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


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

doi: 10.1242/10.1242/dev.00613

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 Araujo, H.
Right arrow Articles by Bier, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Araujo, H.
Right arrow Articles by Bier, E.
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?

Integrins modulate Sog activity in the Drosophila wing

Helena Araujo1,*, Erika Negreiros1 and Ethan Bier2

1 Departamento de Histologia e Embriologia, Universidade Federal do Rio de Janeiro, 21941-970, Rio de Janeiro, Brazil
2 Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, CA 92093-0349, USA



View larger version (109K):

[in a new window]
  		Fig. 1. Components of the Bmp pathway interact genetically with integrins. (A) A wild-type wing. Longitudinal L2, L3, L4 and L5 veins, and anterior (acv) and posterior (pcv) crossveins are indicated. Anterior is towards the top, and proximal towards the left. (B) As previously reported, certain allelic combinations of myospheroid, such as mys1/mysnj42, produce wings with ectopic vein material (asterisks) or vein thickening (arrow). Escapers from mysXR04/mysnj42 exhibit both ectopic veins and vein truncations (not shown). (C) The enhancer piracy-sog line sogEP7 induces small distal truncations of the L4 vein (arrow). Asterisk indicates ectopic vein material. (D) The mys1 null allele enhances the sogEP7 truncation phenotype (arrows), while the mewM6 null allele suppresses this phenotype (E). Note that the mys1 and mewM6 alleles both enhance the amount of ectopic vein material between L2/L3 (asterisk). (F) A scb2 null allele also enhances vein truncation; however, no ectopic veins are seen between L2/L3. (G) Thickened veins are produced in tkv1 mutants (arrows). This phenotype is suppressed by the ßPS allele mysnj42 (H).

 


View larger version (130K):

[in a new window]
  		Fig. 2. Wing veins are poorly defined in ßPS- mutant clones. mysXB87 clones were generated in a sog misexpression background (sogEP7) using FLP/FRT-mediated recombination and were recognized by the marker multiple wing hair (mwh). (A) Dorsal clones that cross over or lie adjacent to longitudinal veins, such as L4 and L5 induce the formation of veins with diffuse boarders (arrows indicate limits of the vein phenotype). Veins in these regions are less compact, less pigmented and wider than normal veins. (B) A higher magnification view of the wing in A showing that veins broaden within two cell diameters from the border of mysXB87 clones, but are unaffected when displaced by greater distances from the clones (e.g. compare vein phenotypes at red versus black arrows). (C) Two dorsal clones running over L3 induce thickened and diffuse vein sections. (D) A ventral clone adjacent to L5 has no effect on vein formation. Broken red lines indicate the limits of dorsal clones; broken purple lines indicate ventral clones; + indicates heterozygous or wild-type tissue; – indicates homozygous mutant clones.

 


View larger version (192K):

[in a new window]
  		Fig. 3. Wing veins deviate toward nearby mew- clones. mewM6 clones were generated in a sog misexpression background (sogEP7) using FLP/FRT-mediated recombination and were scored by the marker forked (f). (A) A dorsal mewM6 clone adjacent to the L2 vein displaces the vein towards the clone. The broken black line indicates the normal trajectory of the L2 vein. (B) A small mewM6 clone adjacent to a distal region of L2 has a similar vein deviating effect. (C) A mewM6 clone between veins L3 and L4 alters the course of the L3 vein. The distance between the tips of L2 and L3 is increased as a function of L3 being displaced posteriorly (arrow) towards the mewM6 clone. The broken black line indicates the approximate location where the L3 vein would normally form. (D) A mewM6 clone that straddles the L2 vein by several cells on each side of the vein does not significantly disrupt the course of the vein. (E) A mewM6 clone adjacent to the posterior crossvein induces formation of ectopic vein material between the normal vein and the clone. (F) Ventral mewM6 clones adjacent to veins have no effect. Broken red lines indicate the limits of dorsal clones, broken purple lines indicate ventral clones.

 


View larger version (48K):

[in a new window]
  		Fig. 4. scab is expressed in pupal intervein cells. scb expression during vein development was monitored by in situ hybridization using a scb antisense RNA probe. scb expression is absent in larval wing imaginal discs (not shown). (A) At 20 hours apf, scb expression is observed in intervein and vein cells. (B) At 25 hours apf scb expression in the interveins is maintained while vein expression becomes weaker. Metalloproteases that cleave Sog are expressed in intervein cells as revealed by RNA probes for tld (C) and tok (not shown). (D) dpp is expressed in the center of provein domains during pupal development, as shown for the L3 vein, while sog is expressed in the intervein cells (E), with peak expression often observed at the border of the provein territory. (F) High magnification view of scb expression in the L3 provein domain at 25 hours apf, showing that staining is excluded from the outermost provein area.

 


View larger version (172K):

[in a new window]
  		Fig. 5. scb- clones alter the width or course of wing veins. scb1 clones were generated using FLP/FRT mediated recombination and scored by the marker pawn. (A) Large clones generate blistered wings, a phenotype characteristic of other integrin- mutants. (B) Two scb1 clones adjacent to veins L2 and L3 result in broadening of the adjacent veins (regions between arrows indicate affected sections of veins), which comprise wild-type cells. (C) A high magnification view of two scb1 dorsal clones surrounding the L3 vein, which are associated with a thickened vein segment (four cells wide). (D) A dorsal scb1 clone located in the center of the L3 provein region divides the vein into two branches, which avoid the clone (arrows). (E) A wing containing several scb1 clones in the proximity of L3. A ventral clone running over the vein has no effect. (F) A dorsal scb1 clone generated in a sogEP11 background lying inside the provein domain splits the L2 vein into two well defined branches. Arrows indicate veins formed outside the clonal boundary (indicated by broken red lines).

