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First published online 30 August 2006
doi: 10.1242/dev.02549

Development 133, 3805-3815 (2006)
Published by The Company of Biologists 2006
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Perlecan and Dystroglycan act at the basal side of the Drosophila follicular epithelium to maintain epithelial organization

Martina Schneider1,{dagger},*, Ashraf A. Khalil1,2,{ddagger}, John Poulton3,{ddagger}, Casimiro Castillejo-Lopez1, Diane Egger-Adam4, Andreas Wodarz5, Wu-Min Deng3 and Stefan Baumgartner1

1 Department for Experimental Medical Science, Section for Developmental Biology, Lund University, 22184 Lund, Sweden.
2 Department of Protein Research, Genetic Engineering and Biotechnology Research Institute, Mubarak City for Scientific Research, Alexandria, Egypt.
3 Department of Biological Science, Florida State University, Tallahassee, FL 32306-4370, USA.
4 Fakultät für Biologie, Universität Konstanz, 78434 Konstanz, Germany.
5 Department of Stem Cell Biology, DFG Research Center for Molecular Physiology of the Brain (CMPB), University of Göttingen, Justus-von-Liebig-Weg 11, 37077 Göttingen, Germany.


Figure 1
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  		Fig. 1. Perlecan is required for integrity of the follicle cell epithelium. (A) Schematic drawing of an ovariole and FCE. (B,B') Pcan accumulates in the BM after the FCE is formed and is expressed in the muscular sheath (asterisk). (C) Pcan stripes run perpendicular to the long axis of the egg chamber. Yellow lines indicate cell borders. The arrow indicates the orientation of the stripes. (D,D') In a trol clone (broken line) expression of Pcan is absent. Red, Pcan; green, GFP. (E) trol clones frequently loose their epithelial organization and become multilayered (arrows). Occasionally mutant cells can be found in aberrantly positioned between the germ cells (arrowhead). Green, GFP; red in E, DNA. Clones are marked by the absence of GFP.

 

Figure 2
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  		Fig. 2. Perlecan is required for cell membrane polarity. trol clones show defects in the distribution of various cell polarity markers (filled arrowheads indicate normal; open arrowheads disrupted protein localization). (A,B) Baz (red) is generally enriched and expanded into the cytoplasm. (C) Patj (red) is not affected by the loss of Pcan. (D) Apical localization of Crb (red) is frequently reduced. (E,F) Arm (red) expression is slightly elevated and expanded to all cell membrane. (G) Dlg (red) is strongly reduced. A'-G' show red channels only. Clones are marked by the loss of GFP and outlined by broken lines. Nuclei of follicle cells in ectopic positions are marked with a circle. Green, GFP; blue, DNA.

 

Figure 3
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  		Fig. 3. Dystroglycan and Perlecan can bind in vitro. Western blots showing co-immunoprecipitation of PcanV with anti-Dgpep. Embryonic glycoprotein extract was mixed with 5 µg PcanV (R1), 0.5 µg PcanV (R2) or 0.05 µg PcanV (R3), and precipitated with anti-Dgpep. Controls were: no glycoprotein extract added to reaction mixture (C1), precipitation reaction with boiled Dg antibody (C2), only primary antibody loaded (C3), primary antibody exchanged for rabbit serum (C4). The precipitates were probed with anti-PcanV (upper panel) or anti-Dgcyto (lower panel). Pure PcanV (PcanV 0.05 µg) and glycoprotein extract (input) were loaded as a positive control. Some of the minor Dg bands in the glycoprotein extract (input) are probably due to protein degradation.

 

Figure 4
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  		Fig. 4. Perlecan is required for basal localization of Dystroglycan. (A,B) Dg (red) is no longer present in the basal cell membrane of trol clones. Occasionally, Dg appears to be redistributed to the apical cell membrane (yellow arrows in B'). (C,D) Pcan localization in the ECM is unchanged in a Dg323 clone (C) and orientation of Pcan-stripes appears normal (D). (E) ßPS Int (red) is normally expressed in trol clones. Blue indicates DNA. (F) In a FCE entirely composed of mutant cells, gaps in ßPS staining can be seen. Green indicates GFP and Dg; arrow indicates gap. (G) ßPS Int (red) is still present (arrows) in the basal membrane of a trol clone that has lost Dg (green). (G') Green channel only, showing both GFP and anti-Dgcyto staining. (G'') Red channel only. (H) Expression of Lam in a wild-type ovariole. Asterisk indicates muscular sheath. (I) A very large trol clone showing fuzzy Lam (red) distribution. Blue indicates DNA. (J) Lam (red) is still present at the basal side of a medium sized lanA clone (arrow). (K) Two egg chambers whose FCE are entirely composed of lanA mutant cells. Lam (red) is almost completely absent. (L) Three egg chambers whose FCE are entirely composed of lanA mutant cells. Dg (red) is still localized at the basal membrane. Clones are marked by the loss of GFP (green) and indicated by broken lines. (A'-L') Red channels only.

