Generation of dGIPC null alleles.

<p><b>A</b>. Schematic of the dGIPC (or kermit) locus adapted from the GBrowser from FlyBase. The blue triangles represent the mapped insertion sites of the P elements EP2431, EP493, and GS2053 used in this study. Overexpression of both EP493 and GS2053 perturbs PCP on the notum and the wing. Imprecise excision of the EP2431 recovered two dGIPC alleles, <i>dGIPC^ex2</i> and <i>dGIPC^ex31</i> represented by the interrupted lines. <i>dGIPC</i> mutants are viable with no obvious PCP defects. <i>dGIPC^ex31</i> homozygous females are sterile and lay round eggs, but this is likely due to a secondary mutation, as <i>kermit^ex2</i> homozygous females, or <i>kermit^ex31</i>/Df(2R)1735 females show only a reduced fertility with normally shaped eggs. <b>B</b>. qPCR on ovarian extracts from different dGIPC mutant combinations. While there is no detectable dGIPC mRNA in <i>dGIPC</i> mutants, the mRNA levels of the neighboring gene CG8709 are normal. <b>C</b>. Western-blot analysis on 3^rd instar larval brain and discs extracts. There are no detectable dGIPC protein in homozygous <i>dGIPC^ex2</i> and <i>dGIPC^ex31</i> (first two lanes). Heterozygous dGIPCex2/Cyo-GFP (3^rd lane), and overexpressed dGIPC under enGal4 control (4^th lane) are shown as control. Armadillo (Arm) is used as loading control. <b>D</b>. <i>dGIPC^ex31</i> homozygous clones in 3^rd instar larval eye disc marked by the loss of LacZ (blue in D and D″) have no detectable dGIPC protein (green in D and D′). There is no defect to cortical actin (red in D and D′″) in <i>dGIPC</i> loss-of-function clones.</p

An overexpression screen for PCP defects identifies dGIPC.

<p><b>A</b>. Schematic of the overexpression screen. Males from EP collections were mated with females expressing the Gal4 transcription factor in the apterous domain (dorsal notum and wing). <b>B–C</b>. Overexpression of the EP line GS2053 gives PCP defects in the notum (C) compared to wild type (B). <b>D–F</b>. Overexpression of dGIPC under the control of engrailed-Gal4 (F) causes PCP defects similar to that of GS2053 (E) compared to control (D). <b>G</b>. Overexpression of GS2053 under enGal4 control leads to the accumulation of dGIPC protein (green in G and G′) and of cortical actin (red in G and G″).</p

dGIPC is enriched in midline glial cells.

<p>In all panels, E-Cadherin is shown in magenta (A–F) and dGIPC in green (A–F) or white (A′–F′). <b>A</b>. dGIPC is enriched in discrete structures in 3^rd instar larval ganglions (yellow arrowhead). <b>B</b>. In ovarian epithelial follicular cells, dGIPC is expressed at low levels and in discrete puncta (yellow arrowhead). <b>C–D</b>. dGIPC is enriched in the midline glia in stage 16 embryos (yellow arrowheads). C: ventral view, D: lateral view. <b>E–F</b>. dGIPC is enriched in the midline glia in 3^rd instar larval brain (yellow arrowhead). E: wild-type larva, F: <i>dGIPC^ex31/Df(2R)ED1725</i> null mutant larva.</p

Genetic modification of the en-Gal4, UAS-dGIPC induced multiple wing hair/PCP phenotype.

<p>*all stocks from Bloomington unless indicated.</p><p>Note that the enhancement seen with <i>fz^R52</i>, <i>mwh¹</i> is due to the <i>mwh¹</i> allele as this enhances by itself and other fz alleles do not interact. Similarly, pk, argos and Egfr alleles only show modification with one allele suggesting second site effects.</p><p>The confirmed strong interactions are highlighted in bold and all belong to the “actin cytoskeleton” remodelers group.</p

Effects of Fz1/2 C-Tail Mutations on Subcellular Localization and PCP Activity

<p>(A) Sequence alignment of Fz1 and Fz2 C-tails. Note high degree of conservation within the membrane proximal shared portion of the Fz1 and Fz2 C-tails. The respective mutations generated and analyzed are indicated above the sequence (see also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020158#pbio-0020158-t001" target="_blank">Table 1</a> for complete data set). As in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020158#pbio-0020158-g002" target="_blank">Figures 2</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020158#pbio-0020158-g003" target="_blank">3</a>, <i>dpp-Gal4</i> was used to drive expression of the respective mutants, and these were detected by anti-Myc staining in third instar wing discs. Examples for Fz1–1-1V559E (V to E substitution) are shown in (B) (localization) and (F) (function). All other mutants analyzed as shown in (A) are listed in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020158#pbio-0020158-t001" target="_blank">Table 1</a>. (C–E, G, and H) show the effects of the Fz2 C-tail-specific sequences. The Fz2 C-tail was truncated at the position of the Fz1 stop codon (amino acid L633), yielding a short Fz2 C-tail (2S). The localization (C and D) and GOF PCP function (G and H) of the respective chimeras, Fz1–2-2S and Fz1–1-2S, is shown. Note that both chimeras localize apically (C and D), and Fz1–1-2S shows a strong PCP GOF phenotype (H), very similar to Fz1–1-1 (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020158#pbio-0020158-g003" target="_blank">Figure 3</a>B). Fz1–2-2S shows only a very weak PCP phenotype (G), mainly occurring at an anterior distal region of the wing (marked by arrow; the rest of the wing is wild-type). (E) Subcellular localization of Fz1–1-1C2. Fz1–1-1C2 is Fz1 with the addition of the Fz2-specific tail extension (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020158#s4" target="_blank">Materials and Methods</a>). Note ubiquitous protein localization within the apical–basal axis (E) and a much reduced PCP activity, as compared to wild-type Fz1–1-1, in the functional assay (I). The phenotype is much weaker than in wild-type Fz1 (compare with [F] and [H] and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020158#pbio-0020158-g003" target="_blank">Figure 3</a>B).</p

Overexpression of Apically Localizing Fz1/2 Chimeras Has an Inhibitory Effect on Canonical Wnt Signaling

<div><p>(A–D) show adult wings of the respective genotypes. Anterior is up and distal to the right.</p> <p>(A) Adult wing of an <i>en-Gal4/+; UAS-fz1–1-1/+</i> fly (<i>en>fz1–1-1</i>). <i>en-Gal4</i> drives UAS reporter genes only in the posterior compartment. Inset shows high magnification of region marked by arrowhead. Some wing margin bristles are missing (arrow) in the posterior compartment. The border between anterior (“a”) and posterior (“p”) compartments is marked with black line.</p> <p>(B) <i>dsh^V26/+; en>fz1–1-1</i> adult wing. Note enhancement of the margin bristle phenotype: all margin bristles are missing from the area between the arrows in the posterior compartment.</p> <p>(C) <i>en>fz2–1-1</i> wing. Most of the wing margin bristles are missing in the posterior compartment. Note also that the posterior compartment is smaller.</p> <p>(D) <i>en>fz2–2-1</i> wing. Again the posterior compartment is smaller and most of the margin is missing.</p> <p>(E–G) show that Fz1–1-1 expression increases apical localization of Dsh-GFP and reduces Dsh-GFP in more basolateral areas of wing cells. (E) and (F) are xy-horizontal optical sections, and (G) is an xz-cross section. The positions of (E) and (F) sections are indicated in (G).</p> <p>(E) Apical xy-optical section of a third instar wing disc. Fz1–1-1 (red) is overexpressed by <i>en-Gal4</i> in the posterior compartment (anterior–posterior border is labeled by white line, and the corresponding compartments are labeled “a” and “p,” respectively). Dsh-GFP (green) accumulates at higher levels apically in the posterior compartment. Single-channel Dsh-GFP staining is shown at right. In wild-type disc, Dsh-GFP is evenly distributed with no anterior–posterior bias (not shown).</p> <p>(F) A more basal xy-section of the same disc as in (E). Note reduction of Dsh-GFP staining in the posterior compartment, except at the apical junctions as seen in folds (arrowhead). In the anterior compartment, where Fz1–1-1 is not overexpressed, Dsh-GFP is only slightly enriched in the apical folds (arrow).</p> <p>(G) xz-section of the same wing disc shown in (E) and (F), with top panel showing double labeling for anti-Myc (red) and anti-Dsh-GFP (green) and bottom panel showing single channel of Dsh-GFP staining.</p> <p>(H) xz-section of a comparable disc expressing Fz2–1-1 in the posterior compartment<i>.</i> Fz2–1-1 overexpression (red) also causes accumulation of Dsh-GFP in apical junctions and reduction of Dsh-GFP along the basolateral region.</p></div

Rescue of the <i>fz⁻</i> Eye Phenotype with <i>tub</i>-Promoter-Driven Fz Chimeras

<div><p>Tangential eye sections with corresponding schematic in lower part of panel reflecting ommatidial polarity (respective genotypes are also marked below each panel). Black arrows, dorsal chiral form; red arrows, ventral chiral form; green arrows, symmetric ommatidia; black circles, ommatidia with missing photoreceptors. Anterior is to the left, dorsal is up, and an area around the equator is shown for each genotype.</p> <p>(A) Section of a wild-type eye (equator is indicated by yellow line).</p> <p>(B) <i>fz^P21/fz^R52</i> (<i>fz</i> null). Note random orientation of ommatidia.</p> <p>(C) <i>fz^P21/fz^R52</i>; <i>tub-fz1–1-1</i>. The <i>fz⁻</i> phenotype is fully rescued (100% with respect to chirality; only a minor rotation wobble is rarely seen).</p> <p>(D) <i>fz^P21/fz^R52</i>; <i>tub-fz1–1-2</i>. Note partial rescue with respect to polarity (approximately 83%) and occasional photoreceptor loss representative of Wg/β-cat signaling.</p> <p>(E) <i>fz^P21/fz^R52</i>; <i>tub-fz1–1-2S</i>. Note 100% rescue, identical to wild-type Fz1 (compare with [C]).</p> <p>(F) <i>fz^P21/fz^R52</i>; <i>tub-fz1–2-1</i>. No rescue due to the presence of the Fz2 7-TM region. This chimera actually shows a mild dominant negative behavior as apparent by the increased percentage of symmetric clusters (approximately 50% as compared to <i>fz⁻</i> [approximately 15%]).</p></div

GOF Planar Polarity Wing Phenotype of Fz1/2 Chimeras

<div><p><i>dpp-Gal4</i> was used to express the respective Fz1/2 chimeras in the wing (same as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020158#pbio-0020158-g002" target="_blank">Figure 2</a>).</p> <p>(A) Wild-type wing. The <i>dpp-Gal4</i> expression domain is highlighted by a thick orange line. In wild-type, all wing hairs are pointing distally.</p> <p>(B) <i>dpp-Gal4; UAS-EGFP/UAS-fz1–1-1</i> wing (<i>dpp>fz1–1-1</i>; the expression domain is again highlighted with light orange). Wing hairs flanking the expression domain point away from it, consistent with previous observations that hair point away from higher levels of Fz1 activity (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020158#pbio-0020158-Adler2" target="_blank">Adler et al. 1997</a>).</p> <p>(C) <i>dpp>fz1–2-2</i> wing. Wing hairs are not pointing away from expression domain, suggesting that Fz1–2-2 is not active for PCP signaling.</p> <p>(D) <i>dpp>fz1–1-2</i> wing. Hairs point away only very slightly (less than 45 ^o; compare with Fz1–1-1, showing a 90 ^o reorientation next to expression domain). Several different lines of <i>UAS-fz1–1-1</i> and <i>UAS-fz1–1-2</i> were compared, showing identical behavior (Fz1–1-1 having a much stronger phenotype), suggesting that the C-tail is required for full PCP Fz activity.</p> <p>(E) <i>dpp>fz2–1-1</i> wing. Most wing hairs point away from expression domain. The phenotype is weaker than Fz1–1-1.</p> <p>(F) <i>dpp>fz1–2-1</i> wing. Wing hair orientation is hardly affected. Since Fz1–2-1 is apically localized (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020158#pbio-0020158-g002" target="_blank">Figure 2</a>E), this result indicates that the presence of the Fz1 7-TM region is important for PCP activity.</p></div

