N ATU RE C ELL BI O LO G Y ADVANCE ONLINE PUBLICATION 1 LETTERS Diego and Prickle regulate Frizzled planar cell polarity signalling by competing for Dishevelled binding Andreas Jenny 1 , Jessica Reynolds-Kenneally 1 , Gishnu Das 1 , Micheal Burnett 1 and Marek Mlodzik 1,2 Epithelial planar cell polarity (PCP) is evident in the cellular organization of many tissues in vertebrates and invertebrates 1–5 . In mammals, PCP signalling governs convergent extension during gastrulation and the organization of a wide variety of structures, including the orientation of body hair and sensory hair cells of the inner ear 6,7 . In Drosophila melanogaster, PCP is manifest in adult tissues, including ommatidial arrangement in the compound eye and hair orientation in wing cells 1,2,4 . PCP establishment requires the conserved Frizzled/Dishevelled PCP pathway 1–5 . Mutations in PCP-pathway-associated genes cause aberrant orientation of body hair or inner-ear sensory cells in mice 6,7 , or misorientation of ommatidia and wing hair in D. melanogaster 1,2,4 . Here we provide mechanistic insight into Frizzled/Dishevelled signalling regulation. We show that the ankyrin-repeat protein Diego binds directly to Dishevelled and promotes Frizzled signalling. Dishevelled can also be bound by the Frizzled PCP antagonist Prickle 8 . Strikingly, Diego and Prickle compete with one another for Dishevelled binding, thereby modulating Frizzled/Dishevelled activity and ensuring tight control over Frizzled PCP signalling. In the D. melanogaster eye, PCP is generated when immature omma- tidial preclusters rotate 90 ° towards the dorso-ventral midline (the equator), adopting opposite chiral forms on either side of it. This results in a mirror-image ommatidial arrangement across the equa- tor (Fig. 1a, b) 2,4 . The Frizzled (Fz) PCP signal — transduced via Dishevelled (Dsh) and potentially a heterotrimeric G protein 9 , Rho- family GTPases and a Jun N-terminal kinase (JNK) cascade 1,2,10 — specifies the cell fates of the photoreceptors R3 and R4. It thus defines the chiral form of each ommatidium and the associated direction of rotation (Fig. 1a, b) 2,4 . Graded Fz activity, higher at the equator than at the poles, results in an initial small difference in Fz activity between R3 and R4. The R3 precursor is thought to receive Fz PCP signalling first or at higher levels, because it is closer to the equator 2,4 . This Fz activity difference between the equatorial R3 and polar R4 precursors is then amplified in a process that involves Delta (Dl) transcription and Notch (N) signalling 1,2,4 . 1 Mount Sinai School of Medicine, Brookdale Department of Molecular, Cellular and Developmental Biology, 1 Gustave L. Levy Place, New York, NY 10029, USA. 2 Correspondence should be addressed to M.M. (e-mail: marek.mlodzik@mssm.edu) Published online: 5 June 2005; DOI: 10.1038/ncb1271 In PCP mutant backgrounds, ommatidia form properly but choose R3/R4 cell fates — and thus chirality — stochastically and rotate to a random extent; a few also remain symmetrical 1,2,4 . Several PCP genes, including prickle (pk), strabismus (stbm), flamingo (fmi) and diego (dgo) are required for the regulation of Fz PCP signalling activity. Whereas fz and dsh are required in R3 (ref. 11), stbm and pk act in R4, where they are required to antagonize Fz 8,12,13 . Dgo is a cytoplasmic protein with six amino-terminal ankyrin repeats that colocalizes with Fz 14,15 . Before the onset of PCP signalling, all PCP proteins initially colocalize apically at the cell membrane. This initial api- cal recruitment depends on Fz and Fmi 14,16 , with Pk also depending on a direct interaction with Stbm for membrane recruitment 12,17 . Furthermore, the Dgo ankyrin repeats can interact with Pk and Stbm and probably contribute to an initial phase of apical localization 14 . Genetic studies show a redundancy at this stage: only double mutants of either dgo, pk or stbm have an effect on Fmi or Fz apical localization. At these early stages, a potential higher-order complex of PCP proteins might function to main- tain Fmi apically 14 . Moreover, overexpression of PCP proteins such as Fz or Pk also leads to increased apical levels of Stbm or Fmi 8,17 . Later, during PCP signalling and asymmetry generation, Fz, Dsh and Dgo become enriched on the R3 side of the R3/R4 cell border, whereas Stbm becomes enriched at the R4 side 10,12 . Likewise in the wing, Fz, Dsh and Dgo sort to the distal end of wing cells, whereas Pk and Stbm, the Fz/Dsh antagonists, become enriched proximally in each cell 12,16–18 . The role of Dgo in Fz PCP signalling, however, remains unclear. To dissect the function of dgo in PCP establishment, we first used mosaic analysis to determine in which photoreceptor it is required (Fig. 1c–e). Our results indicate that dgo is necessary in R3 because 98.4% of mosaic clusters with a wild-type R3 developed correctly. In contrast, ommatidia with a dgo − R3 precursor chose their chirality stochastically or remained symmetrical. We also analysed whether Dgo has the potential to instruct a cell to adopt a specific fate. For example, the cell of the R3/R4 pair with higher Fz levels always adopts the R3 fate 1,2,4 . We thus tested the effect of overexpressed Dgo (sevenless (sev)-Dgo) by analysing ommatidia with mosaic sev-Dgo R3/R4 pairs in an otherwise wild-type background. All R3/R4 mosaic sev-Dgo clusters that showed either a chirality inversion or adopted a symmetrical R3/R3 arrangement carried sev-Dgo in the R4 © 2005 Nature Publishing Group