The proteins encoded by the Drosophila Planar Polarity Effector genes inturned, fuzzy and fritz interact physically and can re-pattern the accumulation of "upstream" Planar Cell Polarity proteins

Abstract

The frizzled/starry night pathway regulates planar cell polarity in a wide variety of tissues in many types of animals. It was discovered and has been most intensively studied in the Drosophila wing where it controls the formation of the array of distally pointing hairs that cover the wing. The pathway does this by restricting the activation of the cytoskeleton to the distal edge of wing cells. This results in hairs initiating at the distal edge and growing in the distal direction. All of the proteins encoded by genes in the pathway accumulate asymmetrically in wing cells. The pathway is a hierarchy with the Planar Cell Polarity (PCP) genes (aka the core genes) functioning as a group upstream of the Planar Polarity Effector (PPE) genes which in turn function as a group upstream of multiple wing hairs. Upstream proteins, such as Frizzled accumulate on either the distal and/or proximal edges of wing cells. Downstream PPE proteins accumulate on the proximal edge under the instruction of the upstream proteins. A variety of types of data support this hierarchy, however, we have found that when over expressed the PPE proteins can alter both the subcellular location and level of accumulation of the upstream proteins. Thus, the epistatic relationship is context dependent. We further show that the PPE proteins interact physically and can modulate the accumulation of each other in wing cells. We also find that over expression of Frtz results in a marked delay in hair initiation suggesting that it has a separate role/activity in regulating the cytoskeleton that is not shared by other members of the group.

Keywords: Drosophila; Planar Cell Polarity; Planar cell polarity effectors; Protein interactions; Wing.

Figure 1

The in/fy gain of function wing phenotype. All images are from the C region of the wing (between the 3^rd and 4^th veins just anterior to the posterior cross vein. This region is in the ptc domain. A-D show the dorsal surface of the wing and E- L the ventral surface. The genotypes are noted in each panel. Ore-R shows the wild type hair pattern.

Figure 2

The over expression of frtz and frtz-GFP result in opposite gain of function PCP phenotypes. All images are from the C region on the dorsal surface of the wing (between the 3 and 4^th veins just anterior to the posterior cross vein. This region is in the ptc domain. The genotypes are noted in each panel.

Figure 3

The gain of function PCP phenotypes associated with over expression of frtz and frtz-GFP can affect the polarity of neighboring wing cells. A and B show a flip out clone that expresses Frtz-GFP. This results in a cell autonomous delay in hair formation. The asterisk marks the region showing hair delay. C and D show a clone in an older wing. The asterisk marks a region of wild type cells distal to the clone that form hairs that point toward the clone. E and F show a flip out clone that over expresses Frtz. Note the delay in hair formation of clone cells. The asterisk marks a region of wild type cells proximal to the clone that form hairs that point away from the clone.

Figure 4

The over expression of PPE genes can alter the accumulation of PCP proteins. A and A’ are from the same ptc-Gal4/+; UAS-frtz/act-Vang-YFP wing. A is from outside the ptc domain and A’ from inside the domain. Vang-YFP immunostaining is shown. Outside the ptc domain Vang accumulates in the typical proximal/distal zig-zag pattern. Inside the ptc domain Vang accumulation is more punctate and is preferentially found on the anterior/posterior sides of cells. B and B’ are from the same ptc-Gal4/+; UAS-frtz wing. B is from outside the ptc domain and B’ from inside the domain. Endogenous Pk accumulation is shown by immunostaining. Outside the ptc domain Pk accumulates in the typical proximal/distal zig-zag pattern. Inside the ptc domain Pk accumulation is more punctate and is preferentially found on the anterior/posterior sides of cells. C shows a ptc-Gal4/dsh-myc; UAS-frtz/+ wing that was immunostained with anti-Myc antibody to show the accumulation of Dsh-myc. E shows a ptc-Gal4 UAS-HA-in UAS-fy-GFP/+ wing immunostained to show the accumulation of the endogenous Pk protein. Note the increased Pk accumulation and altered zigzag accumulation. E shows a ptc-Gal4 UAS-HA-in UAS-fy-GFP/+ wing immunostained to show the accumulation of the endogenous Stan protein. Note the loss of the distinct proximal/distal zigzag in the ptc domain. F shows a ptc-Gal4/+; UAS-frtz-GFP/+ wing immunostained to show the accumulation of the endogenous Pk protein. Note the slightly increased accumulation of Pk and the loss of the proximal/distal zigzag in the ptc domain.

