Coordination of planar cell polarity pathways through Spiny-legs

Abstract

Morphogenesis and physiology of tissues and organs requires planar cell polarity (PCP) systems that orient and coordinate cells and their behaviors, but the relationship between PCP systems has been controversial. We have characterized how the Frizzled and Dachsous-Fat PCP systems are connected through the Spiny-legs isoform of the Prickle-Spiny-legs locus. Two different components of the Dachsous-Fat system, Dachsous and Dachs, can each independently interact with Spiny-legs and direct its localization in vivo. Through characterization of the contributions of Prickle, Spiny-legs, Dachsous, Fat, and Dachs to PCP in the Drosophila wing, eye, and abdomen, we define where Dachs-Spiny-legs and Dachsous-Spiny-legs interactions contribute to PCP, and provide a new understanding of the orientation of polarity and the basis of PCP phenotypes. Our results support the direct linkage of PCP systems through Sple in specific locales, while emphasizing that cells can be subject to and must ultimately resolve distinct, competing PCP signals.

Keywords: D. melanogaster; cell biology; developmental biology; fat; pcp; polarity; prickle; stem cells.

Figure 1.. Localization of Pk and Sple in wing discs, and their interaction with Dachs and Ds.

(A) Schematic diagram illustrating the general direction of PCP protein polarity (arrows), expression gradient of Ds (magenta) and organization of Ds-Fat and Fz PCP pathway components in the Drosophila wing disc. (B) Western blots, using antibodies indicated on the right, showing the results of co-immunoprecipitation experiments between V5-tagged Dachs (lanes 1–3,7), Ds-ICD (lanes 4–6,8) or GFP (lanes 9–11) of Flag-tagged Sple (lanes 1,4,9), Sple-N (lanes 2,5,10), Pk (lanes 3,6,11) or GFP (lanes 7,8). Upper panels (Input) show blots on lysates of S2 cells, lower panels (IP-V5) show blots on proteins precipitated from these lysates by anti-V5 beads. Similar results were obtained in three independent biological replicates of this experiment. (C–G) Portions of wing imaginal discs with clones of cells expressing GFP:Sple (C,E,F) or GFP:Pk (D,G) (green), stained for expression of E-cadherin (blue), and showing either anti-Wg (C–E) or hh-Gal4 UAS-mCD8-RFP (F,G) (red). White arrows indicate direction of polarization of Sple or Pk. DOI: http://dx.doi.org/10.7554/eLife.09946.003

Figure 1—figure supplement 1.. Proteins used in co-immunoprecipitation assays.

Schematics of tagged isoforms of full length Dachs, Ds intracellular domain, full length Sple, Sple N-terminal domain and full length Pk. DOI: http://dx.doi.org/10.7554/eLife.09946.004

Figure 1—figure supplement 2.. Ds and Fj gradients in wing discs.

(A,B) Expression of Ds (magenta) and fj-lacZ (cyan) in a wild-type wing disc. (C) Portion of a wing disc with clones of cells expressing Dachs:Cit (green), stained for Wg (red) and E-cadherin (blue). White arrow indicates the direction of Dachs polarization. DOI: http://dx.doi.org/10.7554/eLife.09946.005

Figure 2.. Localization of Sple in Ds-Fat pathway mutants.

(A–H) Portions of wing imaginal discs with clones of cells expressing GFP:Sple (green) in d^GC13/ d²¹⁰ (A and B), ft⁸/ ft^G-rv (C), ds^36D/ ds^UA071 (D), d^GC13 ds^36D/ d^GC13 ds^UA071 (E and F) and d^GC13 ft⁸/ d^GC13 ft^G-rv (G and H) mutants. Discs were stained for E-cad (blue) and Wg (red). Wg is expressed along the D-V boundary and in proximal rings, the locations of Wg expression shown are indicated. White arrows indicate general direction of Sple polarization. (I) Rose plots summarizing orientation of Sple or Pk in the indicated genotypes, with proximal at left, distal at right, and in the central row, anterior at top. Orientations were scored separately in the three regions depicted in the cartoon, the number of cells scored is indicated by N. DOI: http://dx.doi.org/10.7554/eLife.09946.006

Figure 3.. Additional characterization of Pk and Sple localization in mutants.

Portions of wing discs with clones of cells expressing GFP:Sple (A,F,G) or GFP:Pk (B–E) (green) in d^GC13/ d²¹⁰ (A–C), ft⁸/ ft^G-rv (D), ds^36D/ ds^UA071 (E), d^GC13 pk³⁰ (F), and vang^stbm6(G) mutants. Discs were stained for E-cad (blue) and Wg (red) (B,D,E,F and G) or hh-Gal4 UAS-mCD8-RFP (red) (A,C). The white arrows indicate direction of polarization. DOI: http://dx.doi.org/10.7554/eLife.09946.007

Figure 4.. Contribution of Dachs and Sple to PCP mutant wing phenotypes.

(A) Cartoons depicting inferred protein localization and hair orientation (brown) in wing cells of the indicated genotypes to explain rescue of pk by dachs, and rescue of fat by sple. Faint Sple and Ds indicate lower levels. (B) Schematic adult wing to show approximate location of panels shown in close-up, as indicate by letters. (C–N) Close-ups of portions of wings (as indicated in panel B) to show hair and bristle orientation in the indicated genotypes. Arrows indicate general direction of polarity. (C–F) Show wing margin bristles, (G–N) show wing hairs, in wild type (C,G), pk³⁰ (D,H), d^GC13/ d²¹⁰ (E,I), d^GC13 pk³⁰ (F,J), UAS-RNAi-fat/+; C765-Gal4/UAS-dcr2 (K,M) and sple¹ UAS-RNAi-fat/ sple¹; C765-Gal4/UAS-dcr2 (L,N). Suppression of pk polarity phenotypes by dachs was 100% penetrant. For suppression of fat phenotypes by sple, near the proximal anterior wing margin (region K,L) in 10/10 fat RNAi wings scored hairs point predominantly towards the wing margin, whereas in 7/8 fat sple wings scored hairs point predominantly distally, and in 1/8 wings scored a substantial fraction of hairs (∼1/4) point towards the wing margin. Near the anterior cross-vein (region M-N), in 8/9 fat RNAi wings scored hairs point predominantly proximally, and in 1/9 wings scored hairs point predominantly towards the L3 vein, whereas in 5/8 fat sple wings scored hairs point distally, in 2/8 they point towards the L3 vein, and in 1/8 they point proximally. DOI: http://dx.doi.org/10.7554/eLife.09946.008

Figure 4—figure supplement 1.. Hair polarity in wing is not affected by loss of sple or dachs.

(A) Schematic adult wing to show approximate location of panels shown in close-up, as indicate by letters. (B) Pupal wing with clones of cells expressing GFP:Sple (green) and F-actin in hairs stained by phalloidin (red). (C–J) Close-ups of portions of wings (as indicated in panel A) to show hair orientation in the indicated genotypes. Arrows indicate general direction of polarity. Hair polarity in wild type (C,E), sple¹ mutant (D,F), UAS-dcr2; UAS-RNAi-fat/nub-Gal4 (G), UAS-dcr2; nub-Gal4; UAS-RNAi-dachs (H), UAS-dcr2; UAS-RNAi-fat/nub-Gal4; UAS-RNAi-dachs (I), and fat⁸/fat^G-rv; UAS-wts/tub-Gal4 (J). DOI: http://dx.doi.org/10.7554/eLife.09946.009

Figure 5.. Sple and Pk localization in photoreceptor cells.

Localization of GFP:Sple (A–F) or GFP:Pk (G,H) in cells with expression in R4 or R3 photoreceptor cells in wild type (A,G), d^GC13/ d²¹⁰ (B), ds^36D/ ds^UA071 (C), d^GC13 ds^36D/ d^GC13 ds^UA071 (D), ft⁸/ ft^G-rv (E), d^GC13 ft⁸/ d^GC13 ft^G-rv (F), and sple¹ (H) mutants. Rose plots summarize localization based on the indicated number (N) of examples, with equatorial to the right, polar to the left, and anterior (towards the morphogenetic furrow) at top. DOI: http://dx.doi.org/10.7554/eLife.09946.010

Figure 5—figure supplement 1.. Polarity and gradients in eye discs.

