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First published online 19 July 2006
doi: 10.1242/dev.02488

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Terminal mitoses require negative regulation of Fzr/Cdh1 by Cyclin A, preventing premature degradation of mitotic cyclins and String/Cdc25

BZMB, Department of Genetics, University of Bayreuth, 95440 Bayreuth, Germany.

View larger version (64K): [in a new window]   Fig. 1. Cyclin A is preferentially required for terminal mitoses. (A-J) Mitotic cells were labeled with antibodies against phosphohistone H3 (A-D,G-J; pH3) and ß-galactosidase for genotyping (not shown). Mitotic cells are observed throughout the thoracic and abdominal epidermis in sibling control embryos at the stage of mitosis 16 (A,B; CycA+). By contrast, mitotic cells are restricted mainly to the prospective anterior spiracle region in the CycAC8LR1 mutant epidermis (C,D; CycA-; white arrow). Cells in this restricted epidermal region are exceptional because they progress through an additional division cycle 17 after mitosis 16 during wild-type embryogenesis, as indicated by BrdU pulse labeling (BrdU) of control embryos at the stage of S phase 17 (E,F; white arrow), while essentially all the other epidermal cells exit from the mitotic cycle after mitosis 16. Mitotic divisions were also observed in the central nervous system of stage 14 CycAC8LR1 embryos (I,J; CycA-). B,D,F,H,J present high magnification views from the embryos shown in A,C,E,G,I, respectively. Inset in J illustrates the presence of normal anaphase figures in late CycAC8LR1 embryos, with DNA and anti-phospho-histone H3 staining shown in red and green, respectively. (K) Embryos at the stage of mitosis 16 were immunolabeled with anti-tubulin and anti-ß-galactosidase for genotyping. The number of mitotic cells in thoracic segments 1-3 (see 1, 2 and 3 separated by dashed lines in A) was counted in control (+) and CycA mutants carrying various alleles (C8, CycAC8; 114, CycAneo114; 183, CycA183; C8LR1, CycAC8LR1). Columns represent the average number of mitotic cells (n=10 embryos).

View larger version (155K): [in a new window]   Fig. 2. Restoration of terminal mitosis 16 in the epidermis of CycA mutant embryos requires the simultaneous expression of stabilized B-type cyclins and String/Cdc25-independent Cdk1. (A-X) Heat-inducible transgenes allowing expression of the String/Cdc25 phosphatase-independent Cdk1AF mutant (K1AF), wild-type Cdk1 (K1), String/Cdc25 (Stg), Cyclin B (B), Cyclin B3 (B3), or stabilized versions of these B-type cyclins lacking the degradation signals for APC/C-mediated proteolysis (Bs and B3s) were crossed into the CycAC8LR1 mutant background. Embryos before mitosis 16 were either exposed to a heat shock (+hs) or not (-hs). After ageing to stage 13, embryos were labeled with anti-tubulin (Tub), a DNA stain (DNA) and anti-ß-galactosidase (not shown) for the identification of CycAC8LR1 mutant embryos (CycA-) and sibling control embryos (CycA+). E-L present high magnification views from the embryos shown in A-D. Cell density in CycA mutants after failure of the terminal mitosis 16 is only half of that observed in control embryos (compare A,E,F with B,G,H) and is not increased by Cdk1AF expression (compare B,G,H with D,K,L). Co-expression of Cdk1AF and stabilized B-type cyclins, however, restores entry into the terminal mitosis 16 in CycA mutants and leads to an arrest in this mitosis in both control (M,N) and CycA mutants (O,P), as evidenced by the presence of spindles and condensed chromosomes. Co-expression of wild-type Cdk1 and stabilized B-type cyclins leads to an arrest during mitosis 16 in control embryos (Q,R), but does not restore mitosis 16 in CycA mutants (S,T). Cdk1 activators carrying degradation signals for APC/C-mediated proteolysis also fail to restore mitosis 16 in CycA mutants. Neither expression of a combination of Cdk1AF with wild-type B-type cyclins (U,V) nor expression of wild-type String/Cdc25 phosphatase with stabilized B-type cyclins (W,X) restores the epidermal cell density in the CycA mutants to normal. The numbers in lower right panel corners represent the cell counts in the regions of the right segments shown in E-X.

