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doi: 10.1242/10.1242/dev.00501

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giant nucleiis essential in the cell cycle transition from meiosis to mitosis

Andrew D. Renault^1,*, Xiao-Hua Zhang¹, Luke S. Alphey¹, Lisa M. Frenz², David M. Glover³, Robert D. C. Saunders⁴ and J. Myles Axton^1,

¹ Department of Zoology, University of Oxford, South Parks Road, Oxford, OX1 3PS, UK
² Polgen Division, Cyclacel, Babraham Bioincubator 405, Babraham Institute, Babraham, Cambridgeshire CB2 4AT, UK
³ Department of Genetics, University of Cambridge, Downing Street, Cambridge, CB2 3EH, UK
⁴ Department of Biological Sciences, The Open University, Walton Hall, Milton Keynes, MK7 6AA, UK
^* Present address: Skirball Institute, Developmental Genetics Program, New York University Medical Center, 540 First Avenue, NY 10016, USA

View larger version (104K): [in a new window]   Fig. 1. DNA replication in gnu embryos. (A-D) Fluorescently immunostained eggs and embryos from gnu homozygous mothers. A nuclear lamina surrounded each of the giant nuclei in one embryo (A) but in this same embryo, only one nucleus stained for Drosophila PCNA (B). In another embryo, (C) DNA staining revealed three giant nuclei and, (D) following 5 minutes incubation, only one had incorporated BrdU. This indicated that not all of the nuclei are in S phase. (E,F) A 5 µm confocal section of an unfertilised egg (E) and embryo (F) stained for ß-tubulin in green and DNA in red. Microtubule asters were initiated in the fertilised embryo from duplicating centrosomes, but were not present in the unfertilised egg. Scale bars: 50 µm (A-D), 25 µm (E,F).

View larger version (40K): [in a new window]   Fig. 2. gnu DNA and deduced protein sequence (EMBL Accession Number AJ557828). The genomic sequence of gnu from an isogenic y; cn bw sp stock (Adams et al., 2000) is shown with the exons in upper case and the predicted amino acid sequence shown below the DNA sequence. The consensus splice sites are in italic and the polyadenylation signal is doubly underlined. The site of the C to T mutation on the gnu chromosome (base 709) is indicated by a solid black box. The ESTs GM10421 and LD12084 begin at bases 141 and 151 respectively (denoted by •). On sequencing gnu from OregonR and from our genomic rescue construct the following polymorphisms were found: 91 TC and 113 GA, 165 extra T in our genomic rescue construct (numbers refer to bases with y; cn bw sp version first and polymorphism second). All of these polymorphisms are in untranslated regions except 324 CA in OregonR which is a silent mutation in the coding sequence. Overall there is no polymorphism in the deduced protein sequence. The residues altered in the production of gnuSTOP (boxed), were 692 CA and 694 AT, resulting in a premature TAG stop codon in place of the lysine at 142 and a SpeI site (ACTAGT).

View larger version (40K): [in a new window]   Fig. 3. Developmental expression and post-translational modification. (A) Immunoblot with anti-peptide antiserum detected native Gnu protein in wild type (w[1118]) Drosophila ovaries (O) and 0-3 hour embryos (E) and detected functional Gnu-GFP fusion protein in ovaries from stock GG4c: homozygous gnu, rescued by a homozygous insertion of the genomic gnu-GFP construct (GG). (B) Expression in staged 0- to 4-hour embryos of Gnu (anti-Gnu) in w[1118] embryos (loading control: anti-actin), Gnu-GFP (anti-GFP) in GG4c embryos. (C-E) Functional fusion protein detected with anti-GFP antibody. (C) Proteins extracted from GG4c ovaries in the presence of the protein phosphatase inhibitors fluoride (NaF), orthovanadate (Na3VO4), ß-glycerophosphate (ßGP), okadaic acid (OA) and Inhibitor-2 (I-2Dm). (D) Extracts of ovaries, eggs and embryos from flies containing a single insertion of the genomic gnu-GFP construct in cortex (cort), grauzone (grau) and deadhead (dhd) mutant backgrounds or wild-type control homozygous for the genomic gnu-GFP construct. (E) Extracts of ovaries and embryos from flies containing a single heterozygous insertion of the genomic gnu-GFP construct in homozygous png backgrounds. png1058 and png3318 are null and weak alleles respectively. The wild-type control is from flies homozygous for the genomic gnu-GFP construct.