(A) Cartoon illustrating the arrangement and orientation of photoreceptor cells in eye discs at the 5-cell pre-cluster stage. (B) Schematic illustrating the subcellular localization of Ds-Fat and Fz PCP pathway components at the R4 and R3 interface. (C,D) Expression of Ds (magenta) and fj-lacZ (cyan) in a wild-type eye disc. DOI: http://dx.doi.org/10.7554/eLife.09946.011

Figure 5—figure supplement 2.. Sple and Pk polarity in front of the morphogenetic furrow, and influence of GFP:Pk on PCP.

(A,B) Portions of eye imaginal discs in front of the morphogenetic furrow with clones of cells expressing GFP:Sple (A) or GFP:Pk (B) (green), stained for expression of E-cadherin (blue). The white arrows indicates general direction of Sple or Pk polarization. (C,D) Portions of eye imaginal discs stained for Elav (marks photoreceptor cells) and Prospero (Pros, overlap with Elav marks R7) and expressing GFP:Pk (green). (C) control disc with no GFP:Pk clones. (D) Disc with 1-day-old GFP:Pk clones. (E) Disc with 3-day-old GFP:Pk clones, yellow arrowheads highlight ommatidia with abnormal polarity. DOI: http://dx.doi.org/10.7554/eLife.09946.012

Figure 6.. Localization of Dachs, Sple and Pk in abdominal pleura.

(A–G) Pleura of wildtype (A–C), ft⁸/ ft^G-rv (D,E), d^GC13 ft⁸/ d^GC13 ft^G-rv (F) and sple¹/sple¹ (G) pupae with clones of cells expressing GFP:Dachs (A,D), GFP:Sple (B,E,F) and GFP:Pk (C,G) (green). Posterior compartments are marked by hh-Gal4 UAS-mCD8-RFP (red). Anterior-posterior body axis is indicated at top. (H) Rose plots depicting polarization of GFP:Dachs, GFP:Sple or GFP:Pk in pleural cells of the indicated genotypes; anterior polarization is to the left and posterior polarization is to the right. For wild type and dachs mutants cells were scored separately in A and P compartments. For fat, ds, fat dachs, and ds dachs the anterior compartment was further subdivided into a front region (A1, anterior-most 8 cells), and the remainder of the A compartment (A*). For sple, the A compartment was subdivided into a front region of 5 cells (Af), a back region of 10 cells (Ab), and a middle region comprising the rest of the compartment (Am); P compartment localization is summarized in Figure 6—figure supplement 1. Scoring of sub-regions of the A compartment in wild-type is shown in Figure 6—figure supplement 1. Apparent variations in cell size are mostly due to the flexibility of the pleura, which is easily stretched or compressed. DOI: http://dx.doi.org/10.7554/eLife.09946.013

Figure 6—figure supplement 1.. Gradients influencing PCP in the abdomen.

(A) Schematic illustrating the orientation of hairs and approximate gradients of Ds, Fj and Hh expression in the abdomen (Casal et al., 2002; Struhl et al., 1997). (B) Rose plots depicting polarization of GFP:Sple or GFP:Pk in pleural cells of the indicated genotypes. Additional wild-type analysis is for comparison to mutants shown in Figure 6; anterior polarization is to the left and posterior polarization is to the right. For GFP:Sple the anterior compartment was subdivided into a front region (A1, anterior-most 8 cells), and the remainder of the A compartment (A*). For GFP:Pk the A compartment was subdivided into a front region of 5 cells (Af), a back region of 10 cells (Ab), and a middle region comprising the rest of the compartment (Am). (C) Pleura of d^GC13 sple¹/ d^GC13 sple¹ pupa with clones of cells expressing GFP:Pk (green). Posterior compartments are marked by hh-Gal4 UAS-mCD8-RFP (red). Anterior-posterior body axis is indicated at top. DOI: http://dx.doi.org/10.7554/eLife.09946.014

Figure 7.. Influence of Pk and Sple on hair polarity in the abdomen.

