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Fig. 3. Wee1 depletion inhibits morphogenesis but not zygotic gene expression. (A) Animal cap explants prepared from uninjected embryos and embryos injected with MO-Control, MO-Wee1 or MO-Wee1+WT Wee1 RNA were left untreated or were treated with activin and cultured until stage 22-23. (B) RNA was isolated from animal cap explants prepared as in A and from stage 10.5 whole embryos (WE) that were either uninjected or had been injected with Control-MO or MO-Wee1 at the two-cell stage. Expression of brachyury, goosecoid and chordin was examined by RT-PCR analysis. cDNA levels were normalized to EF-1α, and a sample lacking reverse transcriptase (–RT) was also included. (C) Two-cell embryos were injected with MO-control, MO-Wee1 or MO-Wee1 + WT RNA. β-Gal RNA was injected into the B1 blastomeres at the 32-cell stage and β-gal activity visualized at stage 11.5-12. The B1 clone forms a narrow midline band extending between the blastopore (bottom) and animal hemisphere (top) in uninjected (n=12) and MO-Control injected embryos (n=23), while the B1 progeny form a broad band across the dorsal equator in MO-Wee1 embryos (MO-Wee1; n=36). this defect is significantly reversed by co-injection of WT Wee1 RNA (MO-Wee1+WT RNA; n=16). (D) The embryos shown in C were bisected through the area of β-gal staining. In uninjected embryos, the labeled cells extend from the animal hemisphere (top) to the dorsal blastopore lip (dbl). In Wee1-depleted embryos (MO-Wee1), no epibolic spread towards the vegetal pole (bottom) or involution occurs. However, some of the inner vegetal cells have moved upwards along the inner surface of the blastocoel roof (b.c.; arrow heads). (E) Expression of Xbrachyury (upper two panels, MO-Wee1, n=67) and chordin (MO-Wee1, n=54, lower two panels) was determined by in situ histochemistry (blue staining). |