View larger version (79K): [in a new window]   Fig. 3. Cyclin A prevents a premature disappearance of Cyclin B3 before mitosis 16. (A-C) The prd-Gal4 transgene was used to drive UAS-CycA expression in alternating segments of CycAC8LR1 mutant embryos. Embryos were immunolabeled with anti-Cyclin B3 (CycB3, green in C) and a DNA stain (red in C). The UAS-CycA expressing segments (white arrowheads in A) contain more anti-CycB3-positive cells than do the segments without UAS-CycA expression. Among the segments without UAS-CycA expression, however, the one with most of the prospective anterior spiracle region (white arrow) has an exceptionally high number of CycB3-positive cells. The high magnification views in B and C indicate that mitosis 16 has already been completed in the anti-CycB3-negative regions in the segment with UAS-CycA expression to the right of the dashed white line. These regions (see for example cells around `1+') are characterized by a higher density of cells with smaller nuclei than in regions with CycB3-positive cells in G2 before mitosis 16 (around `2+'). The anti-CycB3-negative region `1-' in the segment without UAS-CycA expression to the left of the dashed line, which corresponds to region `1+', has a low cell density, reflecting the failure of mitosis 16. Importantly, in this segment without UAS-CycA expression, Cyclin B3 is not only absent from region `1-' but also from the region around `2-', indicating that in the absence of Cyclin A, Cyclin B3 disappears prematurely before mitosis 16, as the corresponding region `2+' in the segment with UAS-CycA expression still consists of anti-CycB3-positive cells in G2 before mitosis 16.

View larger version (42K): [in a new window]   Fig. 4. String/Cdc25 phosphatase levels are regulated by the APC/C activator Fizzy-related/Cdh1. The levels of String/Cdc25 phosphatase in embryo extracts after the stage of mitosis 16 were analyzed by immunoblotting with antibodies against String (STG) and tubulin (TUB) to control for loading. Extract from 60 embryos was loaded in each of the lanes except for lanes 3-5, which contain a threefold dilution series of the extract loaded in lane 2. The embryos loaded in lanes 8 and 9 were exposed to a heat shock (+HS) before extract preparation. The following genotypes were analyzed: fzrG0326 (lane 1, fzrG), fzrie28 (lanes 2-5, fzrie28), w1 (lane 6 and 9, control) and Hs-stg (lane 7 and 8, Hs-stg). Comparison of anti-STG signal intensities indicates that the levels of String/Cdc25 phosphatase are at least 10-fold higher in fzr mutant embryos.

View larger version (129K): [in a new window]   Fig. 5. Elimination of zygotic fizzy-related expression restores progression through mitosis 16 and induces an extra division cycle in the epidermis of CycA mutants. (A-D) High magnification views of the embryonic epidermis after anti-tubulin labeling (Tub) at stage 12 when epidermal cells are already post-mitotic in wild-type embryogenesis. The reduced cell density resulting from the failure of the terminal mitosis 16 in CycAC8LR1 mutants (D, CycA-) is not present in fzrie28; CycAC8LR1 double mutants (C, fzr- CycA-), which have the same cell density as sibling control embryos (A, +) and fzrie28 single mutant embryos (B, fzr-). (E-G) High magnification views of the embryonic epidermis after anti-Cyclin B labeling (CycB) at stage 13. Although Cyclin B does not accumulate after mitosis 16 in the post-mitotic epidermal cells of sibling control embryos (E, +), it reaccumulates during an extra division cycle 17 not only in fzrie28 single (F, fzr-) mutants (Sigrist and Lehner, 1997), but also in fzrie28; CycAC8LR1 double mutant embryos (G, fzr- CycA-). Cyclin B is also again degraded during the additional mitosis 17 (for examples see arrows in F,G). (H,I) Mitotic figures observed during the additional cell division 17 in fzrie28 single (H, fzr-) and in fzrie28; CycAC8LR1 double (I, fzr- CycA-) mutant embryos after anti-tubulin (Tub) and DNA labeling (DNA). Normal prophase, prometaphase, metaphase, anaphase and telophase figures are shown from left to right in H and I. In addition, abnormal anaphase and telophase figures with chromatin bridges were also present in the fzrie28; CycAC8LR1 double mutant embryos, as illustrated by the two right most panels in I.

View larger version (160K): [in a new window]   Fig. 6. Prolonged CycE expression during the terminal division cycles restores mitosis 16 in CycA mutants. (A-H) The prd-Gal4 transgene was used to drive UAS-CycE expression in alternating segments of CycAC8LR1 (CycA- prd>CycE) or sibling control (CycA+ prd>CycE) embryos. Embryos were immunolabeled with anti-Cyclin B3 (A,B; CycB3), or with anti-tubulin (C,E,G; Tub) and a DNA stain (D,F,H; DNA). Premature Cyclin B3 degradation before mitosis 16 is prevented in the UAS-CycE-expressing segments of CycAC8LR1 mutants (white arrowheads in A, right side in B). Mitosis 16 is restored in the UAS-CycE- expressing segments of CycAC8LR1 mutants (white arrows in E,F), resulting in a twofold higher cell density in the UAS-CycE-expressing segments of CycAC8LR1 mutants after the stage of mitosis 16 (compare left and right side in G,H).

View larger version (147K): [in a new window]   Fig. 7. Cyclin A and Cyclin E suppress Fizzy-related/Cdh1 lacking all Cdk consensus phosphorylation sites. (A-L) The prd-Gal4 transgene was used to drive various UAS transgenes in alternating segments. Embryos after the stage of mitosis 16 were immunolabeled with anti-tubulin (Tub) and a DNA stain (DNA) for a comparison of the cell densities in segments with (middle segment) and without UAS transgene expression (outer segments). The UAS transgenes were UAS-fzr (C,D), UAS-fzr and UAS-CycA (E,F), UAS-fzrpsm (G,H), UAS-fzrpsm and UAS-CycA (I,J) and UAS-fzrpsm and UAS-CycE (K,L). The UAS-fzrpsm transgene results in expression of a Fzr version in which all of the Cdk consensus phosphorylation sites (S/T P) are eliminated. A control embryo without an UAS transgene is shown in A,B. UAS-fzr and UAS-fzrpsm expression inhibits mitosis 16 and therefore results in a twofold lower cell density. Co-expression of either UAS-CycA or UAS-CycE suppresses this inhibition of mitosis 16.

View larger version (173K): [in a new window]   Fig. 8. Epidermal cell cycle progression in CycA- CycE- and CycA- dap- double mutants. (A-J) Embryos at the stage of mitosis 15 were immunolabeled either with anti-Cyclin B3 (A,B; CycB3), or with anti-tubulin (C-F; Tub) and a DNA stain (G-J; DNA). Comparison of the anti-CycB3 signals in CycEAR95; CycAC8LR1 double mutants (B; CycE-; CycA-) and sibling control embryos (A; +) reveals a premature Cyclin B3 disappearance in the double mutants. The strong anti-CycB3 signals remaining in the head region and along the ventral midline (arrowheads) of double mutants are from cells programmed to progress through mitosis 14 late. They are still in G2 before mitosis 14 at the stage shown and they therefore also still have residual maternal Cyclin A in the double mutants. Mitotic figures (white arrows) reflecting progression through mitosis 15 are observed in sibling control (C,G; +) and CycAC8LR1 (D,H; CycA-) and CycEAR95 (E,I; CycE-) single mutant embryos, but not in CycEAR95; CycAC8LR1 double mutant embryos (F,J; CycA- CycE-). (K-N) Embryos were pulse labeled with BrdU. After the stage of mitosis 16, epidermal cells in dap4 single (M; dap-) and dap4; CycAC8LR1 double (N; CycA- dap-) mutants enter S phase, but not in sibling control (K; +) and CycAC8LR1 single mutants (L; CycA-). Remarkably, epidermal cells in dap4; CycAC8LR1 double mutants enter S phase even though they have not progressed through mitosis 16. The failure of mitosis 16 is also evident from a comparison of the nuclear density in the epidermis (insets in M,N).