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Development 140 (4): 831-842

Signal transduction by the Fat cytoplasmic domain

Guohui Pan, Yongqiang Feng*, Abhijit A. Ambegaonkar, Gongping Sun, Matthew Huff, Cordelia Rauskolb, and Kenneth D. Irvine{ddagger}

Howard Hughes Medical Institute, Waksman Institute and Department of Molecular Biology and Biochemistry, Rutgers The State University of New Jersey, Piscataway, NJ 08854, USA.


Figure 1
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Fig. 1. Rescue of fat PCP phenotypes by FAT4. (A-D) Adult wings from fat8/fatG-rv flies expressing (A) tub-Gal4 UAS-wts and (B) Fat+, (C) Fat:Fat4 or (D) Fat{Delta}EGF. (E-G) Proximal anterior wings from fat8/fatG-rv flies expressing (E) tub-Gal4 UAS-wts and (F) Fat+or (G) Fat:Fat4. (H-J) Abdominal segment from fat8/fatG-rv flies expressing (H) tub-Gal4 UAS-wts and (I) Fat+ or (J) Fat:Fat4. (K-M) The distribution of PCP phenotypes (M) in abdomen (K) and proximal wing (L) for animals of the indicated genotypes. (N) Average distance between cross-veins in animals of the indicated genotypes, normalized to the value in wild-type-rescued animals. Error bars show s.e.m. Abbreviations for Fat transgenes are described in supplementary material Table S3.

 

Figure 2
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Fig. 2. Wing phenotypes associated with Fat intracellular domain motif mutations. (A) The Fat intracellular domain (ICD) showing the locations of mutations examined here (red and green bars), regions that bind Fat-associated proteins (blue bars), and functional regions identified by Matakatsu and Blair (Matakatsu and Blair, 2012Go) (purple). (B-U) Adult wings from fat8/fatG-rv flies expressing (B) Fat+, (C) Fat{Delta}A, (D) Fat{Delta}B, (E) Fat{Delta}C, (F) Fat{Delta}D, (G) Fat{Delta}E, (H) Fat{Delta}F, (I) Fat{Delta}D/Fat{Delta}F, (J) Fat{Delta}D/Fat:Fat4, (K) Fat{Delta}F/Fat:Fat4, (L) Fat+/Fat{Delta}D, (M) Fat+/Fat{Delta}F, (N) Fat{Delta}F/Fat{Delta}F, (O) Fat{Delta}D/Fat{Delta}D, (P) Fat+/Fat:Fat4, (Q) FatP32, (R) FatmIV, (S) FatmI, (T) FatmV, (U) Fat{Delta}D/FatmV. (V) Average wing size in animals of the indicated genotypes, normalized to the value in wild-type-rescued animals. (W) Average distance between cross-veins in animals of the indicated genotypes, normalized to the value in wild-type-rescued animals. Additional statistics on wing measurements are in supplementary material Table S1. Error bars show s.e.m.

 

Figure 3
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Fig. 3. PCP phenotypes associated with Fat ICD motif mutations. (A-C) Proximal anterior wings from fat8/fatG-rv flies expressing (A) Fat+, (B) Fat{Delta}D or (C) Fat{Delta}F. The boxed regions (costa) are magnified in the insets. (D-F) Abdomens from fat8/fatG-rv flies expressing (D) Fat{Delta}E, (E) Fat{Delta}D or (F) Fat{Delta}F. (G-I) Proximal anterior wings, visualized by cuticle refraction microscopy (Doyle et al., 2008Go), from fat8/fatG-rv flies expressing (G) Fat+, (H) Fat{Delta}D or (I) Fat{Delta}F. Yellow lines indicate the estimated angle of ridges relative to the L4 vein. (J-L) The distribution of PCP phenotypes (see Fig. 1M) in (J) proximal wing, (K) costa and (L) abdomen for animals of the indicated genotypes.

 

Figure 4
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Fig. 4. Influence of fat mutations on Fat protein localization and mobility. (A-L) Confocal micrographs of Fat protein staining (green) in wing discs from fat8/fatG-rv flies expressing (A,D) P[acman]V5:fat[68A4], (B,E) P[acman]V5:fat{Delta}EGF[68A4], (C,F) P[acman]V5:fat:FAT4[68A4], (G,J) P[acman]V5:fat{Delta}D[68A4], (H,K) P[acman]V5:fat{Delta}F[68A4] or (I,L) P[acman]V5:fat-mV[68A4]. Upper panels show horizontal sections, lower panels show vertical sections, with E-cadherin staining (red). (M,N) Western blots on wing disc lysates from animals of the indicated genotypes. Upper panel shows Fat antibody staining, lower panel shows a loading control (GAPDH).