(A–D) Hair polarity in pleura revealed by F-actin (phalloidin staining) in wild type (A), and in pk-sple¹⁴ (B), pk³⁰ (C) and sple¹ (D) mutants. Yellow asterisk indicates the position of the spiracle, which forms near the center of the anterior compartment. Yellow arrows indicate the region where hair orientation is normal, and red arrows indicate the region where hair orientation is disrupted. Dashed yellow lines mark approximate boundaries between regions with normal and abnormal polarity. (E–H) Hair polarity in tergites of wild-type (E), pk-sple¹⁴ (F), pk³⁰ (G) and sple¹ (H) mutant animals. Black arrows indicate regions where hair orientation is normal, and blue arrows indicate the region where hair orientation is abnormal. Dashed blue line mark approximate boundaries between regions with normal and abnormal polarity. DOI: http://dx.doi.org/10.7554/eLife.09946.015

Figure 8.. Localization of Dachs, Sple and Pk in abdominal pleura of additional genotypes.

(A–J) Pleura of ds^36D/ds^UA071 (A,B,F), dachs^GC13/dachs²¹⁰ (C,G), ds^36D dachs^GC13/ds^UA071 dachs^GC13 (D,H), ft⁸/ft^G-rv (E), pk³⁰ (I) and ft⁸ dachs^GC13/ft^G-rv d^GC13(D,J) mutant pupae with clones of cells expressing of GFP:Dachs (A), GFP:Sple (B,C,D,I) and GFP:Pk (E-H,J) (green). Posterior compartments are marked by hh-Gal4 UAS-mCD8-RFP (red). (K) Rose plots depicting polarization of GFP:Dachs, GFP:Sple or GFP:Pk in pleural clones of the indicated genotypes; anterior polarization is to the left and posterior polarization is to the right. Clones were scored separately in A and P compartments DOI: http://dx.doi.org/10.7554/eLife.09946.016

Figure 9.. Influence of Ds-Fat PCP on hair polarity in abdominal pleura.

Hair polarity in pleura revealed by F-actin (phalloidin staining) in ft⁸/ft^G-rv (A), d^GC13/d²¹⁰ (B), d^GC13 ft⁸/d^GC13 ft^G-rv (C), ft⁸ sple¹/ft^G-rv sple¹ (D), ds^36D/ds^UA071 (E), d^GC13 ds^36D/d^GC13 ds^UA071 (F), ft⁸ pk³⁰/ft^G-rv pk³⁰ (G), ft⁸ pk-sple¹⁴/ft^G-rv pk-sple¹⁴ (H), d^GC13 pk³⁰ (I) and d^GC13 sple¹ (J) mutant animals. Yellow asterisk indicates the position of the spiracle. Yellow arrows indicate the region where hair orientation is normal, and red arrows indicate the region where hair orientation is disrupted. Dashed yellow lines mark approximate boundaries between regions with normal and abnormal polarity. DOI: http://dx.doi.org/10.7554/eLife.09946.017

Figure 10.. Influence of Ds-Fat PCP on hair polarity in abdominal tergites.

Hair polarity in tergites of ft⁸/ft^G-rv (A), dachs^GC13/dachs²¹⁰ (B), ft⁸ dachs^GC13/ft^G-rv dachs^GC13(C), ft⁸ sple¹/ft^G-rv sple¹ (D), ds^36D/ds^UA071 (E), dachs^GC13 sple¹/dachs^GC13 sple¹ (F), ds^36D dachs^GC13/ds^UA071 dachs^GC13(G), and dachs^GC13 pk³⁰/dachs^GC13 pk³⁰ (H) mutant animals. Black arrows indicate the region where hair orientation is normal, and blue arrows indicate regions where hair orientation is disrupted. Dashed blue line mark approximate boundaries between regions with normal and abnormal polarity. DOI: http://dx.doi.org/10.7554/eLife.09946.018