QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 29 June 2004
doi: 10.1242/jcs.01240

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zhang, X.-H. Articles by Renault, A. D. Search for Related Content PubMed PubMed Citation Articles by Zhang, X.-H. Articles by Renault, A. D. Social Bookmarking                         What's this?

Spatial and temporal control of mitotic cyclins by the Gnu regulator of embryonic mitosis in Drosophila

Xiao-Hua Zhang^*, J. Myles Axton, Jována Drinjákovic, Lena Lorenz, Helen White-Cooper and Andrew D. Renault^,¶

Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK

View larger version (11K): [in a new window]   Fig. 1. Cell cycles at the time of fertilisation. The maternal egg activation sequence takes the Drosophila egg from metaphase I of meiosis to the first mitotic metaphase in the absence of fertilisation. Gnu, Plu and Pan gu proteins are essential to terminate the prezygotic S-phase and to establish the first zygotic mitosis. Fertilisation is required for the first mitotic anaphase because the spindle is organized by the daughters of the centrosome accompanying the paternal (grey) chromosomes; chromosomes only undergo this mitotic anaphase and undertake the subsequent mitotic cycles when attached to a spindle.

View larger version (50K): [in a new window]   Fig. 2. Gnu phosphorylation and expression. (A) Immunoblot with anti-GFP antibody demonstrating the slow-mobility, phosphorylated form of GnuGFP in ovaries from transgenic w; P{w+ gnuGFP}GG4C/+; gnu females (lanes O). Only the dephosphorylated form was found in their embryos (lane E) and in w; P{w+ gnuGFP S/T>A}/+; gnu ovaries from lines DF4, BM1, EM1 and GM3 (lanes 1-4 respectively) containing independent heterozygous insertions of the transgene. (B,C) Confocal sections of (B) w; P{w+ gnuGFP}GG4C/+; gnu/+ and (C) w; P{w+ gnuGFP S/T>A}DF4/+; gnu/+ ovaries showing distribution of GnuGFP (green) and DNA (red). Expression levels of wild-type and mutant Gnu were similar. However, GnuGFP from the wild-type w; P{w+ gnuGFP} transgene was not detected at stage 6 and was noticeably above w[1118] background (not shown) only in stages 9 and 10. By contrast, mutant Gnu from the P{w+ gnuGFP S/T>A} transgene was detectable at stage 6, accumulated in nurse cells but relatively little was found in stage 10 oocyte before the dumping of nurse cell contents.

View larger version (82K): [in a new window]   Fig. 3. Phenotypic series generated by partial rescue of gnu. Phenotypes of 0-3 hours embryos, from w; P{w+ gnuGFP S/T>A}/+;gnu lines (quantified in Table 2) stained for DNA. (A) fully rescued. (B) mitotic and giant nuclei. (C) >5 giant nuclei. (D) <5 giant nuclei.

View larger version (30K): [in a new window]   Fig. 4. Translational control at egg-activation conferred on gnu by bcd3' UTR. (A) Immunoblot with anti-Gnu antibody of extracts from ovary (O) and embryo (E). w[1118]: Native Gnu in wild-type is mostly phosphorylated (slow mobility) in ovaries and dephosphorylated in embryos. gnu3': w;P{w+ gnuGFP}GG4C/+;gnu, the 55 kDa GnuGFP from the rescuing transgene with gnu 3' UTR was translated in ovaries and persisted in embryos. bcd3': w;P{w+ gnuGFP-bcd3'UTR}S2/+;gnu[+] ovaries contained no GnuGFP, but it was translated in embryos. (B) Consequence of Gnu expression on cyclins A and B. Immunoblots of homozygous gnu mutant ovaries (O) revealed reduced cyclin A and B expression, but embryos (E) from homozygotes had much lower levels than the w[1118] wild type. The P{w+ gnuGFP-bcd3'UTR} transgene restored cyclin A and B levels to embryos from w;gnu P{w+ gnuGFP-bcd3'UTR}T1 females but not to their ovaries. Anti-actin antibody was used to verify comparable protein loading on the blot.

View larger version (54K): [in a new window]   Fig. 5. Gradients of functional Gnu protein from localized anterior source, conferred on gnu by bcd3'UTR. Phenotypes of embryos from w; P{w+ gnuGFP-bcd3'UTR}S2/+; gnu (quantified in Table 2). (A) Nuclei showing a gradient of Gnu mitotic activity. (B) Gradient of GnuGFP fluorescence. (C-F) Merged images of DNA (red) and GFP (green). (C) Merged images of A,B. (D) Mitotic and over-replicated nuclei. (E) Fully rescued. (F) Unfertilized egg. Mean length of embryos 570 µm.

View larger version (55K): [in a new window]   Fig. 6. cycB mRNA in gnu ovaries and embryos. In situ hybridization to cycB mRNA in ovaries and 0-3 hours embryos from gnu mutants (A-E) or w[1118] wild type embryo (F). Just as in wild type, cycB mRNA was seen in nurse cell cytoplasm at stage 8 of oogenesis (A) and accumulated in the oocyte cytoplasm, localizing to the anterior of the oocyte from stage 9 (B) to stage 12 (C). At stage 14 (D) the mRNA moved to the posterior where it remained throughout the cytoplasm and concentrated at the posterior pole in the embryo (E).

View larger version (88K): [in a new window]   Fig. 7. Stabilisation and loss of cyclin B in a Gnu gradient. (A) cyclin B in a w; gnu P{w+ gnuGFP-bcd3'UTR}T1 (Gnu gradient) embryo. (B) Merged images of cyclin B (green), -tubulin (blue) and DNA (red). (C,D) Detail of threshold region at approximately 25% egg length. (C) Metaphase -tubulin (blue), DNA (red). (D) Metaphase spindle-associated and cytoplasmic cyclin B. (E) Posterior region of Gnu gradient embryo showing loss of phosphorylated histone H3 (green) on anaphase chromosomes (DNA: red). Compare with the wild type in the inset F. (G) Posterior region of Gnu gradient embryo stained for centrosomal -tubulin (green), spindle -tubulin (blue) and DNA (red). The posterior-most mitotic nucleus is tetraploid, with four centrosomes organising a tetrahedral spindle array. Scale bar: 10 µm.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter