Article

Components of Intraflagellar Transport Complex A Function Independently of the to Regulate Canonical Wnt Signaling in Drosophila

Graphical Abstract Authors Sophie Balmer, Aurore Dussert, Giovanna M. Collu, Elvira Benitez, Carlo Iomini, Marek Mlodzik

Correspondence [email protected]

In Brief Intraflagellar transport (IFT) form multi-subunit complexes to ensure bidirectional transport within the cilium. Balmer et al. identify through a reverse genetic screen that a subset of IFT complex A proteins promotes canonical Wnt signaling independent of their ciliary function by modulation of b-catenin/Armadillo activity.

Highlights d Ciliary proteins can modulate the canonical Wnt pathway independently of the cilium d IFT-A protein knockdown leads to loss of canonical Wnt- specific target expression d IFT-A proteins modulate Wnt signaling through the stabilization of b-cat/Arm

Balmer et al., 2015, Developmental Cell 34, 705–718 September 28, 2015 ª2015 Elsevier Inc. http://dx.doi.org/10.1016/j.devcel.2015.07.016 Developmental Cell Article

Components of Intraflagellar Transport Complex A Function Independently of the Cilium to Regulate Canonical Wnt Signaling in Drosophila

Sophie Balmer,1,4 Aurore Dussert,1,3 Giovanna M. Collu,1 Elvira Benitez,1 Carlo Iomini,1,2 and Marek Mlodzik1,2,* 1Department of Developmental & Regenerative Biology and Graduate School of Biomedical Sciences 2Department of Ophthalmology Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA 3Present address: CNRS, UMR 6290, Institut de Ge´ ne´ tique et De´ veloppement de Rennes, Universite´ de Rennes 1, 35043 Rennes, France 4Present address: Sloan-Kettering Institute, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.devcel.2015.07.016

SUMMARY bule-based protrusions arising from a modified centriole: the (Pazour and Witman, 2003). Cilium biogenesis and The development of multicellular organisms requires function require a complex network of cilia-associated proteins the precisely coordinated regulation of an evolu- that serve, for example, to anchor the basal body to the cell tionarily conserved group of signaling pathways. membrane or to selectively allow protein entry and exit, thus Temporal and spatial control of these signaling cas- creating a distinct subcellular compartment (Gherman et al., cades is achieved through networks of regulatory 2006; Ishikawa and Marshall, 2011; Nachury et al., 2010; Pazour proteins, segregation of pathway components in and Witman, 2003; Pedersen et al., 2008; Reiter et al., 2012; Taschner et al., 2012). The ability to function as a separate specific subcellular compartments, or both. In verte- compartment appears key to the primary cilium acting as an brates, dysregulation of primary cilia function has environmental sensor and signaling hub (Berbari et al., 2009). been strongly linked to developmental signaling Cilia are critical for Sonic Hedgehog (Shh)/Hh signal transduc- defects, yet it remains unclear whether cilia tion, and potential roles in other developmental pathways are sequester pathway components to regulate their emerging (Berbari et al., 2009; Goetz and Anderson, 2010). The activation or cilia-associated proteins directly modu- Hh ligand binds to its receptor Patched (Ptc) to relieve Ptc inhibi- late developmental signaling events. To elucidate tion of Smoothened (Smo), which then activates the downstream this question, we conducted an RNAi-based screen transcription factor Ci (Gli in vertebrates). Ptc, Smo, and Gli all in Drosophila non-ciliated cells to test for cilium- localize to the cilium in vertebrates in a ligand-regulated manner, independent loss-of-function phenotypes of ciliary and ciliary mutants disrupt Hh signaling (Briscoe and The´ rond, proteins in developmental signaling pathways. 2013; Goetz et al., 2009). It is unclear whether cilia facilitate Hh signaling by providing a specialized subcellular compartment Our results show no effect on Hedgehog signaling. or whether (specific) ciliary components are directly required to In contrast, our screen identified several cilia- transduce the Hh signal. In contrast, the cilium has been sug- associated proteins as functioning in canonical gested to limit the response to Wnt signaling by affecting the sta- Wnt signaling. Further characterization of specific bility and localization of b-catenin/Armadillo (b-cat/Arm) (Oh and components of Intraflagellar Transport complex A Katsanis, 2013). When the Wnt/Wg ligand binds to its receptor uncovered a cilia-independent function in potenti- complex of Frizzled (Fz)/LRP5-6 (Arrow [Arr] in Drosophila) it in- ating Wnt signals by promoting b-catenin/Armadillo hibits the function of the b-cat destruction complex, allowing activity. b-cat accumulation and its nuclear translocation, as well as tran- scriptional activation via T-cell factor/lymphoid enhancer-bind- ing factor (TCF/LEF) transcription factors (Clevers and Nusse, INTRODUCTION 2012; MacDonald et al., 2009; Niehrs, 2012). Although roles for ciliary proteins in degrading b-cat and limiting its nuclear entry Signaling pathways that regulate embryonic development and have been reported (Corbit et al., 2008; Gerdes and Katsanis, adult homeostasis are highly conserved from Drosophila to hu- 2008), there are many contradicting conclusions from analyses mans. Many of these pathways were elucidated in Drosophila, of Wnt signaling in the context of ciliary mutants, ranging from in particular the Hedgehog (Hh), Wnt/Wingless (Wg), Notch (N), inactivation to overactivation of the pathway (Oh and Katsanis, and receptor tyrosine kinase (RTK) pathways, making the fly an 2013). Given the interest sparked by the primary cilium func- ideal organism to identify new pathway regulators. tioning in Hh and Wnt signaling, other signaling pathways, The primary cilium is an organelle involved in sensing the including N, RTK, and transforming growth factor b (TGF-b), extracellular environment, and it is required throughout develop- have been examined. However, a specific requirement for ment and for homeostasis in most eukaryotes. Cilia are microtu- cilia during signaling by each of these pathways remains to be

Developmental Cell 34, 705–718, September 28, 2015 ª2015 Elsevier Inc. 705 confirmed (Christensen et al., 2012; Clement et al., 2013; Ezratty significant developmental defects and a secondary screen ex- et al., 2011; Leitch et al., 2014; Ten Dijke et al., 2012). amining specific molecular target to identify the res- The discovery that mutations in ciliary proteins can lead to pective signaling pathways affected by each KD (Figure 1; Fig- several genetic disorders, termed , has led to great ure S1; Table 1; Table S1). The Gal4-upstream activating interest in this cellular compartment. It remains unclear, how- sequence (UAS) system (Brand and Perrimon, 1993) was used ever, whether the affected ciliary proteins are directly required to drive RNAi expression (KD), and wherever possible, we for signal transduction or only to maintain the cilium as a func- used at least two independent non-overlapping RNAi lines to tioning compartment. Interestingly, different roles for ciliary eliminate false-positive off-target effects. In the wing, we used proteins in mitotic spindle orientation and immune synapse recy- engrailed (en)-Gal4 in the posterior compartment and nubbin cling that are independent of their function within the cilium have (nub)-Gal4 in the entire wing pouch. We additionally used recently been described (Delaval et al., 2011; Finetti et al., 2009; apterous (ap)-Gal4 in the thorax (Figures 1A–1G, S1A, and Sedmak and Wolfrum, 2010). The main barrier to understanding S1B and Table S1). KDs of 38 genes of the 62 tested genes how cilia-associated proteins could function independently of induced reproducible and specific phenotypes in the wing, tho- the cilium stems from the crucial role that most of these proteins rax, or both, and these were categorized according to their play in biogenesis and maintenance of the cilium; thus, it is diffi- appearance (Figures 1B–1G; Table S1; Figures S1A and S1B). cult to distinguish cilia-dependent and cilia-independent effects. These effects suggested cilia-independent functions for several To overcome this problem, we have used Drosophila as a model, cilia-associated proteins in the development of the wing, thorax, because here all cell types are non-ciliated, with the exception of or both. sensory neurons and sperm (Gogendeau and Basto, 2010). Con- To assess which signaling pathways were affected by candi- sequently, any phenotypes associated with the loss of cilia- date gene KDs, we first established control RNAi lines directed associated protein function should be due to cilia-independent to receptors of the respective developmental signaling path- functions. ways: Frizzled/Frizzled2 (Fz/Fz2-Wnt pathways), Notch (N We thus performed a two-step candidate RNAi-based screen pathway), Smo (Hh pathway), EGFR-RTK pathway, and Thick- in Drosophila, in vivo, to test for roles of cilia-associated proteins veins (Tkv-Dpp/TGF-b pathway) and compared these effects in developmental signaling pathways independent of their ciliary to the candidate KD phenotypes in adults (Figures 1B–1G; Ta- function. We first assayed adult phenotypes in the wing and ble S1; Figures S1A–S1C). Based on the adult wing and thorax thorax for developmental defects. The respective hits were phenotypes from the primary screen, 38 candidates were then analyzed using molecular markers in developing wing discs selected as potential regulators of developmental signaling to determine which specific signaling pathways were altered. pathways. We tested these in a secondary screening strategy Wg signaling was predominantly affected (17 of 63 tested genes) to monitor, at the molecular level, the expression of target by knockdown (KD) of cilia-associated proteins, with small genes of the respective signaling pathway. In developing wing numbers modulating the N and epidermal growth factor recep- imaginal discs, Senseless (Sens) was used as marker for canon- tor (EGFR)-RTK pathways (4 of 63 tested genes and 2 of 63 ical Wg activity, Wg ligand expression was used to monitor N tested genes, respectively). Importantly, none of the candidates activity, Ptc expression reflected Hh signaling activity, and ar- affected Hh signaling. We selected components of the Intrafla- gos-lacZ (aos-lacZ) was used as an EGFR-signaling reporter. gellar Transport complex A (IFT-A) for further study, because KDs of Fz/Fz2, Notch, Smo, and EGFR were used as they can form a defined complex, and the majority of IFT-A com- pathway-specific controls (Table 1 and Figures 1H–1L; also Fig- ponents (four of five) appeared to have a comparable effects on ures S1D–S1I). Because N signaling controls the transcription of Wg signaling. Strikingly, the IFT-A proteins act as positive regu- Wg (which activates the canonical Wnt/Wg pathway), N and Wg lators of Wg signaling. Epistasis experiments in vivo suggest that signaling were always assayed simultaneously. Depending on they function at the level of b-cat/Arm stabilization. Together, our the target genes’ expression pattern, we used either enGal4 data argue for a role of a specific subset of IFT-A proteins in the or apGal4 to knock down the 38 candidate genes (and control Wg pathway, distinct from their role in ciliary biogenesis and genes) identified in the primary screen (Table 1 and Figures function. S1D–S1I). The secondary screen revealed that none of the candidates RESULTS had an impact on Ptc expression, implying Hh signaling was un- affected by impairing cilia-associated protein expression (see An In Vivo RNAi-Based Screen Identifies a Role for Discussion). Only a small number of candidate genes showed Ciliary Proteins in Wg/Wnt Signaling in Non-ciliated either loss- or gain-of-function effects for N or EGFR signaling Cells (four and two genes, respectively). However, a significant frac- To compile a list of candidate genes for the cilia-associated pro- tion displayed a Wg-signaling loss-of-function-like phenotype, tein screen, we started with a total of 103 genes that either were manifested by decreased Sens expression, without affecting previously characterized in cilium and centriole biogenesis and Wg expression (17 genes, Table 1). Our results thus strongly sug- function in vertebrates or had been linked to ciliopathies in hu- gest that several ciliary proteins are involved in regulating as- mans. Of these genes, 63 homologs are highly conserved in pects of Wg signal transduction, independent of their function Drosophila and expressed during the imaginal disc patterning within the cilium. stages; these homologs were thus selected (Table S1 shows Of particular note were the IFT-A proteins, because their KD the full listing). We then conducted an RNAi-based KD screen (with the exception of IFT144, see Discussion) led to highly in two steps: a primary screen examining adult phenotypes for reproducible phenotypes with loss or reduction of Sens (Figures

