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 Hannus, M.
Right arrow Articles by Eaton, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hannus, M.
Right arrow Articles by Eaton, 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?

Planar cell polarization requires Widerborst, a B' regulatory subunit of protein phosphatase 2A

Michael Hannus*, Fabian Feiguin{dagger}, Carl-Philipp Heisenberg and Suzanne Eaton{ddagger}

Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse-108, 01307 Dresden, Germany
* Present address: Cenix Biosciences, Pfotenhauerstrasse-108, 01307 Dresden, Germany
{dagger} Present address: Institute for Molecular Pathology, Vienna, Austria



View larger version (65K):

[in a new window]
  		Fig. 1. Widerborst is a B' subunit of PP2A required for planar polarization. (A) Wild-type wing epithelial cells each make a single hair. (B) Driving expression from EP3559 on the dorsal wing blade with Apterous GAL4 causes multiple wing hairs to form. (C) Expression of a cDNA (LD3434) corresponding to CG5643 (the locus downstream of EP3559) produces a similar phenotype. (D) Dendritic tree depicting the relationships between different PP2A B' subunits from fungi (Sp T11663, Sp Ydd2, Sc Rts.1, Nc CAC28812.1), C. elegans (Ce C13G3.3a, Ce RTS.1), Drosophila (Dm CG7901, Dm Wdb), Xenopus (Xl B' {epsilon}), zebrafish (zWdb1, zWdb2) and Homo sapiens (Hs B' {alpha}, ß, {gamma}, {delta} and {epsilon}). The bar in the upper right -hand corner depicts the branch length corresponding to 0.05 amino acid substitutions per residue. (E) Location of EMS mutations within the Wdb transcript, and structure of the Wdb dominant-negative construct. Grey regions are non-coding. The red region is strongly conserved between all members of the B' subfamily. Green indicates sequences conserved only between Widerborst and the {alpha} and {epsilon} subfamily members. Blue indicates divergent sequence unique to Widerborst. Arrows indicate the positions at which, wdbdw, wdb12, wdb14, and wdbIP are mutated and the resulting sequence changes. wdbdw and wdb12 probably cause short N-terminal truncations (see Materials and Methods) because they make protein that is detectable with an antibody against the C terminus. (F) The ventral wing blade of a wdbdw/wdbIP transheterozygote shows regions where hair formation fails or is compromised. Stunted hairs are often misoriented (arrows). (G) The ventral wing blade of a fly that expressed dominant-negative Wdb along the anteroposterior compartment boundary, under the control of PatchedGAL4. The expressing region is indicated in red. Expressing cells form hairs that are stunted and misoriented. Occasionally, hairs fail to form. Posterior to the expression domain, hair formation and orientation are affected non-autonomously (asterisk and arrows). (H) The ventral wing blade of a fly that expressed dominant negative Wdb along the anteroposterior compartment boundary (red). Even hairs of normal morphology display orientation defects.

 


View larger version (124K):

[in a new window]
  		Fig. 2. Widerborst activates the catalytic subunit of PP2A, which promotes hair formation. (A) The multiple wing hair phenotype caused by Widerborst overexpression is suppressed by removing one copy of mts. Each bar represents the average number of wing hair duplications in the region between veins 4 and 5, distal to the posterior crossvein. At least 12 wings were averaged. (B) Wing from a fly that expressed the wild-type catalytic subunit of PP2A along the AP compartment boundary under the control of PatchedGAL4. Each overexpressing cell makes prodigious numbers of hairs that point in all directions. (C) Wing from a fly that expressed a dominant negative catalytic subunit along the AP compartment boundary. Wing hairs are stunted or fail to form. (D) Dominant-negative catalytic subunit expression wing showing a milder phenotype than in C; hairs display stunting and polarity defects.

 


View larger version (148K):

[in a new window]
  		Fig. 3. Localization of Widerborst. (A-C) Widerborst (red), Flamingo (green) localization at 26 hours after puparium formation. Widerborst (B,C) is present on cell-internal spots and forms a distoproximal concentration gradient, unlike Flamingo (A,B), which is present on the proximal and distal cortex. (D-F) Nine optical sections separated by 0.25 µm were deconvolved and projected to give these images. Widerborst (D, green) is concentrated distally in the region of planar microtubules (red, F). (G-I) A single section from the projection shown in D-F. Widerborst (green) colocalizes exactly with microtubules (red). Coracle (blue) indicates cell boundaries. In all panels, distal is towards the right.

