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


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

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 Andrioli, L. P. M.
Right arrow Articles by Small, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Andrioli, L. P. M.
Right arrow Articles by Small, 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?

Anterior repression of a Drosophila stripe enhancer requires three position-specific mechanisms

Luiz Paulo Moura Andrioli, Vikram Vasisht, Ekaterina Theodosopoulou, Adam Oberstein and Stephen Small*

Department of Biology, New York University, 1009 Main Building, 100 Washington Square East, New York, NY 10003-6688, USA



View larger version (25K):

[in a new window]
  		Fig. 1. A map of the eve locus is shown at the top. The positions of five enhancers that control early stripe formation (1-7) are shown. Two other enhancers that control the refinement of the initial stripes (LE), and later expression in inter-stripe regions (ftz-like) are also indicated. A map of the eve 2 minimal stripe element (MSE) is shown in the middle with positions of defined binding sites for transcription factors. Activator and repressor sites are closely linked, especially in two clusters, each of which contains two pairs of overlapping sites. Regions tested by deletion analyses are marked (D1-D5). A model for eve 2 regulation is presented at the bottom. Activation is mediated by Bcd and Hb, while Gt and Kr are involved in repression mechanisms that form the stripe borders.

 


View larger version (97K):

[in a new window]
  		Fig. 2. Deletion analysis of the eve 2 MSE. The lacZ reporter construct used in these assays is shown schematically at the top, and contains the eve 2 MSE and the eve 3+7 MSE to control for levels of expression. Whole-mount lacZ mRNA expression patterns directed by this construct are shown for the wild-type eve 2 MSE (A,B), and for eve 2 MSEs containing deletions (D1-4; C-J) as shown in Fig. 1. Unless otherwise indicated, all embryos in this paper are oriented with anterior towards the left and dorsal upwards. Expression patterns are shown for embryos early (left column) or mid-way through (right column) cleavage cycle 14. The wild-type eve 2 enhancer is first activated in a broad anterior domain (marked by the broad line, A), which is then refined to a stripe (B). Deletions 1, 3 and 4 each cause a failure to activate or maintain wild-type expression levels of eve 2 (C,D,G-J). D2 causes a premature strengthening of eve 2 with a more extensive anterior expansion (E). This expansion refines to form an ectopic stripe (*) in mid-cycle 14 (F).

 


View larger version (27K):

[in a new window]
  		Fig. 3. Evolutionary conservation of the eve 2 D2 region. (A) Sequences of the eve 2 enhancer region between the Bcd2 and Kr4 sites are shown for D. melanogaster (mel), D. erecta (ere), D. yakuba (yak), D. pseudoobscura (pse) and D. picticornis (pic). Blocks of identical sequences at least three bases long are shaded in blue. (B) Sequences of similar repeats of the GTTT motif in three different eve enhancers.

 


View larger version (124K):

[in a new window]
  		Fig. 4. The (GTTT)4 repeat is critical for anterior repression of eve 2. lacZ mRNA expression patterns are shown for early (left) and mid cycle 14 (right) embryos containing the wild-type eve 2-lacZ transgene (A,B) or an identical construct in which the (GTTT)4 sequence was either deleted (C,D) or mutated (E,F).

 


View larger version (122K):

[in a new window]
  		Fig. 5. Genetic analysis of anterior eve 2 repression. lacZ expression patterns are shown for embryos containing the eve 2-lacZ and eve2{Delta}(GTTT)4-lacZ transgenes in a wild-type embryo background (A,B), and in mutant embryos lacking gt (C,D) or tor activity (E,F). Both genetic mutants expand the derepression caused by the deletion of the (GTTT)4 site.

 


View larger version (49K):

[in a new window]
  		Fig. 6. Gel shift analysis of the (GTTT)4 sequence. (A) Incubation of an 32P-labeled oligo containing the (GTTT)4 sequence with Drosophila embryonic extracts causes the formation of several complexes (arrows). Most of these complexes can be competed by excess cold probe (lane 2), but not by the mutant probe (lane 3). Complexes are not formed with the mutant probe (lane 4). (B) Slp1 protein binds specifically to the (GTTT)4 repeat. Increasing concentrations of a bacterial extract containing full-length Slp1 protein (lanes 4-6) causes formation of a specific complex (arrow). Binding can be competed by excess cold probe (lane 7), but not by the mutant probe (lane 8).

 


View larger version (72K):

[in a new window]
  		Fig. 7. Effects of ectopic slp1 or gt expression on the eve striped pattern. (A) Wild-type expression patterns of slp1 (black) and gt mRNA (red) in an early cleavage cycle 14 embryo. Embryos carrying activated sna-slp1 (B) or sna-gt (C) transgenes express low levels of ectopic mRNA along the entire ventral surface in addition to their endogenous patterns. (D-I) eve RNA expression patterns in wild-type embryos (D,G) and those containing either ectopic slp1 (E,H) or ectopic gt (F,I). Images in A-F are lateral views with anterior towards the left and dorsal upwards. Those in G-I are ventral views.

 


View larger version (102K):

[in a new window]
  		Fig. 8. Ectopic Slp1 represses reporter gene expression driven by the eve 1+5 and the eve 3+7 enhancers, but not the eve 2 enhancer. lacZ mRNA expression patterns (blue or black) driven by the eve 1+5 enhancer (A,B), the eve 3+7 enhancer (C-F) or a 7.8 kb 5' regulatory fragment that contains the eve 2 and 3+7 enhancers (G,H) are shown in wild-type embryos (left column) or embryos that ventrally misexpress Slp1 (right column). Stripe numbers are indicated on each panel, and repression events are marked with asterisks. (E,F) Ventral views of embryos double stained to detect lacZ (black) and sna mRNA (red), which is expressed in ventral-most regions.

 


View larger version (35K):

[in a new window]
  		Fig. 9. (A) Three different mechanisms control repression of eve 2 in anterior regions of the embryo. The activity of Gt and at least one other factor (X) is required for repression very near the anterior border. Anterior to this domain, Slp1 and at least one other factor (Y) mediate eve 2 repression. At the anterior pole, Tor activity may downregulate the activity of Bcd, the primary activator of eve 2. (B) A model for Bcd coordination of eve 2 regulation. Bcd activates transcription of the eve 2 enhancer even at quite low concentrations. Bcd also is required for activation of the repressors of eve 2 (Gt and Slp1). We propose that these genes are positioned based on their decreased sensitivity to the Bcd gradient. At the anterior tip of the embryo, the activity of Bcd may be disrupted by the Tor phosphorylation cascade, which prevents activation of several Bcd target genes in this region.

 

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 2002