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:

The dorsal two blastomeres of the four cell stage embryos were each injected with combinations of: 250pg DN-XRhoA N-19 + 18.4pg Active-XRhoA V-14 RNAs, or 250pg DN-XRhoA N-19 + 765pg XRhoA wildtype RNAs, or 300pg DN-XRhoA N-19 + 918pg XRhoA wildtype RNAs; just below the marginal zone and as close to the first cleavage septum as possible. During and up to three hours after injection, the embryos were kept in 1X MBS containing 4% Ficoll to allow them to heal the puncture site. The uninjected controls embryos 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them in exactly the same way as injected embryos, after all ficoll is known to offer some hindrance in gastrulation (unpublished, personal observation). After around two to three 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:

The injected as well as control embryos were fixed in 5% formaldehyde at around stage 11. The embryos were then fractured along the mid-sagittal plane with the help of a sharp blade, while submerged in 1/10^th XMBS. The fractured sections were then photographed at 66X magnification with a digital camera.

Myosin Inhibition Assay using Blebbistatin:

Embryos were transferred to 1X MBS containing 4% Ficoll where they were injected with 23nl of 16.67mM Blebbistatin (a myosin inhibitor) solution in DEPC, into the blastocoel at stage 9. The embryos were then kept in ficoll for one hour at room temperature, after which they were transferred back to 0.1X MBS and allowed to recover from the effects of ficoll and develop till stage 11 at room temperature. Later they were fixed and fractured as described for RNA-injected embryos. These fractured embryos were also photographed as before.