View larger version (119K): [in a new window]   Fig. 4. Gnu localization in ovaries and embryos. Confocal sections of ovaries from gnu females rescued by the homozygous genomic gnu-GFP construct (stock GG4c). (A) Gnu-GFP fluorescence (green; DNA stained red) was first observed in oocytes of stage 11 egg chambers. (B) In subsequent stages, the fusion protein accumulated in the oocyte but was not observed in nurse cells. (C) Gnu-GFP fluorescence was not found to be specifically associated with the polar bodies (D). (E-F) Syncytial embryo in interphase of cycle 10. The Gnu-GFP fluorescence was cytoplasmic (E) and not excluded from nuclei (F). (G-I) Confocal sections of a giant nucleus from flies homozyogus for png3318 and also containing a single heterozygous copy of the genomic gnu-GFP construct. (G) DNA, (H) Gnu-GFP fluorescence, (I) merged image with DNA in red and Gnu-GFP fluorescence in green. Scale bars: 100 µm (A,B), 10 µm (C-F), 20 µm (G-I).

View larger version (79K): [in a new window]   Fig. 5. Gnu mis-expression in ovaries results in sterility. Confocal sections of ovaries and egg chambers from females containing (A,D-F) the maternal 4 tubulin>GAL4:VP16 and UASp gnu-GFP, (B) maternal 4 tubulin>GAL4:VP16 with UASp EGFP constructs in a wild-type background, (C) maternal 4 tubulin> GAL4:VP16 with UASp gnu-GFP in a homozygous png1058 null background. (A) Gnu-GFP mis-expressed in ovaries is nuclear and disrupts oogenesis with inappropriate nurse cell degeneration and fragmented chromatin (arrow). (B) Ectopic GFP is cytoplasmic and nuclear and does not disrupt ovary morphology. (C) Ectopic Gnu-GFP is largely nuclear but oogenesis is not disrupted and ovarian morphology is normal. DNA is red and GFP fluorescence, green. (D-F) A single stage 8 egg chamber. (D) Propidium iodide-stained DNA, (E) GFP fluorescence, (F) merged image of D and E, with DNA in red and Gnu-GFP fluorescence in green. Not all nurse cell nuclei contain Gnu-GFP (arrowhead). Scale bars: 50 µm. (G) Anti-GFP blot shows that Gnu-GFP expressed from the GAL4-UAS system is unmodified, in contrast with that expressed from the genomic gnu-GFP construct.

View larger version (105K): [in a new window]   Fig. 6. Abnormal actin reorganisation in ovaries mis-expressing Gnu. (A) Propidium-stained DNA, (B) FITC-phalloidin stain for F-actin. (C) Merged image of A and B. DNA red, F-actin green. (D) Gnu-GFP in nurse cell nuclei. (E) Rhodamine-phalloidin stain for F-actin. (F) Merged image of D and E Gnu-GFP green, rhodamine-phalloidin red. Gnu-GFP mis-expression was compatible with some aspects of F-actin organisation, such as ring canals (E arrowhead, F). At the equivalent of wild-type stage 10, the actin cytoskeleton aggregated (E arrow) and did not develop the contractile meshwork (B arrowhead, C) that in the wild type ovary dumps nurse cell cytoplasm into the oocyte and retains nurse cell nuclei in oogenic stages 10B and 11. Confocal sections were taken of egg chambers from females containing the maternal 4 tubulin>GAL4:VP16 (A-C) and UASp gnu-GFP (D-F) constructs in wild-type background.