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First published online 15 April 2009
doi: 10.1242/dev.035261

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Start of the embryonic cell cycle is dually locked in unfertilized starfish eggs

Masatoshi Hara¹, Masashi Mori^1,*, Tadashi Wada², Kazunori Tachibana¹ and Takeo Kishimoto^1,

¹ Laboratory of Cell and Developmental Biology, Graduate School of BioscienceTokyo Institute of Technology, Nagatsuta, Midoriku, Yokohama 226-8501, Japan.
² Integrated Research Institute, Tokyo Institute of Technology, Nagatsuta, Midoriku, Yokohama 226-8501, Japan.

View larger version (39K): [in this window] [in a new window]   Fig. 1. Inactivation of Rsk in G1-arrested starfish eggs leads to S phase, but further progression into M phase requires inactivation of MAPK. (A) Inhibition of either MAPK or Rsk causes DNA replication. Unfertilized mature eggs were treated with U0126 or control DMSO, or injected with a neutralizing antibody against Rsk (anti-Rsk) or control IgG. Forty minutes later, eggs were fixed and inspected for DNA replication by incorporation of BrdU. DNA was stained with DAPI. Scale bar: 10 µm. (B) Inhibition of MAPK causes NEBD, but inhibition of Rsk does not. Unfertilized mature eggs were injected with a neutralizing antibody against Rsk (anti-Rsk) or control IgG, along with GST-IBB-GFP. At the indicated times (minutes) after addition of U0126 or control DMSO, NEBD was scored by the dispersal of GST-IBB-GFP from the nucleus. The number of eggs examined is indicated in parentheses. Scale bar: 200 µm. (C) U0126 inhibits the activities of both MAPK and Rsk, but the Rsk antibody inhibits only Rsk, not MAPK. Unfertilized mature eggs were injected with a neutralizing antibody against Rsk (anti-Rsk) or control IgG, and/or treated with either U0126 or control DMSO. Sixty minutes after applying U0126 or DMSO, extracts were prepared for immunoblotting with anti-MAPK. Upper and lower bands of MAPK correspond to the active and inactive forms, respectively. Rsk activity in the same extracts was measured by phosphorylation of GST-S6 (S6K activity).

View larger version (22K): [in this window] [in a new window]   Fig. 2. Inactivation of MAPK can induce M phase in unfertilized starfish eggs in which S phase is prevented. (A) Prevention of S phase by Rsk. Unfertilized mature eggs were injected with either CA-Rsk-EE (CA, constitutively active form) or control KD-Rsk-EE (KD, kinase dead form), or were not injected, and were then treated with U0126 or control DMSO (–). Forty minutes later, eggs were fixed and DNA replication was detected by BrdU incorporation. DNA was stained with DAPI. Scale bar: 10 µm. (B) Entry into M phase in S phase-inhibited eggs. Unfertilized mature eggs in A were co-injected with GST-IBB-GFP. After treatment with U0126, NEBD was scored by dispersal of fluorescence of GST-IBB-GFP, as shown in Fig. 1B. The number of eggs examined is indicated in parentheses. (C,D) Requirement of MAPK inactivation for entry into M phase. Immature oocytes were injected with GST-IBB-GFP without (–) or with GST-Mos, and then treated with 1-MeAde. After completion of meiosis II, eggs were inseminated, and NEBD was examined, as in B, at the indicated times (minutes after insemination). Some eggs not injected with GST-Mos were inseminated in the presence of aphidicolin (Aph). Arrows and arrowheads indicate female and male pronuclei, respectively. Scale bar: 100 µm.

View larger version (64K): [in this window] [in a new window]   Fig. 3. Inactivation of Rsk in unfertilized starfish eggs does not result in cyclin B-Cdk1 activation. (A) Dynamics of cyclin A, cyclin B and Cdk1 after fertilization. Mature eggs arrested at G1 phase were inseminated, and egg extracts prepared at 10-minute intervals were immunoblotted with anti-cyclin A, anti-cyclin B, anti-Cdk1-pY15 and anti-PSTAIR for Cdk1, and anti-MAPK. Cdk1 activity associated with immunoprecipitates of anti-cyclin A or anti-cyclin B was measured as histone H1 kinase activity. As a control, extracts were prepared from unfertilized mature eggs at indicated times corresponding to fertilized eggs. (B,C) Dynamics of cyclin A, cyclin B and Cdk1 after Rsk inhibition. Unfertilized mature eggs were injected with a neutralizing antibody against Rsk (anti-Rsk) and recovered at indicated times (B) or at 60 minutes (C) after injection. As controls, unfertilized mature eggs that were not injected (–) were recovered at times corresponding to the injected eggs (B); alternatively, unfertilized mature eggs were either injected with control IgG or treated with U0126 or DMSO, and then recovered 60 minutes later (C). Extracts prepared from these eggs were processed for immunoblots or measurement of whole histone H1 kinase activity (H1K activity) as in A. Im, immature oocytes.