 

Figure 5
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  		Fig. 5. Perlecan-dependent Dystroglycan lacks the mucin domain. (A) Schematic drawing of the Dg forms A, B and C, which are generated through differential splicing of exon 8 (red box) and exon 9 (black box). The transmembrane-domain is indicated with a yellow line. (B) Graphic showing the potential of mucin type O-linked glycosylation for each position of the Dg-C sequence. A stretch of 80 amino acids (positions 424-503, indicated by the blue bar) contains a cluster of 52 high potential o-glycosylation sites, which constitute the mucin-like domain. The red bar indicates the region encoded by exon 8 (position 243-507). (C) Western blots of embryonic protein extract (0-20 hours) probed with anti-Dgcyto (cyto), anti-Dgpep (pep) and anti-Dgex8 (ex8). (D-F) Wild-type ovaries stained with anti-Dgcyto (D,F) and anti-Dgex8 (E,G). F and G are higher magnification of D and E, respectively. Dg is strongly concentrated in the basal membrane of the FCE throughout oogenesis (D, arrow in F). Dg-C is expressed in the muscular sheath (yellow asterisks in E and G) but absent from the basal membrane of the FCE (arrow in G). Red, Arm; green, Dg. (H-J) Ectopic expression of Dg-B (H,J) and Dg-C (I) induces ectopic accumulation (arrows) of Lam (H) and Pcan (I,J). Cells expressing the Dg construct are marked by GFP (green). Red indicates Lam (H) and Pcan (I,J). (H'-J') Red channels only.

 

Figure 6
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  		Fig. 6. Dystroglycan and Dsytrophin are mutually dependent for basal membrane localization. (A) In a wild-type ovariole, Dys is expressed in the muscular sheath (star) and at the basal membrane of the follicle cells. (B) In a Dg323 follicle cell clone, Dys (red) is lost from the basal membrane. (C) Expression of dsDys (Dys-hairpin) efficiently reduces Dys expression (red). (D) Reduction of Dys leads to a reduction of Dg (red) in the basal membrane. (E) In a trol clone, Dys is no longer concentrated at the basal membrane but diffusely localized in the cytoplasm. All follicle cell clones are marked with broken lines. Filled arrowheads indicate the wild type; open arrows indicate the mutant expression pattern. (B'-E') Red channels only. Blue indicates DNA.

 

Figure 7
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  		Fig. 7. Distribution of NrxIV, Con and Nrg in the FCE. All pictures show antibody staining of wild-type ovaries. (A,B) NrxIV (red) gradually concentrates at the basal side of the lateral membrane of follicle cells during development. (C-E) Cont (red) can bee seen in a punctate staining during early and mid-oogenesis (D). In older egg chambers (E), Cont concentrates apicolaterally. (F) Nrg (red) is expressed in the lateral cell membrane. (G) Surface views of the FCE showing NrxIV (green, G'), Cont (red, G'') and Nrg (blue, G'''). All three proteins co-localize at tricell junctions. Green indicates actin in A-D,F. (B',D',E') Red channel only.

 

Figure 8
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  		Fig. 8. Dg is required to exclude NrxIV and Cont from the basal cell membrane. (A,B) NrxIV (red) is ectopically expressed in the basal membrane in Dg323 clones. (C,D) A similar phenotype is observed in trol clones. (E,F) In a Dg clone, Cont (red) accumulated together with NrxIV (green) at the basal membrane, whereas a change in Nrg (blue) distribution was not observed. Clones are marked by loss of GFP and indicated by broken lines. (B,D,F) Surface views of the FCE. (A'-F') Red channels; (E'',F'') green channels; (E''',F''') blue channels.

 




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