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Biochem Biophys Res Commun
2002 May 31;2941:120-6. doi: 10.1016/S0006-291X(02)00447-3.
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The APC regulator CDH1 is essential for the progression of embryonic cell cycles in Xenopus.
Zhou Y
,
Ching YP
,
Ng RW
,
Jin DY
.
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The orderly progression of cell cycle depends on timely destruction of key regulators through ubiquitin-mediated proteolysis. The anaphase-promoting complex (APC) is a major component of this degradation machinery and its activation is regulated by CDC20 and CDH1. We demonstrate here that CDH1 mRNA is ubiquitously expressed in Xenopus embryos of all developmental stages. Loss of CDH1 function during early embryonic cell cycles leads to an immediate and prolonged arrest with low cyclin-dependent kinase activity. In contrast, ectopic overexpression of CDH1 induces cell cycle arrest during the first G(1) phase at the midblastula transition. CDH1-dependent degradation of cyclin A is likely involved in this G(1) arrest. Our findings establish the essential roles of CDH1 in embryonic cell cycles.
Fig. 1.
Expression of CDH1 mRNA in Xenopus oocytes and embryos. RT-PCR was performed with sub-saturating amounts of templates and the amplification was in the linear range. Histone H4 was co-amplified as an internal control. Amplification signals for Xenopus CDC20 from the same samples were shown for comparison. Results are representative of duplicate amplifications of three independent preparations of RNA. Sequences for the PCR primers are: H4: 5â²-CGGGATAACATTCAGGGTA TCACT-3â²+5â²-ATCCATGGCG GTAACTGTCTTCCT-3â² (188 bp fragment); CDH1: 5â²-ACAG ACTGAAGATAGACGAC-3â²+5â²-AAGATCATCCT GTCCCTACTC-3â² (551 bp fragment); CDC20: 5â²-CGTCTTCGTAATATGATCAG -3â²+5â²-AGACCATACTATG GAGCAC-3â² (449 bp fragment).
Fig. 2.
Xenopus CDH1 is essential for early embryonic cell cycles. (A) Depletion of endogenous XCDH1 transcript by as-XCDH1 RNA. Both blastomeres of the 2-cell stage embryos were injected with 4 ng as-XCDH1 RNA (lanes 1–3), 4 ng as-MCDH1 RNA (lanes 4–6), or PBS (mock, lanes 7–9). Injected embryos were collected at indicated time (30, 45, and 70 min after injection) or stages (4-cell, 8-cell, and 64-cell), and RT-PCR was performed as in Fig. 1. Sequences for the XCDH1 primers are 5′-CTACGTGTCT CTATCTGCAA-3′ and 5′-AAGATCATCCT GTCCCTACTC-3′ (900 bp fragment). Arrows indicate the annealing positions of the primers. It is noteworthy that the forward primer anneals to the endogenous XCDH1 mRNA, but not to the injected as-XDH1 RNA. (B) Loss of XCDH1 function induces cell cycle arrest: representative images of embryos. One blastomere of the 2-cell stage embryos was injected with PBS (mock; panel 1), 4 ng as-MCDH1 RNA (panel 2), 4 ng as-XCDH1 RNA (panel 3), or 4 ng as-XCDH1 RNA + 4 ng MCDH1 RNA (panel 4). Images were photographed at the 4-cell stage. Arrows indicate the injected side. (C) Persistent cell cycle arrest induced by XCDH1 depletion. An as-XCDH1-injected embryo was followed for 4 consecutive divisions (4-cell, 8-cell, 16-cell, and 32-cell). (D) Graphic quantitation of the cell cycle arrest phenotype induced by XCDH1 depletion. Each of the 7 indicated groups had 20–30 embryos. Percentages of embryos that showed the phenotype of cell cycle arrest as in panel 3 of B were calculated. Each bar represents the average values from four experiments.
Fig. 3.
Loss of CDH1 function in early embryonic cell cycles leads to prolonged arrest with low CDK activity. (A) Time course of histone H1 kinase activity immuno-precipitated with an antibody to CDK1. Both blastomeres of the 2-cell stage embryos were mock-injected with PBS (mock; upper panel), or injected with 4 ng as-XCDH1 RNA (lower panel). Five embryos from each group were collected and lysed every 15 min. The lysates were immunoprecipitated with anti-CDC2/CDK1 antibody and the precipitates were assayed for relative histone H1 kinase activity. Cell numbers of the embryos are indicated. Experiments were repeated twice with similar results. (B) Western blot analysis of cyclins. Five embryos were collected at each of the indicated stages. B1: cyclin B1. A2: cyclin A2. n.s.: non-specific band. Experiments were repeated twice with similar results.
Fig. 4.
Overexpression of CDH1 induces post-MBT arrest. (A) Representative images of embryos. One blastomere of the 2-cell stage embryos was mock-injected with PBS (mock; panel 1) or injected separately with 4 ng of the indicated RNAs: as-MCDH1 (panel 2), XCDH1 (panel 3), and MCDH1 (panel 4). Images were photographed at stage 8.5â9.5. The arrow indicates the arrested large cells on the injected side. (B) Graphic quantitation. For each indicated group 25â30 embryos were counted. Each bar represents the average values from three experiments.
