation of transplantation pipettes
Pull the pipette from the glass capillary using the micropipette puller (Fig.2B, left).
Break the glass to an appropriate diameter (OD of about 50 μm) using watchmaker’s forceps while viewing under the stereomicroscope (Fig.2B, right).
Attach the micropipette tightly to the micropipette holder fixed on the micromanipulator.
Check ejection of Fluorinert from the tip of the micropipette under pressure.
Preparation of a V-shaped well plate
Pour liquefied 1.5% agarose/Yamamoto’s Ringer’s solution into a 100-mm plastic Petri dish.
Allow an acrylic mold to float on into the agarose (Fig.2C, left).
When the agarose is completely solidified, gently lift the mold using the watchmaker’s forceps and then pour Yamamoto’s Ringer’s solution (Fig.2C, right).
Inject 4 ng of control MO and 4% rhodamine-dextran or 4 ng of β4GalT2 MO and 4% FITC-dextran into 1-cell stage embryos, and then maintain them at 16.5oC until the developmental stage is adjusted (Fig.3A).
Dechorionate the injected embryos with the hatching enzyme immediately before the shield stage (step 4-4).
Place them in the V-shaped well in a 1.5% agarose gel and then orient them using a fine probe.
Puncture the donor blastoderm with micropipette and slowly draw up donor cells from deep within the blastoderm core (Fig.2D, left).
Pull the micropipette out of the donor blastoderm and wait a few minutes till aspirated cells descend and gather at the tip of the micropipette.
Insert the micropipette into the host blastoderm and inject the harvested cells slowly (Fig.2D, right).
Place embryos in a 60-mm plastic dish coated by 1.5% agarose/Yamamoto’s Ringer’s solution containing penicillin and streptomycin, and maintain them at 26oC until they reach the segmentation stage.
Take photographs under a fluorescent stereomicroscope (Fig.3B-D).
At the segmentation stage, cell populations from both donor embryos were incorporated into the embryonic body axis, and they consequently formed a band of mediolaterally elongated cells known as “cellular strings” in the midline tissue (n = 8/8, Fig.3B). In contrast, when MO-injected cells were transplanted into wild-type host embryos, the β4GalT2 MO-injected green cells failed to be incorporated into the midline tissue including the notochord and instead remained round (Fig.3C, white arrows), whereas the control MO-injected red cells migrated into the midline tissue and showed an elongated shape (n = 10/10). As a complementary experiment, we next transplanted donor cells into β4GalT2 MO-injected host embryos. The control cells were incorporated into the midline tissue and seemed to form an organized axial structure to some extent. In contrast, some donor cells injected with β4GalT2 MO were also incorporated into the midline tissue, but other cells were excluded from this structure (n = 10/13, Fig.3D, white arrows). These results suggest that β4GalT2 functions cell-autonomously, and not non-cell-autonomously, in the axial cells that take part in mediolateral cell intercalation during late gastrulation.
Figure & Legends
Fig.1 Setting up transplant apparatus
(A) The placement of equipment for transplantation (B) Preparation of microinjector
Fig.2 Tools for transplantation
(A) Micropipettes for transplantation. Prepared micropipettes should be stored on the adhesive stage in a box to avoid chipping. (B) Shape of micropipette tip. A glass capillary is heated and pulled (left). Then, it is snapped properly. The outer diameter of the pipette tip is about 50 μm (right). (C) An acrylic mold (left) and a V-shaped well plate (right) (D) Scheme for cell transplantation at shield stage.
Fig.3 β4GalT2 functions cell-autonomously during cell intercalation in late gastrulation
(A) Schematic representation of the transplantation experiments. (B) Donor cells in embryos injected with FITC-dextran or rhodamine-dextran were transplanted into the deep layer of the shield in wild-type hosts. Both types of donor cells were incorporated into the embryonic body at 1 dpf. (C) Donor cells from embryos injected with β4GalT2 MO or control MO were transplanted into wild-type hosts. The β4GalT2 MO-injected cells failed to polarize and become incorporated into the midline, although the control MO-injected cells migrated into the axial midline tissue. (D) Donor cells were transplanted as shown in (C). (B–D) Dorsal view, Animal pole is facing the top. Bar: 100 μm.
* Reprinted from Mech Dev., 126(7), Tonoyama Y, Oka S. et al., Essential role of beta-1,4-galactosyltransferase 2 during medaka (Oryzias latipes) gastrulation, 580-94, 2009, with permission from Elsevier. doi:10.1016/j.mod.2009.03.004.
Copyright 2009. Elsevier, for Fig.3 in Figure & Legends
Copyright 2010. Ritsumeikan University, JCGGDB & AIST. for the rest of the contents
Tonoyama Y, Anzai D, Ikeda A, Kakuda S, Kinoshita M, Kawasaki T, Oka S. (2009) Essential role of beta-1,4-galactosyltransferase 2 during medaka (Oryzias latipes) gastrulation. Mech Dev. Jul;126(7):580-94. [PMID : 19324086]
Kobayashi D, Shimada A and Maruyama K. (2009) “Transplantation” Kinoshita M, Murata K, Naruse K, Tanaka M (Eds). Medaka, Biology, Management, and Experimental Protocols. pp. 362-367, Wiley-Blackwell.