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 21 December 2006
doi: 10.1242/dev.02738

Development 134, 467-478 (2007)
Published by The Company of Biologists 2007
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplementary Material
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 Shibutani, S.
Right arrow Articles by Duronio, R. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shibutani, S.
Right arrow Articles by Duronio, R. J.
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?

Rbf1-independent termination of E2f1-target gene expression during early Drosophila embryogenesis

Shusaku Shibutani1,*, Lisa M. Swanhart1,* and Robert J. Duronio1,2,3,{dagger}

1 Department of Biology, University of North Carolina, Chapel Hill, NC 27599, USA.
2 Program in Molecular Biology and Biotechnology, University of North Carolina, Chapel Hill, NC 27599, USA.
3 Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA.


Figure 1
View larger version (86K):
[in this window]
[in a new window]

  		Fig. 1. Rbf1 activity is controlled by phosphorylation in the early embryo. (A-G) In situ hybridization of stage 10 embryos with an RnrS probe. (A) Sibling control embryo from a collection expressing UAS Rbf1 with prd-Gal4. (B) UAS Rbf1/prd-Gal4. Arrow marks paired-expressing segment. (C) UAS Rbf-280/prd-Gal4. Arrow denotes the precocious termination of RnrS expression in a paired-expressing segment. (D) UAS Rbf-280/prd-Gal4 embryo pulse labeled for 15 minutes with BrdU (green). RnrS expression was detected by FISH (red). Arrow and arrowhead indicate cells in cycle 15 and 16, respectively. (E) Sibling embryo from a collection expressing UAS Rbf-280 with arm-Gal4 VP16. (F) UAS Rbf1/arm-Gal4 VP16. (G) UAS Rbf-280/arm-Gal4-VP16. (H) Rbf1 was immunoprecipitated from 0- to 4-hour-old and 5- to 8-hour-old w1118 embryo extracts, and the IPs were probed for the presence of E2f1 and Dp by western blotting. (I) Schematics of the embryonic cell cycle program and the regulation of E2f1 activity. Scale bar: 200 µm.

 

Figure 2
View larger version (128K):
[in this window]
[in a new window]

  		Fig. 2. E2f1-target gene expression is terminated in mutants containing ectopic CycE-Cdk2. Embryos were pulse-labeled with BrdU for 5 minutes (A-D) or 15 minutes (E) and were stained for BrdU incorporation (green) and phospho-tyrosine to highlight cell boundaries (cyan). RnrS expression was detected by FISH (red). (A) Stage 10 w1118 control embryo. The bar denotes S16 in the dorsal epidermis and the bracket marks cycle 15 in the ventral epidermis. (B) Stage 10 dap4454/dap4454 embryo. Note that phospho-tyrosine staining was omitted because anti-ß-galactosidase was used to distinguish CyO P[wg-lacZ]-containing embryos from the dap mutants. (C) Control embryo that is a sibling of the embryo in D. The arrow in the BrdU image denotes cells of the anterior spiracle primordium that normally enter S17. (D) Stage 11 dap4454/dap4454 embryo. (E) Df(1)biD3/Df(1)biD3 fzr mutant embryo. Scale bars: 50 µm.

 

Figure 3
View larger version (78K):
[in this window]
[in a new window]

  		Fig. 3. E2f1 protein accumulation during embryogenesis. w1118 embryos were pulse-labeled with BrdU for 5 minutes and stained for E2f1 (green), BrdU incorporation (red) and phospho-tyrosine (cyan). Embryos undergoing (A) S13; (B) S14; and (C) G214. (D) S15 cells are indicated by arrows in the BrdU image; the remainder are still in G214. Note that entry into M14 is not synchronous throughout the embryo, resulting in groups of cells called mitotic domains that proceed through the cycle coordinately and that generate a reproducible and stereotypic pattern of BrdU incorporation [e.g. the top arrow indicates mitotic domain 11 (Foe, 1989Go)]. (E) Cycle 15 in the ventral epidermis (bracket) and S16 in the dorsal epidermis (bar). (F) S16 in the ventral epidermis (bracket) and G216-G117 in the dorsal epidermis (bar). Arrowheads in E-H indicate amnioserosa cells. (G,H) Although most cells of the epidermis have entered G117 (see Fig. 2C), some cells continue into cycle 17 (arrows in the BrdU image). (I) G117. Scale bar: 50 µm.

