spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online 10 June 2003
doi: 10.1242/jcs.00614

This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jang, J. K.
Right arrow Articles by McKim, K. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jang, J. K.
Right arrow Articles by McKim, K. S.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Relationship of DNA double-strand breaks to synapsis in Drosophila

Janet K. Jang, Dalia E. Sherizen, Rajal Bhagat, Elizabeth A. Manheim* and Kim S. McKim{ddagger}

Waksman Institute and Department of Genetics, Rutgers, the State University of New Jersey, 190 Frelinghuysen RD, Piscataway, New Jersey 08854-8020, USA



View larger version (112K):

[in a new window]
  		Fig. 1. {gamma}-HIS2AV staining in wild-type female meiosis. (A) Projection of an image stack containing a single germarium stained for ORB (green), {gamma}-HIS2AV (red) and DNA (blue). Before region 2a, four incomplete premeiotic divisions produce a cyst of 16 cells interconnected by ring canals. Because developing cysts are propelled to the posterior end of the germarium (in direction of arrow), they are usually arrayed in order of their relative developmental ages (Carpenter, 1975aGo). The ORB protein (Lantz et al., 1994Go) was used as a cytoplasmic marker to identify the 16-cell cysts, which are numbered in order of developmental stage. When cysts with two pro-oocytes are initially formed, they are in region 2a (cysts 1-4) and have a round shape. This region contains early zygotene and pachytene oocytes. Several cells within each cyst show evidence of entering meiosis, and two cells, the pro-oocytes, have four ring-canals and reach pachytene with complete assembly of synaptonemal complex (SC) (Carpenter, 1975aGo). One of these cells will become the oocyte. When the cysts flatten out and become encircled by follicle cells, they are in region 2b (cyst 5 and 6) and 3 (cyst 7). Region 3 oocytes are in mid-late pachytene. ORB becomes enriched in the oocyte in regions 2b and 3 (arrows, cysts 5-7), but initially (more anterior) it appears uniformly in all 16 cells of a cyst (region 2a, cysts 1-4). In projections such as this, it is difficult to determine which nuclei contain foci. It is clear from this view, however, that abundant {gamma}-HIS2AV foci are not observed until cysts 3. C(3)G staining is usually present in the first ORB staining cysts (1 and 2) (Page and Hawley, 2001Go) (data not shown), suggesting that {gamma}-HIS2AV staining appears after SC formation (see Fig. 2). Single optical sections show {gamma}-HIS2AV foci in nuclei from cyst 3 (B) and cyst 5 (C) but not later stage nuclei from cysts 6 and 7 (D). Arrowheads mark the oocytes. Bar, 10 µm.

 


View larger version (24K):

[in a new window]
  		Fig. 2. {gamma}-HIS2AV staining in wild-type female meiosis appears after the initiation of SC formation. All images are single sections, with {gamma}-HIS2AV (red), C(3)G (green) and DNA (blue) staining. (A) An early region 2a pro-oocyte in zygotene with no nuclear {gamma}-HIS2AV staining. (B) A pachytene nucleus with five {gamma}-HIS2AV foci associated with the C(3)G-staining. A color merge and the {gamma}-HIS2AV channel are shown. (C) A pro-nurse cell with {gamma}-HIS2AV foci and fragments of C(3)G staining at the same developmental stage as the cell in B. The three {gamma}-HIS2AV foci are all associated with segments of C(3)G staining. Next to this cell and in the same cyst is a pro-oocyte in pachytene. Bars, 1 µm.

 


View larger version (76K):

[in a new window]
  		Fig. 3. Increased {gamma}-HIS2AV in late pachytene oocytes of DSB repair defective mutants. All oocytes were stained for {gamma}-HIS2AV (red), ORB (green) and DNA (blue) and, unless otherwise noted, were in region 3 (late pachytene). (A) {gamma}-HIS2AV foci are rare in region 2a nuclei (a lower magnification image to show more nuclei) and (B) abundant in a region 3 oocyte from a spnBBU female. (C) Region 2b and (D) region 3 oocytes from the same okrWS mutant germarium, showing that the foci become brighter in more posterior oocytes, which are in a later stage of pachytene. In both spnB and okr mutants, large numbers of foci were present in every region 3 oocyte examined (n>20 for each genotype). By contrast, nuclear {gamma}-HIS2AV foci were absent in region 3 oocytes from (E) His2Av{Delta}CT; spnBBU His2Av810 (F) and okrWS mei-W681 females. (G) Nuclear {gamma}-HIS2AV foci were present in okrWS; c(3)G68 double mutants, suggesting that a few DSBs are made in the absence of SC. (H) In a region 3 oocyte from an okrZ0682 mutant female, {gamma}-HIS2AV foci are present, but less abundant than in an okrWS mutant. All images are maximum projections of the sections needed to view the entire nucleus. Bar, 5 µm; images B-H are approximately the same scale.

 


View larger version (66K):

[in a new window]
  		Fig. 4. Defects in DSB repair cause a delay in {gamma}-HIS2AV staining. Wildtype and DSB repair mutant germaria were stained for {gamma}-HIS2AV (red), DNA (blue) and C(3)G (green). Also shown are the single channels for {gamma}-HIS2AV and C(3)G staining. (A) A region 3 oocyte from a wild-type female that stains for C(3)G but not for {gamma}-HIS2AV foci. (B) A region 3 cyst of an okrWS mutant germarium showing {gamma}-HIS2AV foci in the oocyte (with extensive C(3)G staining, arrow) and nurse cells (arrowheads). (C) Pro-oocytes in region 2a and 2b cysts of the a spnBBU mutant germarium. In wildtype, these stages would normally have the maximum number of {gamma}-H2AX foci for the germarium. Instead, these pro-oocytes have just begun to form foci associated with the SC (arrows). (D) A Region 3 cyst of the spnBBU mutant shown in (c) that has a large number of foci in the oocyte (arrow) and nurse cells (arrowheads). In this germarium, the previous cyst (region 2b, not shown) was the first one to strongly stain for {gamma}-HIS2AV and the sixth in the germarium that stained with C(3)G (see C). As in Fig. 3, all images are maximum projections. Bar, 5 µm.

 


View larger version (58K):

[in a new window]
  		Fig. 5. {gamma}-HIS2AV foci persist into late stages of meiotic prophase in okr and spnB mutants. Following region 3 in the germarium, each cyst enters the vitellarium, which is divided into several developmental stages (numbered 2-14). The meiotic nucleus condenses into a tight mass, the karyosome, meiosis arrests and the SC dissolves from the nucleus by stage 6. The remaining cells become polyploid nurse cells. The oocyte (arrows) is marked by the ORB protein (green), DNA is stained blue and the {gamma}-HIS2AV foci are red. (A) A merge of five confocal sections from an okrWS vitellarium showing foci in a stage 3 oocyte and their absence at stage 5. (B) A merge of three confocal sections from a spnBBU vitellarium. The {gamma}-HIS2AV foci are present in a stage 4 cyst but absent in the stage 6 cyst (see inset). Bar, 10 µm.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2003