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???displayArticle.abstract??? PTEN phosphatase mediates several developmental cues involving cell proliferation, growth, death, and migration. We investigated the function of the PTEN gene at the transition from the cell proliferation state to morphogenesis around the midblastula transition (MBT) and gastrulation in Xenopus embryos. An immunoblotting analysis indicated that PTEN expresses constantly through embryogenesis. By up- or down-regulating PTEN activity using overexpression of the active form or C terminus of PTEN before MBT, we induced elongation of the cell cycle time just before MBT or maintained its speed even after MBT, respectively. The disruption of the cell cycle time by changing the activity of PTEN delayed gastrulation after MBT. In addition, PTEN began to localize to the plasma membranes and nuclei at MBT. Overexpression of a membrane-localizing mutant of PTEN caused dephosphorylation of Akt, whereas overexpression of the C terminus of PTEN caused phosphorylation of Akt and inhibited the localization of EGFP-PTEN to the plasma membranes and nuclei. These results indicate that an appropriate PTEN activity, probably regulated by its differential localization, is necessary for coordinating cell proliferation and early morphogenesis.
Fig. 1. Changes in expression and activity of PTEN during embryogenesis. (A) The anti-Xenopus PTEN antibody (anti-PTEN) recognized PTEN protein by Western blot, but pre-immune serum (pre) did not. (B) PTEN antibody specificity. The anti-PTEN recognized endogenous PTEN (endo), exogenous PTEN (PTEN-WT), and bacterially expressed PTEN (PTEN-His) whereas the anti-PTEN that had been mixed with PTEN-His (anti-PTEN+PTEN-His) did not recognize them. (C) The constant expression of PTEN proteins. Proteins equivalent to one embryo were analyzed by Western blot with anti-Xenopus PTEN antibody. (D) The changes in PTEN activity. Akt expression and Akt phosphorylation (p-Akt) were analyzed by Western blot with anti-Akt antibody and anti-P-Ser474 of Akt antibody. PTEN was activated during gastrulation (stages 106). (E) The relative change in the amount of PTEN, Akt, p-Akt, and a ratio between p-Akt and Akt, based on the amount at stage 8 (n = 3). Nieuwkoopaber (N/F) stages are shown at the bottom.
Fig. 2. Overexpression of PTENtail mutants altered the level of Akt phosphorylation. (A) A schematic illustration of mRNA constructs used for exogenous PTEN expression by mRNA injection into one-cell stage embryos. Myc, three Myc tags; T, phosphorylation site of Thr382 that is equivalent to Thr383 in mouse PTEN; A, substitution of Ala for Thr. (B) Ser474 phosphorylation of Akt (p-Akt). Embryos ectopically expressing PTEN-PD, PTEN-T382A, or PTEN-Cter-T382A were analyzed as in Fig. 1B at the indicated stages. The Akt was dephosphorylated at stages 102 in PTEN-PDexpressed embryos, as in the non-injected embryos. In PTEN-T382A-expressed embryos, Akt was more dephosphorylated, whereas in PTEN-Cter-T382Aexpressed embryos, Akt dephosphorylation was inhibited. Nieuwkoopaber (N/F) stages are shown at the bottom.
Fig. 3. Correlation between cell size and cell cycle time in dissociated
blastomeres. PTEN-T382A (red circles) or PTEN-Cter-T382A (blue
triangles) shows the cell cycle times of dissociated blastomeres derived
from embryos that had been injected with mRNA of PTEN-T382A or
PTEN-Cter-T382A at the one-cell stage, respectively. All embryos were
collected from the same female, blastomeres of nearly the same size were
selected at the same time, and three types of blastomeres (none, PTENT382A,
and PTEN-Cter-T382A) were dropped into the same droplet for
culture. The cell radius was measured just before each cell division, and the
cell cycle time was recorded between the points at which the cleavage
furrows appeared. Open shapes represent the cell size-independent phase,
and closed shapes represent the cell size-dependent phase. (A) The increase
of PTEN activity (PTEN-T382A) enhanced elongation of the cell cycle times
just before MBT (closed red circles). Conversely, the decrease of PTEN
activity (PTEN-Cter-T382A) caused delay in the increase of the cell cycle
after MBT (closed blue triangles). (B) The enhancement of the elongation of
the cell cycle times by PTEN-T382A occurred dose-dependently. The earlier
elongation of the cell cycle times was induced by high concentration of
PTEN-T382A (4.6 ng, red circle) than by low concentration of PTENT382A
(920 pg, orange circle).
Fig. 4. Alteration of PTEN activity caused a delay in progression of gastrulation.
The embryos were injected with mRNA of either PTEN-PD, PTEN-WT, PTENT382A,
or PTEN-Cter-T382A at the one-cell stage. The progression of
gastrulation was determined by measuring the ratio between the size of the
blastopores and that of the whole embryos (number of embryos: none, n = 95;
PTEN-PD, n = 93; PTEN-WT, n = 84; PTEN-T382A, n = 41; PTEN-Cter-
T382A, n = 77) at 11 h, 14 h, and 15.5 h after fertilization. No lip, embryos
before gastrulation; lip, embryos with crescent-like blastopore lips. The
progression of gastrulation significantly delayed in the embryos injected with
PTEN-T382A, PTEN-WT, or PTEN-Cter-T382A in comparison with noninjected
embryos (*P < 0.00001, Student's t test), but not in the embryos
injected with PTEN-PD (**P > 0.01) at 15.5 h after fertilization.
Fig. 5. Localization of endogenous PTEN proteins during embryogenesis. PTEN localization was determined by confocal fluorescent microscopy with anti-Xenopus-
PTEN antibody. Optical cross sections of embryos at morula (N/F stage 6) (A, F, K, P, U), at MBT (stage 8.5) (B, G, L, Q, V), at early gastrula (stage 10) (C, H, M, R,
W), at mid-gastrula (stage 12) (D, I, N, S, X), and at tail bud (stage 29/30) (E, J, O, T, Y). Left panels (A) showed anti-PTEN antibody-staining embryos. Panels in
the right row (F) showed higher magnification of squares than in panels in the left row (A), and inserts (g) showed localization of PTEN in the nuclei. PTEN
localization near the plasma membrane (arrow) appeared before MBT, and the accumulation in the nuclei (arrowhead) was observed at MBT. Double staining with
anti-PTEN antibody and Sytox green (anti-PTEN+Sytox) showed dotlike localization of PTEN (red) in the nuclei (green). Scale bar, 100 μm (A, white), 50 μm
(F, white), 10 μm (green).
