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First published online October 13, 2005
doi: 10.1242/10.1242/dev.02057

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Loss of Drosophila borealin causes polyploidy, delayed apoptosis and abnormal tissue development

MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK

View larger version (46K): [in a new window]   Fig. 1. borr is required for mitosis in embryonic VNC cells. (A) Schematic representation of the borr locus (black, coding; white, non-coding). The position of the E133 mutation is indicated by an arrowhead. (B,C) Ventral views of stage 12 (B) wild-type and (C) borr mutant embryos; nuclei are stained with Hoechst dye to visualise DNA. (D) Average volumes of wild-type and borr mutant VNC nuclei; numbers of pixels were calculated for each genotype based on outline tracings of 50 nuclei from five different embryos (see Materials and methods).

View larger version (23K): [in a new window]   Fig. 2. Mitotic progression is affected in the VNC of borr mutant embryos. (A) Counts of mitotic cells, as judged by P-H3 staining and chromatin morphology (see also Fig. 4B,D), in the VNC of wild-type or borr mutant embryos at stage 12 (n=6 or 7, respectively), and at stage 14 (n=12 or 10, respectively). (B,C) Relative frequencies of the four main mitotic stages (see D), expressed in percentages at stage 12 (B) and stage 14 (C). Asterisks in A-C indicate statistical significance (**P<0.0005) of the observed differences. (D) Examples of mitotic cells in which chromatin has been visualised by P-H3 staining, selected to illustrate the four main mitotic stages of wild-type and borr mutant VNC cells. There is abnormal chromatin segregation in the mutant anaphase.

View larger version (83K): [in a new window]   Fig. 3. Subcellular localisation and function of Borr in cultured Drosophila cells. (A-G) Mitotic DmD8 cells transiently expressing GFP-Borr, stained with Hoechst dye and antibodies against ß-tubulin, Aurora B or Incenp, as indicated. (H-M) Kc167 cells, (H) mock-treated or (I-M) treated with borr dsRNA. Staining is with Hoechst dye (blue) and phalloidin (red), and with antibody against ß-tubulin (green). Arrows indicate DNA fragments in the cytoplasm of a multi-nucleate Borr-depleted cell (M).

View larger version (83K): [in a new window]   Fig. 4. Reduced P-H3 levels in VNC cells of borr mutant embryos. (A-D) Ventral views of stage 13 (A,B) wild-type or (C,D) borr mutant embryos, stained with Hoechst dye and antibody against P-H3; projections of z-stacks of confocal sections with identical settings are shown, revealing lower numbers of mitotic cells in the mutant and reduced P-H3 levels (see also Fig. 2A,D). (E-J) Individual mitotic VNC nuclei prior to anaphase from (E-G) wild-type or (H-J) borr mutant stage 13 embryos; arrow in I indicates a mutant nucleus with a degree of chromatin condensation similar to that in wild type (F). There are reduced levels of P-H3 staining in both mutant nuclei (J, compare with G).

View larger version (97K): [in a new window]   Fig. 5. Aurora B and Incenp fail to localise to mitotic spindles in borr mutant imaginal disc cells. Wing discs of late third instar larvae bearing borr mutant clones, triple-stained with Hoechst dye and antibodies against ß-tubulin and (A-E') Aurora B or (F-J') Incenp. Areas containing wild-type or mutant mitotic cells are boxed, and the corresponding individual channels are shown below (B'-E',G'-J', wild-type cells; B-E,G-J, borr mutant cells). Localised Aurora B (C) and Incenp (H) staining is missing in the abnormal borr mutant spindles. Scale bars: in A, 5 µm for A,F; in B, 5 µm for B-E',G-J'.

View larger version (73K): [in a new window]   Fig. 6. Delayed apoptosis of large borr mutant cells. (A) Third larval instar wing disc, bearing early-induced borr mutant clones (marked by absence of GFP, indicated by blue arrow) that are invariably small; red arrow indicates large twin spot (showing strong GFP fluorescence), which lacks an associated borr mutant clone. (B) Third instar larval Minute/+ wing disc, bearing early-induced borr mutant clones (that are Minute+ and thus have a proliferative advantage), stained with Hoechst dye, showing numerous large borr mutant cells that have apparently survived multiple abnormal mitoses. (C,D) Wing disc as in A, showing DNA (blue), ß-tubulin (red, to mark cellular outlines) and Aurora B staining (green, to mark normal mitotic cells within the apical plane, indicated by arrowheads). Dying mutant cells are extruded basally (bottom). Both images are optical 3D reconstructions through the pouch regions of the disc along the y (C) or x (D) axis, generated from a z-stack of 0.25 µm confocal sections. (E) Wing disc as in A, stained with Hoechst dye and antibody against active caspase. Arrows in C-E indicate large borr mutant cells that are healthy (grey), apoptotic (white) or intermediate (red).

View larger version (110K): [in a new window]   Fig. 7. Early-induced borr mutant clones cause non-autonomous defects. (A-C) Adult wings (A) without or (B,C) with borr mutant clones; (C) enlargement of wing nick in B, with twin spot (marked with ck) outlined in white. (D-G) Third instar wing discs (D) without or (E-G) with borr mutant clones (marked by absence of GFP in F), stained with antibody against Wg; (F,G) magnifications of the boxed region in E. Red arrows indicate giant borr mutant cell expressing Wg, blue arrows indicate Wg expansion in wild-type cells that are not associated with a borr mutant clone. (H-M) Marginal regions of wing discs with borr mutant clones, stained as indicated; (LA) arrow indicates cell rearrangement that may have resulted from a dying borr mutant clone.

View larger version (108K): [in a new window]   Fig. 8. Survival and cell-autonomous defects of late-induced borr mutant clones. (A) Adult wing blade, with borr mutant clone (marked by f, outlined in yellow) and associated twin spot (marked by ck, outlined in grey). (B-D) Single confocal section through the epithelial sheet of a pupal wing blade with borr mutant clones (marked by absence of GFP), grazing the bases of the cell nuclei within the epithelial plane, stained as indicated in the panels; arrows indicate individual giant borr mutant cells. (E) Margin area from a wing bearing borr mutant clones with a giant bristle. (F-H) Single confocal section through a pupal wing, stained as in B-D and also with Hoechst dye. An individual large borr mutant cell (lacking GFP) gives rise to a giant bristle. ß-Tubulin staining is absent at the base of the bristle, indicating the lack of a socket.

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