View larger version (93K): [in this window] [in a new window]   Fig. 4. Inactivation of MAPK can induce cyclin B-Cdk1 activation in unfertilized starfish eggs in which Rsk is maintained active. (A) MAPK inactivation results in activation of cyclin A-Cdk1 and cyclin B-Cdk1. Unfertilized mature eggs were treated with U0126 or control DMSO. Egg extracts prepared at the indicated times were immunoblotted with anti-cyclin A, anti-cyclin B, anti-Cdk1-pY15 and anti-PSTAIR for Cdk1, and anti-MAPK. Cdk1 activity associated with immunoprecipitates of anti-cyclin A or anti-cyclin B was measured as histone H1 kinase activity. (B) MAPK inactivation in the presence of Rsk activity results in activation of cyclin A-Cdk1 and cyclin B-Cdk1. Unfertilized mature eggs were uninjected (–) or injected with either CA-Rsk-EE or control KD-Rsk-EE, and then treated with U0126. Egg extracts were prepared at the indicated times. Protein levels of CA- or KD-Rsk-EE were detected by immunoblots with anti-GST. Rsk activity was assayed as S6 kinase (S6K) activity. (C) Maintenance of MAPK activity prevents cyclin A-Cdk1 and cyclin B-Cdk1 from activation induced by insemination. Immature oocytes were injected with GST-Mos or control GST, treated with 1-MeAde and then inseminated after completion of meiosis II. Egg extracts were prepared at the indicated times after insemination.

View larger version (36K): [in this window] [in a new window]   Fig. 5. Inactivation of MAPK can induce activation of protein synthesis of cyclin A and cyclin B in unfertilized starfish eggs in which Rsk remains active. (A,B) Incorporation of 35S label into overall proteins or cyclins A and B. Mature eggs were inseminated (Fe) or uninseminated (UF), and recovered at the indicated times. For 5 minutes before recovery, eggs were incubated in seawater containing [35S]Met/Cys. Incorporation of radioactivity into overall proteins (A, top) or immunoprecipitates of cyclins A and B (A, middle) was assayed by autoradiography. Extracts were immunoblotted with anti-MAPK, anti-cyclin A, anti-cyclin B and anti-PSTAIR (A, bottom). Relative incorporation of radioactivity into overall proteins or cyclin B was measured by Multi Gauge (Fuji Film), and considered as 1 at 0 minutes (B). (C) Uptake of 35S label into eggs. Mature eggs with or without insemination, or U0126 treatment, were pulse-labeled with [35S]Met/Cys as in A, washed and dissolved in LSB, followed by liquid scintillation counting. Relative uptake of radioactivity into eggs is considered as 1 at 0 minutes. (D) MAPK prevents synthesis of cyclins A and B. Immature oocytes were uninjected (–) or injected with either GST-Mos or control GST, and then treated with 1-MeAde to reinitiate meiosis. Mature eggs were inseminated (Fe) or uninseminated (UF), pulse-labeled with [35S]Met/Cys as in A and recovered at the indicated times after insemination. Cyclins A and B were immunoprecipitated from egg extracts and incorporation of radioactivity into immunoprecipitates was assayed by autoradiography. Extracts were also immunoblotted with anti-MAPK to confirm successful insemination or effect of GST-Mos. (E) MAPK inactivation induces synthesis of cyclins A and B. Unfertilized mature eggs were treated with U0126 or control DMSO, pulse-labeled with [35S]Met/Cys as in A, and recovered at the indicated times after U0126 addition. (F) MAPK inactivation-induced synthesis of cyclins A and B in the presence of Rsk. Unfertilized mature eggs were injected with CA-Rsk-EE (CA) or KD-Rsk-EE (KD) and then treated with U0126. Thirty-five minutes after U0126 addition, eggs were pulse-labeled with [35S]Met/Cys as in A, and then recovered. In E and F, immunoprecipitation of cyclins A and B, autoradiography and immunoblots for MAPK were performed as in D.