 

Figure 4
View larger version (58K):
[in this window]
[in a new window]

  		Fig. 4. E2f1 protein persists through mitosis into early S phase. (A) Stage 8 w1118 embryo labeled with E2f1 (green), phospho-histone H3 (red) and phospho-tyrosine (cyan). Prophase (arrowhead), metaphase (large arrow), anaphase (small arrow), and daughter cells in early interphase (double arrow) are indicated. (B) Stage 11 w1118 embryo labeled with E2f1 (green), BrdU (red; 5 minute pulse) and phospho-tyrosine (cyan). Early, mid, and late S phase are marked by a large arrow, a small arrow, and an arrowhead, respectively. Scale bars: 20 µm.

 

Figure 5
View larger version (105K):
[in this window]
[in a new window]

  		Fig. 5. E2f1 destruction is replication-dependent. (A-E) Stage 11 embryos were pulse-labeled with BrdU for 5 minutes, and stained for E2f1 (green), BrdU incorporation (red) and phospho-tyrosine (cyan). (A) stg7B/stg7B. (B) dupa1/dupa1. (C) dupa3/dupa3. (D) dap4454/dap4454; arrow indicates epithelial cells expressing low levels of E2f1. (E) UAS dap/arm-Gal4 VP16. (F) Stage 11 Df(1)biD3/Df(1)biD3 fzr-mutant embryos were pulse-labeled with BrdU for 15 minutes, and stained for E2f1 (green) and BrdU incorporation (red); arrow indicates epithelial cells expressing low levels of E2f1 similar to dap mutants. Scale bars: 50 µm.

 

Figure 6
View larger version (91K):
[in this window]
[in a new window]

  		Fig. 6. The initial termination of E2f1-target gene expression does not require Rbf1. Rbf114 maternal and zygotic null embryos were pulse-labeled with BrdU for 15 minutes and stained for E2f1 (green) and BrdU incorporation (cyan). RnrS expression was detected by FISH (red). (A) Stage 10 embryo; bracket marks the dorsal epidermis in S16 and the bar indicates cycle 15. (B) Stage 11 embryo; the epidermal cells are in G216. (C) Stage 13 embryo; arrow indicates epidermal cells arrested in G117 and the bracket denotes epidermal cells inappropriately incorporating BrdU. Scale bar: 50 µm.

 

Figure 7
View larger version (132K):
[in this window]
[in a new window]

  		Fig. 7. Dap expression activates Rbf1. Embryos were stained for Dap (green) and phospho-tyrosine (cyan). RnrS expression was detected by FISH (red). (A) Stage 10 stg7B/stg7B embryo. (B) Stage 12 stg7B/stg7B embryo. Brackets mark the epidermal cells and asterisks denote G214-arrested aminoserosa cells. (C-E) Embryos from dap4454/+; stg7B/+ parents. (C) Stage 11 embryo with the stg7B/stg7B phenotype. (D) Stage 11 dap4454/dap4454; stg7B/stg7B embryo. C and D are siblings that are stage-matched based on age, morphology and phospho-tyrosine staining. Bracket in C indicates epidermal cells in which RnrS is starting to decline and Dap is expressed at high levels. Bracket in D shows the corresponding region in which RnrS levels remain high. Asterisk in D denotes aminoserosa cells. (E) Stage 12 dap4454/dap4454; stg7B/stg7B embryo. Scale bar: 50 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 2007