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First published online 13 April 2005
doi: 10.1242/dev.01797

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Cell cycle diversity involves differential regulation of Cyclin E activity in the Drosophila bristle cell lineage

UMR 7622, CNRS-University Paris VI. 9, Quai Saint Bernard, 75005 Paris, France

View larger version (73K): [in a new window]   Fig. 1. Characterization of the S-phase in pIIa and pIIb precursor cells (A-C) and pIIIb precursor cells (D-F). A strategy combining time-lapse imaging (A,D) and BrdU incorporation (B,E) was used to define the duration of the S-phase in these cells. (A,D) Time-lapse analysis of living neuP72> H2B::YFP pupae at 17 hours APF (A) and 19 hours APF (D). Time (h:min) before the end of the time-lapse recording is shown at the top of each frame. (B,E) BrdU incorporation in the same clusters as in A and D, respectively (numbered from 1 to 4). BrdU incorporation is revealed in red, and lineage cells were identified by specific anti-Sens immunoreactivity (green). In B, the time following the end of the corresponding pI metaphase, determined by time-lapse recording, was 19 minutes for cluster 1, 27 minutes for cluster 2, 59 minutes for cluster 3 and 1 hour 56 minutes for cluster 4. Note that both precursor cells exit the S-phase at the same time, as suggested by the punctuated BrdU labelling (B, cluster 3). In E, the time after the end of the corresponding pIIb metaphase was 7 minutes for cluster 1, 19 minutes for cluster 2, 39 minutes for cluster 3 and 1 hour 15 minutes for cluster 4. Note that BrdU incorporation was observed only in the pIIIb cell, recognized by the size of the nucleus and its anterior position in the cluster. (C,F) Histograms showing the percentage of BrdU-positive clusters as a function of time after pI (C) and pIIb (F) metaphase. Black bars correspond to cell pairs in which BrdU incorporation was homogeneous throughout the nuclei. Hatched bars correspond to cell pairs in which punctuated BrdU incorporation was observed. Note in F that no BrdU incorporation was observed before 15 minutes after pIIb division. In both histograms, bars were calculated after analysis of at least 5 clusters. Scale bar: 2 µm.

View larger version (103K): [in a new window]   Fig. 2. S-phase entry in the secondary and tertiary precursor cells. BrdU incorporation (red) in A101 pupae at 17 hours APF during pI division (A-B) and in pupae at 19 hours APF during pIIb division (C-D). Bristle lineage cells were identified by specific anti-ß-gal or anti-Sens immunoreactivity (blue). Cells in telophase were identified by incomplete formation of the nuclear envelope, as suggested by lamin immunoreactivity in the cytoplasm (A) and by the presence of the midbody (green) detected by -Tub immunoreactivity (B,C). Note in C that that midbody is observed along the axis of division that follows an apico-basal orientation. In D, interphasic cells showing well-defined Lamin-labelling (green) around the nuclei. In each panel, overlay is on the right. Note in B that BrdU incorporation takes place in pIIa and pIIb cells, while the ß-gal was still present in the cytoplasm. Note also that there is no BrdU incorporation late in pIIb telophase (C) and after formation of the pIIIb nucleus (D). Scale bar: 2 µm. g, glial cell.

View larger version (47K): [in a new window]   Fig. 3. Two rounds of endoreplication occurred in the socket and the shaft cells. Staining of nota from A101 pupae between 21 hours 30 minutes and 30 hours APF (A-D). ß-Gal staining is in green, BrdU detection in red and Su(H) in blue. During this period, BrdU, when incorporated, was located in the shaft and/or the socket cells. Four types of clusters were observed: clusters with no BrdU labelling (A); clusters in which the shaft cell incorporated BrdU (B); clusters in which the socket cell incorporated BrdU (C); and clusters in which both the shaft and socket cells incorporated BrdU (D). (E) Histogram showing the percentage of clusters harbouring a BrdU-labelled shaft cell (black bars) or a BrdU-labelled socket cell (white bars) as a function of time after pupal formation. Note the bimodal distribution for each cell. Bars were calculated after analysis of 25 clusters. Scale bar: 2 µm. n, neuron; s, sheath cell; sh, shaft cell; so, socket cell.

View larger version (10K): [in a new window]   Fig. 7. Schematic representation of cell cycle phases and expression pattern of the CycE, Dap and Ttk proteins in the bristle cell lineage. The bristle lineage is represented by an arborisation where cell cycle phases are shown in different colours (see code). The length of each colour bar is proportional to the duration of each phase. The expression pattern of CycE, Dap and Ttk are represented by horizontal bars above or below the lineage branches. The cross over the glial cell indicates that this cell enters apoptosis. Time scale in hours APF.