The Cytoplasmic Region of Fz Regulates Subcellular Localization

<div><p>All Fz1/2 chimeras shown are Myc-tagged (the tag being inserted right after the CRD of Fz1 or Fz2; see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020158#s4" target="_blank">Materials and Methods</a>; <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020158#pbio-0020158-Boutros2" target="_blank">Boutros et al. 2000</a>). The respective Fz1/2 chimeras, with their schematic structure shown under each photomicrograph, were expressed under <i>dpp-Gal4</i> (expression domain marked with <i>UAS-EGFP</i> in example in [A]) and analyzed by confocal microscopy xz-sections (perpendicular to the stripe of expression in the wing pouch region).</p> <p>(A) Subcellular localization of wild-type Fz-Myc (Fz1–1-1, in green; red channel shows coexpressed GFP to mark expressing cells). Single-channel black-and-white staining of Fz-Myc is shown on right.</p> <p>(B–F) Anti-Myc staining of different Fz1/2 chimeras: (B) Fz1–2-2, (C) Fz1–1-2, (D) Fz2–1-1, (E) Fz1–2-1, and (F) Fz2–2-1.</p> <p>(G) Fz2–2-2. Note the correlation of apical Fz localization with the presence of the Fz1 C-tail.</p></div

Subcellular Localization of the Fz1 Protein

<div><p>(A) Anti-GFP staining of Fz-GFP in <i>arm-fz-GFP</i> third instar wing disc (<i>arm</i> drives ubiquitous expression); xz-section is shown.</p> <p>(B) Illustration of a cross section of a third instar wing disc. Wing epithelium forms several folds in the hinge region, where apical–basal localization can be visualized in a horizontal xy-section. The purple line in (B) indicates the position of the xy-optical section in such folds shown in (C–F).</p> <p>(C) Staining of a <i>dpp-Gal4/UAS-fz1–1-1(myc)</i> third instar wing disc. Localization of DE-Cad (in red), Dlg (green), and Fz1–1-1 (anti-Myc, blue) is shown. Apical region of the epithelium faces the lumen in the fold, and the basolateral regions are away from the lumen.</p> <p>(D) same staining as in (C) with two channels shown: DE-Cad and Fz1–1-1. DE-Cad (red) and Fz1–1-1 (blue) largely overlap.</p> <p>(E) Dlg (green) and Fz1–1-1 (blue) from (C) are shown. Fz1–1-1 localizes generally more apical than Dlg (with only a very slight overlap).</p> <p>(F) Fz1–1-1 single-channel staining. In summary, Fz1–1-1 is mainly localized in the apical adherens junctions and strong punctae inside cells (probably intracellular vesicles). Low levels of Fz1–1-1 also exist more ubiquitously in the basolateral region.</p> <p>(G) Schematic illustration of relative positions of DE-Cad, Dlg, and Fz1–1-1 along the apical–basal axis epithelial cells. DE-Cad marks the adherens junctions, whereas Dlg localization correlates with septate junctions.</p></div