 

Figure 5
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Fig. 5. Mapping Dco phosphorylation sites in Fat. (A) Deletion constructs used for mapping phosphorylation sites within the Fat ICD; truncations are indicated by dotted boxes, the open rectangle indicates the transmembrane domain and the black box indicates the epitope tags; P indicates the approximate position of the phosphorylation sites that influence Fat ICD mobility. To the right are shown portions of western blots of lysates of S2 cells expressing these constructs and Dco or Dco3, with detectable mobility shift indicated by +. (B) Western blot on S2 cells expressing Fat-STI-4:FVH and point mutant derivatives, together with Dco or Dco3, as indicated. The amino acids mutated are indicated in supplementary material Fig. S5; D indicates a Ser to Asp mutation; in other cases Ser to Ala mutations were employed. (C) Western blots showing results of co-immunoprecipitation experiments from lysates of S2 cells expressing Dco:V5 and the indicated FLAG-tagged Fat isoforms.

 

Figure 6
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Fig. 6. Influence of targeted fat mutations on Dachs localization. (A-E') Examples of wing discs from animals of the indicated genotypes, with clones of cells expressing Dachs:Cit (green) from an AyDachs:Cit transgene. These discs are stained for expression of Wg (red; marks dorsal-ventral boundary) and E-cad (blue; outlines cells). The polarity of Dachs localization is indicated by the arrows pointing in the direction of Dachs:Cit membrane localization; white arrows indicate normal polarity, yellow arrows abnormal polarity; asterisks indicate lack of polarity. (A'-E') The Dachs:Cit channel only. (F) The distribution of Dachs polarization phenotypes in animals of the indicated genotypes. Distributions were scored blind and over 100 Dachs:Cit clones were scored per genotype. (G-K) Rose plots depicting the vectors of Dachs:Cit polarization identified within polarized clones in discs of animals of the indicated genotypes. Polarities were scored blind; the number scored is shown top left. The diagrams are oriented with proximal at the top, anterior right and distal at bottom (defined by Wg expression). Normal distal polarization is in gray. (L) Anisotropy of Fat staining along proximal-distal versus anterior-posterior interfaces. ImageJ was used to calculate average staining intensities along all cell interfaces within the central region of the wing pouch, from five to six different discs, that could be defined as predominantly proximal-distal or anterior-posterior based on comparison with Wg staining. Error bars indicate s.e.m.

 

Figure 7
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Fig. 7. Influence of membrane-tethered Dachs on wing growth and PCP. (A-F) Adult wings from animals expressing nub-Gal4 and (B) UAS-Zyx, (C) UAS-dachs, (D) UAS-Zyx UAS-dachs, (E) UAS-myr:dachs or (F) UAS-Zyx:dachs. (G-J) Localization of membrane-tethered Dachs constructs, showing clones of cells expressing (G,H) Myr:Dachs:V5 (green) or (I,J) Zyx:Dachs:V5 (green) in (G,I) horizontal or (H,J) vertical section. (K) Western blot on lysates of wing discs expressing tub-Gal4 and UAS-dachs, UAS-Zyx, UAS-Zyx:dachs, or UAS-RNAi-fat, or mutant for fat, as indicated. Loss of Fat activity reduces Wts protein levels (Cho et al., 2006Go). GAPDH serves as a loading control. Mean Wts levels from four independent experiments, normalized to Wts levels in fat mutants, were: UAS-dachs 3.3, UAS-Zyx 3.6, UAS-Zyx:dachs 3.3, UAS-RNAi-fat 1.8, wild type 5.0, and fat mutant 1.0. (L-Q') Wing discs from animals expressing (L) en-Gal4 (marked by UAS-GFP, green) and (M) UAS-Zyx, (N) UAS-dachs, (O) UAS-Zyx UAS-dachs, (P) UAS-myr:dachs or (Q) UAS-Zyx:dachs, stained for ex-lacZ (red). (L'-Q') ex-lacZ channel only. (R-T) Anterior proximal wing from animals expressing (R) nub-Gal4 and (S) UAS-Zyx UAS-dachs and (T) UAS-Zyx:dachs.

 


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