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First published online 7 January 2004
doi: 10.1242/dev.00971

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Morphogenesis during Xenopus gastrulation requires Wee1-mediated inhibition of cell proliferation

¹ Cellular Growth Mechanisms Section, Regulation of Cell Growth Laboratory, NCI-Frederick, Frederick, MD 21702, USA
² Developmental Signal Transduction Section, Regulation of Cell Growth Laboratory, NCI-Frederick, Frederick, MD 21702, USA
³ Department of Anatomy and Cell Biology, The George Washington University Medical Center, 2300 I Street, NW, Washington, DC 20037, USA

View larger version (68K): [in a new window]   Fig. 1. Wee1-depletion using antisense morpholino oligonucleotides (MO). (A) MO-Wee1 was injected into both cells of a two-cell embryo. Uninjected and MO-Wee1-injected embryos were collected at various stages and lysates examined by immunoblot analysis using anti-Wee1 antibodies (* indicates a nonspecific band). (B) Two-cell embryos were injected with MO-Control, MO-Wee1 or co-injected with MO-Wee1 and WT Wee1 RNA (MO-Wee1+RNA). Embryos were collected at stage 10 and lysates examined as in A. (C) Embryos injected as in B were collected at stage 8, 10 or 12. Lysates were examined by immunoblot analysis using anti-phospho-Cdc2 or anti-Cdc2 antibodies. (D) The mitotic nuclei of injected stage 11 embryos were visualized by whole-mount immunostaining using phospho-histone H3 (PH3). Mitotic index (n=10-12 embryos): MO-control, 8.9% (4739 nuclei); uninjected, 9.9% (1221 nuclei); MO-Wee1, 24.5% (4734 nuclei); MO-Wee1+RNA, 11.4% (2736 nuclei).

View larger version (49K): [in a new window]   Fig. 2. Wee1 is required for Xenopus gastrulation. (A) Embryos injected with MO-Control, MO-Wee1 or MO-Wee1+WT Wee1 RNA were examined for blastopore formation at stage 10.5 and 11. MO-Wee1 disrupts blastopore formation, which is rescued by WT-Wee1 RNA. (B) Percentage of embryos with gastrulation defects: MO-control (n=64), MO-Wee1 (n=243) and MO-Wee1+WT RNA (n=47).

View larger version (78K): [in a new window]   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-1, 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. ß-Gal RNA was injected into the B1 blastomeres at the 32-cell stage and ß-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 ß-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).

View larger version (31K): [in a new window]   Fig. 4. Wee1 is tyrosine phosphorylated at the MBT and gastrulation. (A) Lysates prepared from stage VI oocytes (oocyte), eggs arrested at metaphase of meiosis II (egg), embryos in the first mitotic cell cycle (egg 30 minutes), cleavage stage embryos (stage 7 and 8) and embryos after the MBT (stage 9), the early gastrula stage (stage 10), and at mid-gastrula stage (stage 11.5) were examined by immunoblot analysis using antibodies recognizing Wee1, Cdc25A, Cdc25C, phospho-Cdc2 and total Cdc2. One oocyte or embryo equivalent was loaded per lane. Tyrosine-phosphorylated Wee1 was detected by probing Wee1 immunoprecipitates with anti-phosphotyrosine antibody. (B) Depicted are developmental expression profiles of members of the Wee1 kinase and Cdc25 phosphatase families and the developmentally regulated tyrosine phosphorylation (pY) of Cdc2 and Wee1.

View larger version (56K): [in a new window]   Fig. 5. Upregulation of Wee1 activity by tyrosine autophosphorylation is required for normal gastrulation. (A) Both cells of a two-cell embryo were injected with RNAs encoding KD-Wee1, YYY/FFF-Wee1 or WT-Wee1. Anti-Flag immune complexes isolated from lysates prepared at stages 5, 8, 9 and 10 were examined by immunoblot analysis using anti-phosphotyrosine and anti-FLAG antibodies. (B,C) Embryos injected with MO-Wee1 alone, MO-Wee1+RNA (WT), MO-Wee1+RNA (KD) or MO-Wee1+RNA (YYY/FFF) were scored for gastrulation defects at stage 11. Number of embryos examined: MO-Wee1 (n=17); MO-Wee1+WT Wee1 (n=34); MO-Wee1+KD-Wee1 (n=42); MO-Wee1+YYY/FFF-Wee1 (n=35). Rescue RNAs were injected at 2 ng/embryo. Lysates from the injected embryos were examined by immunoblot analysis using anti-Wee1 antibody.

View larger version (51K): [in a new window]   Fig. 6. KD-Wee1 and YYY-FFF-Wee1 act as dominant-inhibitors to disrupt gastrulation. The two dorsal blastomeres of four-cell embryos were injected with 5-6 ng of RNA encoding ß-gal, KD-, YYY/FFF-, Shift- or Stop-Wee1 and embryos were examined for blastopore formation at stage 11.5-12. Co-injection of Wee1 and ß-gal (100 pg) and the subsequent staining for ß-gal activity (red) shows the area of the embryo expressing the exogenous RNAs. (B) RNAs used in A were analyzed by agarose gel electrophoresis and ethidium bromide staining (top panel). Lysates prepared from embryos in A were examined by immunoblot analysis using the FLAG antibody (middle panel). Percentage of embryos with gastrulation defects (bottom panel). Number of embryos examined: ß-gal (n=55), Stop- (n=78), Shift- (n=72), KD- (n=82) and YYY/FFF-Wee1 (n=86).

View larger version (59K): [in a new window]   Fig. 7. Cdc25C overexpression disrupts gastrulation. (A,B) The two dorsal blastomeres of four-cell embryos were injected with ß-gal RNA (4 ng) or co-injected with ß-gal (100 pg) and His-Cdc25C (3 ng) and embryos were scored for gastrulation defects at stage 11.5-12. Number of embryos examined: Cdc25C (n=141), ß-Gal (n=162). (A, lower panels) Stage 10.5-11 embryo lysates were examined by immunoblot analysis using anti-His-epitope, Cdc2 and phospho-Cdc2 antibodies. (C) Following injection as in A, mitotic nuclei of stage 11 embryos were visualized. Mitotic index (n=10-12 embryos): ß-gal, 8.3% (6928 nuclei), Cdc25C, 28.1% (5550 nuclei). (D,E) Embryos were injected with 4 ng of Cdc25C RNA and either 0, 0.2 or 0.5 ng of Wee1 RNA. Number of embryos examined: Cdc25C + 0 ng Wee1 (n=74), Cdc25C + 0.2 ng Wee1 (n=100) and Cdc25C + 0.5 ng Wee1 (n=86). Note that increased expression of Wee1 counteracts the defects induced by Cdc25C overexpression.