 


View larger version (107K):

[in a new window]
  		Fig. 4. Widerborst polarizes by 18 hours after puparium formation. (A-C) Widerborst (A; green in B), microtubules (C; red in B) and coracle (blue) in wings from pupae aged for 5 hours at 29°C. Widerborst is localized to the proximal side of the cell. The arrow in B points distally, and anterior is upwards. (D-F) Widerborst (D; red in E) is polarized distally by 18 hours after puparium formation (pupae aged at 25°C). Flamingo (F; green in E) is only slightly polarized at this stage, compared with 24 hours (see Fig. 3A). Coracle (blue) outlines cell boundaries. The arrow in E points distally.

 


View larger version (119K):

[in a new window]
  		Fig. 5. Widerborst polarizes independently of Dsh and Fmi and is needed for polarization of cortical domains. Arrows point distally. (A) Widerborst (green) and Coracle (red) in a dsh1 pupal wing (aged 27 hours after puparium formation). Wdb is normally polarized. (B) Widerborst (green) and coracle (red) in a stan3 pupal wing (aged 27 hours after puparium formation). Wdb is normally polarized. (C) Frizzled, Flamingo, and Wdb staining in a pupal wing expressing Frizzled under the control of PatchedGAL4. Both Frizzled and Flamingo proteins were detected with mouse monoclonal antibodies and the red channel is a composite of their two patterns. In adjacent cells, Flamingo is repolarized perpendicular to the normal proximal-distal axis, but Wdb is normally polarized. (D) Flamingo and Frizzled, from the same image shown in C. (E) Wdb, from the same image shown in C. Fz-overexpressing cells are shaded red. (F) Wdb and Coracle, from the same image shown in C. (G-I) Wdb (G; red in H) and Fmi (I; green in H) in wings expressing dominant negative Wdb under the control of PatchedGAL4. Expression of dnWdb disrupts the polarization of wild-type Wdb up to five cells away from the overexpression domain. Fmi fails to polarize in dnWdb-expressing cells (H,I). Fmi also fails to polarize in cells near the dnWdb-expressing domain (asterisks in I). (J-L) Wdb (J; red in K) and Dsh (L; green in K) in wings expressing dominant negative Wdb under the control of PatchedGAL4. Expression of dnWdb disrupts the polarization of wild type Wdb in adjacent cells. Dsh fails to polarize in dnWdb-expressing cells (K,L). Dsh also fails to polarize in cells adjacent to the dnWdb-expressing domain (asterisks in L).

 


View larger version (124K):

[in a new window]
  		Fig. 6. Widerborst is required to organize planar microtubule webs. (A-C) dnWdb was expressed along the AP compartment boundary under the control of Ptc:GAL4. Pupal wings were fixed with paraformaldehyde and stained with phalloidin, to detect filamentous actin (A; red in B), and with anti-Wdb to detect dnWdb (C; blue in B). Cortical actin accumulates normally in dnWdb-expressing cells. The stripe of smaller cells with brighter actin staining correspond to the fourth wing vein. DnWdb-expressing cells should be compared with the larger intervien cells. Note that paraformaldehyde fixation does not preserve the polarized localization of Wdb. (D-F) dnWdb was expressed along the AP compartment boundary under the control of Ptc:GAL4. Pupal wings were fixed with methanol and stained with anti-Coracle (F; blue in E), anti-tubulin (D; green in E) and anti Wdb (red in E). The cortical organization of Coracle is normal, but microtubule structure is disturbed in dnWdb-expressing cells.

 


View larger version (70K):

[in a new window]
  		Fig. 7. Injection of morpholino antisense oligonucleotides (MO) against zebrafish wdb 1 and wdb2 interferes with dorsoventral patterning and convergent extension movements during gastrulation. (A,B) The notochord, outlined by expression of ntl, is shortened and broadened in MO (1 ng)-injected (B) versus mock-injected (A) embryos. (C,D) The shape of the prechordal plate, marked by the expression of hgg is elongated in MO (1 ng)-injected (D) versus mock-injected (C) embryos. (E,F) the expression of gsc within the shield is expanded ventrally in embryos injected with 4 ng of wdb1 and wdb2 MOs (F) when compared with mock-injected embryos (E) at shield stage, indicating that these embryos are dorsalized.

 

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