View larger version (44K): [in this window] [in a new window]   Fig. 6. Elongation of poly(A) tail in cyclin A and B mRNAs occurs during meiotic maturation but does not occur after fertilization of mature eggs. (A,B) Total RNA was isolated either from immature oocytes (Im) or unfertilized mature eggs (F0 lane in A; 0 minute lane in B). Alternatively, mature eggs were inseminated or treated with U0126 and then total RNA was isolated at the indicated times (F30 and F60 in A and B). Poly(A) tail length was measured by the PAT assay. In B, the first embryonic cell cycle progression was confirmed by the dynamics of various cell cycle markers.

View larger version (41K): [in this window] [in a new window]   Fig. 7. Rsk promotes destruction box-dependent proteolysis of cyclin A and cyclin B in unfertilized starfish eggs. (A) Inhibition of overall protein synthesis. Unfertilized mature eggs were incubated in the presence (+) or absence (–) of emetine for 20 minutes and then recovered. For 5 minutes before recovery, eggs were pulse-labeled with [35S]Met/Cys. Immunoprecipitation of cyclins A and B, autoradiography and immunoblots were performed as in Fig. 5D. (B) Disappearance of cyclins A and B by inhibition of their synthesis is prevented by D-box peptide. Unfertilized mature eggs were co-injected with morpholino antisense oligonucleotides (MOs) against cyclins A and B, and wild-type (WT, lane 2) or mutant (mt, lane 3) cyclin B destruction box peptide (D-box pep). As controls, eggs were uninjected (lane 4) or injected with MOs against cyclins A and B only (lane 1). After incubation for 30 minutes, eggs were recovered and the extracts were immunoblotted with anti-cyclin A, anti-cyclin B and anti-PSTAIR for Cdk1. (C) Disappearance of cyclins A and B by prevention of their synthesis requires Rsk. Unfertilized mature eggs were co-injected with CA-Rsk-EE (CA) or KD-Rsk-EE (KD) and MOs against cyclins A and B in the presence of U0126 (lanes 4 and 5). As controls, eggs were uninjected (lanes 1 and 6) or injected with MOs against cyclins A and B in the presence (lane 3) or absence (lane 2) of U0126. After incubation for 30 minutes, egg extracts were prepared and immunoblotted with anti-cyclin A, anti-cyclin B, anti-MAPK and anti-GST for CA- and KD-Rsk-EE.

View larger version (23K): [in this window] [in a new window]   Fig. 8. Dual-lock to the start of the embryonic cell cycle in unfertilized mature eggs of the starfish Asterina pectinifera. Unless fertilization occurs, mature eggs of the starfish Asterina pectinifera arrest at the G1 phase of the pronuclear stage after completion of meiosis II. To block the start of the embryonic mitotic cycle, two separate pathways emerge downstream of the Mos-MAPK pathway. One is the established Rsk-mediated pathway that leads to prevention of entry into S phase (Mori et al., 2006). The other is a novel Rsk-independent pathway that prevents entry into M phase. Such a dual-lock downstream of MAPK is necessary for G1 arrest, as the lack of a DNA replication checkpoint allows entry into M phase in the absence of S phase. The Rsk-independent pathway causes inhibition of protein synthesis of cyclin A and cyclin B (although the direct target of MAPK remains unclear) thus preventing accumulation of both cyclins that are required for entry into M phase. Cyclin A-Cdk1 contributes to activation of cyclin B-Cdk1 via inhibition of Wee1/Myt1, inhibitory kinases for cyclin B-Cdk1 (Okano-Uchida et al., 2003). By contrast, the Rsk-dependent pathway causes destruction of cyclin B and possibly cyclin A, thus also preventing accumulation of both cyclins. However, the Rsk-dependent destruction is dispensable for prevention of entry into M phase because suppression of MAPK can induce M phase even in the presence of Rsk. In the physiological condition, fertilization induces degradation of Mos to shut down both of its downstream pathways, resulting in the cell cycle progression into S phase and M phase.

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