January 1, 2017;
Chk1 Inhibition of the Replication Factor Drf1 Guarantees Cell-Cycle Elongation at the Xenopus laevis Mid-blastula Transition.
The early cell divisions of many metazoan embryos are rapid and occur in the near absence of transcription. At the mid-blastula
transition (MBT), the cell cycle elongates and several processes become established including the onset of bulk transcription and cell-cycle checkpoints. How these events are timed and coordinated is poorly understood. Here we show in Xenopus laevis that developmental activation of the checkpoint kinase Chk1
at the MBT results in the SCFβ-TRCP-dependent degradation of a limiting replication initiation factor Drf1
. Inhibition of Drf1
is the primary mechanism by which Chk1
blocks cell-cycle progression in the early embryo
and is an essential function of Chk1
at the blastula
stage of development. This study defines the downregulation of Drf1
as an important mechanism to coordinate the lengthening of the cell cycle and subsequent developmental processes.
[+] show captions
Figure 1. Chk1 Inhibition Does Not Affect the Cell Cycles at the MBT(A) Western blot of Chk1 and β-actin from staged embryos at the indicated number of hours post fertilization (hrs.p.f). Embryos were injected at the one cell stage either with water (control) or with mRNA of the four limiting replication factors (treslin, drf1, recq4, and cut5) or the chk1 dominant-negative mutant (D148A). The extracts from embryos over-expressing Chk1 D148A were diluted 1 in 20 to allow a direct comparison between endogenous and over-expressed Chk1. See also Figure S1A.(B) Still images from time-lapse movies of embryos injected in both blastomeres at the 2-cell stage as in (A) The fourth division, generating the 16-cell embryo, was set to time zero. See also Movie S1.(C) The division of embryonic cells from (B) were followed throughout the movie. Each time point represents the division of a single cell. The cell divisions for the three conditions are displayed side by side for each cleavage cycle. Cleavages 4–7 are excluded for simplicity. n = 16 cells from four embryos for each condition.(D) Total number of divisions undergone by each cell in (C) until the end of the time-lapse movie.(E) The DNA content of embryos, injected as in (A), was quantified on agarose gels using ImageJ. The DNA content of control embryos was set to 1. Data are presented as mean ± SD, n = 5. See also Figure S1B.
Figure 2. Chk1 Inhibits Drf1 at the MBT(A) Western blot as in Figure 1A. For only the Chk1 blot from the over-expression of chk1 D148A, extracts were diluted 1 in 20 to allow a direct comparison between endogenous and over-expressed Chk1. All other samples are undiluted.(B–D) As for Figures 1B–1D. For (C) and (D), n = 15 cells from four embryos for each condition.See also Movies S2 and S3; Figure S2.
Figure 3. Chk1 Blocks the Cell Cycle by Inhibiting DDK(A) Images of pre-MBT embryos (6 hr post fertilization), not expressing (Control) or expressing increasing amounts of chk1 mRNA (pg), with or without co-expression of 500 pg of drf1 or dbf4. See also Figures S1C–S1E.(B) Analysis of the division of individual cells, as in Figure 1C, from movies of embryos expressing 50 pg of chk1 mRNA, with or without co-expression of 500 pg of drf1 or dbf4. The second division, generating the 4-cell embryo, was set to time zero. n = 12 cells from three embryos for each condition. See also Movie S5.(C) The average duration of cell cycle 5, generating the 32-cell embryo. Red dashed line shows mean time of cycle 5 for control embryos. n = 12 cells from three embryos for each condition. Data are presented as mean ± SD, which indicates the level of synchrony of cell division. See also Figure S4.
Figure 4. β-TRCP Controls Drf1 Levels(A and B) Western blots as in Figure 2A (left for Drf1/Dbf4, right for β-TRCP at 8 hrs.p.f). For (A) the control was injection of a control morpholino. Asterisk denotes non-specific band.(C–E) As for Figures 1B–1D. For (C) and (D), n = 20 cells from five embryos for each condition. MO, morpholino.See also Movie S4 and Figure S6.
Figure 5. Chk1 Phosphorylates Drf1 for β-TRCP-Dependent Degradation(A and B) Western blots after immunoprecipitation (IP) of myc-tagged Drf1 or unrelated myc-tagged protein of the same size (Smicl) from MBT-stage extracts co-expressing HA-tagged β-TRCP. Asterisk denotes immunoglobulin heavy chain.(C) Anti-myc Western blot of myc-tagged Drf1 from MBT-stage embryos resolved on a phos-tag SDS-PAGE gel.(D) Anti-myc Western blot of myc-tagged Drf1 fragments expressed in embryos and harvested at the indicated times.(E) As in (D). For only the Chk1 blot from the over-expression of chk1 D148A, extracts were diluted 1 in 20 to allow a direct comparison between endogenous and over-expressed Chk1. All other western blots are of undiluted samples.(F) Scale diagram of Xenopus laevis Drf1, showing the three conserved Dbf4 domains (N, M, and C) and the degenerate potential β-TRCP binding domains (pink) within the region 1–467. Top: alignment of potential β-TRCP binding sites between X. laevis and Xenopus tropicalis. Asterisks denote residues mutated to alanine in the Drf1 6A mutant.(G and H) Left: immunoprecipitations as in (A). Right: western blot as in (D) and (E). WT, wild-type. δ indicates Drf1 with both potential β-TRCP binding sites deleted. 6A denotes full-length Drf1 with the six residues marked by asterisks in (F) mutated to alanine.(I) As in (E).
Figure 6. Inhibition of Drf1 Is Important for Development(A) Images of staged embryos, injected at the 1-cell stage with water (control) or mRNA (over-expression). Percentages underneath the images represent the number of embryos that survived beyond that stage. n = 50 embryos for each condition.(B) As in (A); 500 pg of cdk1-AF and 1 ng of drf1 were injected at the 1-cell stage.
Figure 7. Inhibition of Drf1 Is a Crucial Function of Chk1(A) As for Figures 6A and 6B. The control was injection of a control morpholino (MO). n = 50 embryos for each condition.(B) Measurement of the appearance of cell death (white, extruded cells) from the start of stage 10 (9.5 hr post fertilization). n = 100 embryos for each condition.(C) Western blot from stage-11 embryo extracts showing the partial knockdown of Drf1 and Cdc6 after the morpholino (MO) injections.(D) The nuclear to cytoplasmic ratio ensures the lengthening of the cell cycle at the MBT both by out-titration of limiting replication factors (yellow box) and by inducing Chk1 activation, leading to Drf1 downregulation (red circle). See also Figure S7. Inhibition of Drf1 is the primary mechanism by which Chk1 elongates the cell cycle in the early embryo. Chk1-dependent degradation of Drf1 ensures a switch to Dbf4 as the regulatory subunit of DDK in post-MBT cycles. Downregulation of Drf1 is critical for developmental processes from the blastula stage onwards. Stages 1–6 are excluded for simplicity.