Figure 5

In accumulation is altered in wing cells by the over expression of frtz and fy. A and B show a ptc-Gal4/+; UAS-frtz-GFP/+ wing immunostained to show the endogenous In protein. The arrow marks the approximate location of the edge of the ptc-domain. Note the increased accumulation of In and its altered accumulation pattern associated with the over expression of frtz-GFP. C and D show a ptc-Gal4/+; UAS-fy-GFP/+ wing immunostained to show the endogenous In protein. The arrow marks the approximate location of the edge of the ptc-domain. Note the decreased accumulation of In associated with the over expression of fy-GFP. E and F show a ptc-Gal4/UAS-fy-GFP; UAS-frtz-GFP/+ wing immunostained to show the endogenous In protein. The arrow marks the approximate location of the edge of the ptc-domain. Note the increased accumulation of In and its altered accumulation pattern associated with the over expression of frtz-GFP. G shows a ptc-Gal4/ UAS-frtz wing immunostained to show the endogenous In protein. The arrow marks the approximate location of the edge of the ptc-domain. Note the increased accumulation of In where frtz was overexpressed. H shows the quantitation of the changes in In immunostaining inside and outside of the ptc domain. Average grey scale values in the ptc domain are normalized to the level outside of the ptc domain. A t-test was used to compare the grey values inside and outside of the ptc domain. * p = 0.05-0.01, ** p = 0.01-0.001, *** p<0.001.

Figure 6

The over expression of one PPE protein affects the accumulation of the others. A, B and C show a ptc-Gal4/UAS-HA-in; ubi-myc-frtz-GFP/+ wing immunostained for both In (red) and GFP (green-Frtz). This ubi-myc-frtz-GFP transgene gives variegated expression. D shows the quantitation of GFP (Frtz) staining inside (2) and outside (1) of the ptc domain. (Note the quantitation was done on the peripheral accumulation in cells that expressed high levels of Frtz). The increase in Frtz accumulation was significant (p<0.05). E, F and G shows a ptc-Gal4/UAS-fy-Flag-ollas; ubi-myc-frtz-GFP/+ wing immunostained for both Ollas (red-Fy) and GFP (green-Frtz). H shows the quantitation of GFP (Frtz) staining inside (2) and outside (1) of the ptc domain. The decrease in Frtz accumulation was significant (p<0.05). I, J and K show the results of Western blots of wing disc samples where the expression of UAS-HA-in and UAS-myc-frtz were driven either singly or together using ptc-Gal4. I and J show the quantitation and K the blot. M, N and L show the results of Western blots of wing disc samples where the expression of UAS-fy-Flag-Ollas and UAS-myc-frtz were driven either singly or together using ptc-Gal4. L and M show the quantitation and N the blot. O, P and Q show the results of Western blots of wing disc samples where the expression of UAS-HA-in and UAS-fy-GFP were driven either singly or together in varying doses using ptc-Gal4. O and P show the quantitation and Q the blot.

Figure 7

The over expression of fy and frtz modifies a hypomorphic in phenotype. Panel B is a control showing the in-ts phenotype at 25°C. The wings in A and C used actin-Gal4 to drive the expression of myc-frtz and fy-GFP respectively.

Figure 8

Co-immunoprecipitation of PPE proteins. A shows an experiment where HA-In and Fy-Flag were co-immunoprecipitated by either anti-In or anti-Flag antibodies. In this experiment a heat shock was used to induce the expression of transgenes subcloned behind the hsp70 promoter. B shows the co-immunoprecipitation of In and Frtz. This experiment used UAS-HA-in and UAS-myc-frtz transgenes driven by ptc-Gal4. Wing disc samples were immunoprecipitated using Rabbit anti-HA antibody. Arrows point to myc-Frtz and HA-In on the Western blots. C shows an experiment where we tested the co-immunoprecipitation of Fy and Frtz. Samples from ptc-Gal4/UAS-fy-Flag-Ollas; UAS-myc-frtz/+ wing discs were IP with anti-Myc antibody and then assayed by western blotting. Lane 3 and 4 (on the left side) are duplicates. The arrows point to Fy-Flag-Ollas (left) and myc-Frtz (right). Note that Fy was not preciptiated with Frtz. D shows an experiment where all three of the PPE proteins were co-expressed (ptc-Gal4/UAS-HA-in UAS-fy-GFP; UAS-frtz-GFP/+). Samples were made from wing discs and immunoprecipitated using anti-Fy antibody. The Western blots were probed with anti-GFP (to detect both Fy-GFP and Frtz-GFP) and anti-In. Control experiments established that anti-Fy antibody could not pull down either HA-in or Frtz-GFP (data not shown).