First published online 7 January 2004
doi: 10.1242/dev.00971
Development 131, 571-580 (2004)
Published by The Company of Biologists 2004
Morphogenesis during Xenopus gastrulation requires Wee1-mediated inhibition of cell proliferation
Monica S. Murakami1,*,
Sally A. Moody3,
Ira O. Daar2 and
Deborah K. Morrison1
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.
|
|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
© The Company of Biologists Ltd 2004