 


View larger version (29K):

[in a new window]
  		Fig. 6. A truncated form of Sog binds to {alpha}PS1 integrin. Co-immunoprecipitation of Sog with various anti-integrin antibodies. Protein lysates were prepared from wild-type pupae (24 hours apf) and then incubated with protein A-Sepharose bound anti-ßPS, anti-{alpha}PS1 or anti-{alpha}PS2 antibodies. Lysates (lys), unbound supernatants (unb) and bound (bd1 and bd2) protein samples were run on 10% SDS-PAGE, immunoblotted and detected by the polyclonal 8A anti-Sog antiserum, which recognizes an epitope following CR1. (A) The Sog antibody reacts strongly with a large 120 kDa fragment in pupal lysates. A smaller 50 kDa reactive fragment is also present at very low levels. After co-immunoprecipitation with anti-{alpha}PS1, the 50 kDa band is greatly enriched and small amounts of the full-length band are detected (arrowhead). Sog protein does not coimmunoprecipitate with the anti-ßPS antibody, but does co-immunoprecipitate to a much lesser extent with {alpha}PS2. No binding was observed for short or full-length Sog with the protein ASepharose beads alone. (B) The structure of Sog protein indicating the transmembrane domain (TM), four cysteine repeats (CR1-CR4) and putative Tolloid cleavage sites (arrows). The blue bar indicates the predicted fragment corresponding to the 50 kDa Sog band that co-immunoprecipitates with anti-{alpha}PS1. The red bar indicates the location of the epitope recognized by the 8A anti-Sog antibody.

 


View larger version (70K):

[in a new window]
  		Fig. 7. Sog protein diffuses into the provein territory. Staining with the 8B anti-Sog antiserum reveals that Sog protein is initially expressed in a patchy intervein pattern at 18-22 hours apf (A). At this stage, there are more labeled intervein cells on the dorsal than ventral surface. (B) At 22-26 hours apf, Sog staining is still patchy but expands evenly to all intervein cells towards the end of this period. Staining is also visible in provein domains on the dorsal surface of the wing. (C) At 26-30 hours apf, Sog protein is present throughout the entire provein domain on the dorsal surface of veins, with increased staining at the provein/intervein border. (D) Later (>30 hours apf), anti-Sog staining again becomes restricted to intervein cells. Hemocytes running through the center of the vein also label. Staining fades away in subsequent stages. (E) High magnification of the L3 vein of a 24-26 hours apf pupal wing shows that Sog protein is present in provein cells on the dorsal surface of the wing except for the most central cells. This pattern is not synchronous for all veins. (F) On the corresponding ventral surface of the same region no provein (bar) staining is observed. (G) High magnification of the L3 vein of a wing as in C, showing Sog protein localized over the entire vein competent domain of all veins on the dorsal surface, but excluded from the provein regions of veins on the ventral (H) surface. Arrows indicate increased staining at the provein/intervein border. Note that the texture of reticular staining in intervein regions (asterisk) on the dorsal surface is different from the more punctate staining on the ventral surface of the wing. (I-N) High magnification confocal images of a 22-26 hours apf wild-type wing double labeled for Sog (red) and ßPS integrin (green). This optical section, which is focused on the basolateral region of the dorsal wing epithelium, reveals that Sog (I,K) and integrin receptors (J,M) are co-localized, staining the cell perimeter. Sog staining is also observed inside intervein cells and entering the provein area (arrows, I,K), where integrins are absent. No Sog staining inside the provein area is observed on the ventral wing epithelium (L-N), as shown by the double arrows (L,N).

 


View larger version (131K):

[in a new window]
  		Fig. 8. Integrins regulate Sog protein distribution on the dorsal wing surface. The distribution of Sog and integrin proteins was examined in wings containing integrin- clones. In all panels, integrin staining is green and Sog staining is red. mys- clones were generated to analyze the distribution of Sog protein in the absence of ß-integrin (A-F). Clones generated on the dorsal surface induce a patchy pattern of Sog distribution (A-C). In addition (C), adjacent to a dorsal mys- clone Sog protein does not enter the provein area (double arrow), while on the opposite side of the same vein, Sog enters the vein competent domain (arrow). Integrin staining defines the limit of the intervein territory (B,C). A ventral mys- clone does not alter the distribution Sog (DF), while inside a small dorsal and ventral clone, Sog staining is reduced and patchy in appearance. (G-L) mew- clones result in similar effects on Sog protein distribution. (G-I) A dorsal mew- clone running between L4 and L5 veins induces patchy Sog distribution. The arrow in I indicates that Sog enters the provein area on the opposite side of the vein. A high magnification confocal optical section localized at the basolateral region of the dorsal wing epithelium reveals that Sog (J,L) and {alpha}PS1 (K,L) co-localize at the intervein area where the integrin is expressed, while a Sog is distributed in a patchy fashion at the same cell level inside a mew- clone.

 


View larger version (25K):

[in a new window]
  		Fig. 9. A model for Sog/integrin interactions in the wing. The primary proposed function of {alpha}PS1ßPS integrin in modulating Sog activity in the wing. In wild-type wings, {alpha}ß integrin heterodimers enhance the delivery or diffusion of an active Bmp inhibitory form of Sog into the provein domain. This non-autonomous source of Sog limits peak Bmp signaling to the center of the provein territory. In the absence of {alpha}PS1ßPS, a repulsive form of Sog protein is unable to enter the provein territory, while a remaining unaffected Bmp promoting Sog activity (not shown) attracts the vein towards the mutant clone.

 

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