View larger version (83K): [in a new window]   Fig. 4. Cyclin E and Dacapo expression pattern in the microchaete cell lineage. Staining of nota from A101 pupae between 16 and 24 hours APF. (A-I) CycE expression in bristle lineage cells at progressive stages of development. CycE staining is in red and ß-gal staining in green. Combined time-lapse imaging of living pupae followed by CycE detection was performed in parallel to order the panel sequence. (J-R) Dap expression in bristle lineage cells at progressive stages of development. Dap staining is in red and ß-gal staining in green. In R, the neuron is identified by Elav staining (blue). (S,T) Coexpression of CycE (red) and dap (blue) at a two (S) and three-cell (T) cluster stage as shown in E and L, in N and an intermediate stage between F and G, respectively. Sensory organ cells are stained by anti-Sens immunoreactivity (green). In S and T, the overlay is on the right. (U) Temporal localisation of each panel (A-T) during the cell lineage. Scale bar: 2 µm. g, glial cell, s, sheath cell; n, neuron; sh, shaft cell; so, socket cell.

View larger version (105K): [in a new window]   Fig. 5. The activity of CycE controls the G1 phase in secondary and tertiary precursor cells. (A) dap4 null mutant pupae at 19 hours APF, BrdU incorporation (red) occurs during pIIb telophase, identified by the presence of the midbody (-Tub, green). (B) After overexpression of CycE, BrdU incorporation (red) occurs during pIIb telophase, revealed by the midbody staining (-Tub; green). Note that the BrdU-positive cell is the larger of the pair, suggesting that it corresponds to the pIIIb cell. Nota from neuP72> CycE pupae at 19 hours APF. (C) Overexpression of CycE induces precocious BrdU incorporation (red) in the shaft and the socket cells. Nota from neuP72> CycE pupae at 19 hours 30 minutes APF. Note that simultaneous BrdU incorporation in the pIIIb, the shaft and the socket cells was never observed in control animals. (D) Overexpression of Dap triggered a G1-phase in pIIa and pIIb cells. By contrast to control animals (see Fig. 2A), no incorporation of BrdU (red) was observed during pI telophase, identified by the presence of the midbody (-Tub; green). Nota from neuP72> Dap pupae around 17-18 hours APF. In all panels, sensory organ cells are identified by anti-Sens immunoreactivity (blue in A,B,D and green in C) and the overlay is on the right. g, glial cell; sh, shaft cell; so, socket cell. Scale bar: 2 µm.

View larger version (121K): [in a new window]   Fig. 6. Ttk69 repressed CycE expression in pIIa cells. (A-C) Expresssion pattern of Ttk in the bristle cell lineage. Ttk immunoreactivity (red) of nota from A101 pupae between 16 and 24 hours APF. Sensory organs were identified with anti ß-gal antibodies (green). Note that Ttk expression preceded pIIa cell division. (D) Ttk overexpression repressed CycE expression in pIIb cells. A sensory organ from hs-Gal4>Ttk69 pupae. CycE detection is in red and Sens in green. Note that in three-cell clusters, CycE was not present in pIIIb cells (compare with control animals, Fig. 4F,T). (E-G) ttk1e11 somatic clones. Clones were detected by the lack of GFP staining (green), their limits are shown with a white dotted line. Sensory organs were detected with anti-Cut (E,F) and anti-Sens (G) antibodies (green). (E) Ttk69 protein is not detected in clusters in ttk1e11 somatic clones. Ttk protein is detected in red. Nota from pupae at 23 hours APF. (F) Socket cells are present in sensory organs inside ttk1e11 clones. Socket cells are identified by the high expression of Su(H) (red). Nota from pupae at 23 hours APF. In E and F, arrows indicate clusters located in the clone. (G) Ectopic CycE expression was observed in the progeny of pIIa cells in ttk1e11 somatic clones. Each panel is shown separately in G', G", G"'. (G') ttk1e11 somatic clones identified by the lack of GFP staining, outlined by a white dotted line. Sensory organs were detected with anti-Sens antibodies. (G") Prospero expression revealed the pIIb progeny (arrowheads). (G"') CycE accumulates in all cells of clusters inside the clone. Note that even pIIa daughter cells, identified by the lack of prospero staining (arrows), are CycE positive. Nota from pupae around 21 hours APF. g, glial cell; s, sheath cell; n, neuron; sh, shaft cell; so, socket cell. (A-D) Scale bars: 2 µm in A-D; 10 µm in E-G.