MATERIALS
& METHODS:
Embryos:
Unfertilized
wild type Xenopus eggs were obtained from females that had been injected
with HCG (human chorionic gonadotrophin). The eggs were fertilized using a
small piece of a testis that had been taken from a mature male. Tiny pieces of
testis (as a source of sperm) were mixed with eggs in small amount of 1X MBS in
a petridish. The eggs were then allowed to sit for a few minutes, after which
the petridish was filled up with 0.1X MBS (Modified Barth’s Solution). The
fertilized eggs were then treated with 2% L-Cysteine solution, at pH 8, for ~5
minutes to remove the jelly coat from the eggs. After cysteine treatment, the
eggs were washed twice with 0.1X MBS and culture in the same solution in a
chilling incubator at 15°C. Throughout all the above detailed steps, the eggs
& embryos were kept at 15°C, except for brief intervals at room temperature (~25°C),
for manipulations such as cysteine treatment and washes etc. The embryos were
staged according to Nieuwkoop and Faber (Nieuwkoop and Faber, 1967), and
used at appropriate stages in the experiments as required.
mRNA Microinjections:
Messenger RNA encoding a dominant negative form of Xenopus RhoA (DN-XRhoA N-19) was synthesized as described by Ren et al (2006). The dorsal two blastomeres of the four cell stage embryos were injected with 184 pg, 368 pg, 552pg, or 250pg/blastomere of the above-mentioned RNA, just below the marginal zone and as close to the first cleavage septum as possible. Each embryo was also co-injected with 200pg/blastomere of b-Gal mRNA, to serve as an injection marker (confirming a successful injection) and lineage tracer (illustrating the final position of affected cells when fixed). b-Gal mRNA was provided by Dr. Hiromasa Ninomiya (University of Toronto). The control embryos were either not injected at all or were injected only with the b-Gal mRNA. During and up to four hours after injection, the embryos were kept in 1X MBS containing 4% Ficoll to allow them to heal the puncture site. The uninjected controls were also kept in ficoll for the same amount of time in order to treat them in exactly the same way as injected embryos, after all ficoll is known to offer some hindrance in gastrulation (unpublished data). After around four hours, the injected as well as uninjected embryos were transferred to 0.1X MBS and allowed to grow to the desired stage. Except for the half hour period it took to inject the embryos at room temperature, they were maintained at 15°C until they reached the desired stage.
Fixation, Mid-sagittal
Fracture & b-Gal Staining:
The injected as well as control embryos were fixed in 5% formaldehyde at around stage 10.5. The embryos that had been injected with b-Gal mRNA (both DN-RhoA embryos as well as b-Gal controls) were removed from formaldehyde after around one and a half hours and transferred to PBS (Phosphate Buffered Saline). The embryos were then fractured in mid-sagittal plane with the help of a sharp blade, while still in PBS; the whole procedure took less than half an hour. The purpose of keeping embryos in PBS for half an hour was to wash away excess formaldehyde that could affect the activity of the b-Gal protein. Fractured embryos were then transferred to the staining solution containing 976ml of PBS, 8ml Potassium Hexacyanoferrate (II) K4Fe(CN)6, 8ml Potassium Hexacyanoferrate (III) K3Fe(CN)6, 4ml Magnesium Chloride MgCl2, and 4ml b-Gal substrate (Red or Blue). The stained embryos were then removed from the staining solution after one hour and photographed, at 66X magnification, along with the uninjected controls that had also been similarly fractured. The embryos that had not been injected appropriately (as determined by b-Gal-staining) were rejected and not photographed.
ROK (Rho Kinase) Inhibition:
A set of embryos that had just reached (or about to reach) stage 10 was transferred from the main culture (0.1X MBS) to 1X MBS. Their vitelline membranes were torn open with the help of forceps and their blastocoel roofs were cut away at the level of blastocoel floor, with the help of an eyelash tool. These BCR-less embryos were then treated with the ROK (Rho activated Kinase) inhibitor, and left at room temperature for about one and a half hours while still in 1X MBS. Later, these BCR-less embryos were fixed in ~5% formaldehyde for two hours and then fractured in the mid-sagittal plane as described earlier for RNA-injected embryos. These were also photographed at 66X magnification as described earlier.
Whole Embryos:
A second set of embryos, from the same batch as the BCR-less experiment, was transferred to 1X MBS containing 4% ficoll. These embryos were injected with 11.5 nanolitres of pharmacological ROK inhibitor into the blastocoel cavity at stage 9. The embryos were left at room temperature (in 1X MBS with ficoll) for about four hours after which they were fixed and fractured as described earlier for RNA-injected embryos. A set of control embryos that had not been injected in the blastocoel were also kept in ficoll for the same amount of time and fixed and fractured in the same way. They were also photographed as described above.
A third set of embryos from the same batch as above was transferred the ficoll solution where they were injected with 23nl of ROK inhibitor into the blastocoel at stage 8. The embryos were then kept in ficoll for four hours at 15°C, after which they were transferred back to 0.1X MBS and allowed to recover from the effects of ficoll for one hour at 15°C. Later they were fixed and fractured at stage 10.5 as described for RNA-injected embryos. These fractured embryos were also photographed as before.