706 Developmental Cell 34, 705–718, September 28, 2015 ª2015 Elsevier Inc. Figure 1. Primary and Secondary Screening Outline and Examples (A) Schematic representation of the screening strategy. (B) WT control adult wing expressing enGal4. Adult wings are oriented proximal left and anterior up. (C–E) Control phenotypes induced by KD of components of the different signaling pathways using en>. (C) en-driven Fz/Fz2 RNAi phenotype displaying wing margin notches in the posterior compartment, a phenotype characteristic of canonical Wg/Wnt signaling. (D) Notch KD showing notches at the margin and vein thickening. (E) Smo RNAi (Hh pathway) displaying characteristic vein defects. (F and G) Examples of phenotypes of the primary screen. (H–L) WT and control examples of phenotypes observed in the secondary screen. Wing discs are oriented anterior left and dorsal up. (H) WT patterns of Sens (red, monochrome in H0) and Wg (blue, monochrome in H00), used as markers for activation of canonical Wg and N pathways, respectively, in flies expressing GFP under the control of en>. (I and J) fz/fz2-IR driven in the posterior compartment leads to loss of Sens but not Wg (I), whereas N-IR leads to loss of both Wg and Sens (J), compared to WT patterns (H). (K) WT pattern of Ptc staining (red, monochrome in K0), used as a marker for Hh pathway activation, in flies expressing GFP under the control of ap>. (L) Loss of Hh signaling using ap>smo-IR, inducing loss of Ptc staining in GFP-marked compartment. See also Figure S1.

2A–2F). Therefore, the components of the IFT-A protein complex specific isoforms of cytoplasmic (Pazour et al., 1999; were selected for further functional analyses. Signor et al., 1999). More specifically, a subgroup of IFT, the IFT-A protein complex, controls retrograde protein transport IFT-A Proteins Are Necessary for Wg Signal from the tip to the base of the cilium (Behal et al., 2012; Iomini Transduction et al., 2001; Iomini et al., 2009; Piperno et al., 1998). There are Cilia assembly and function is dependent on the conserved six IFT-A proteins in vertebrates, of which five are conserved in bidirectional IFT complexes. IFT complexes carry axonemal Drosophila: Oseg1/IFT122, Oseg3 or RempA/IFT140, Oseg4/ and membrane proteins in and out of the cilium through anter- IFT121, Oseg6/IFT144, and CG5780/IFT43. Adult wings of ograde transport driven by heterotrimeric kinesin II (Cole et al., silenced IFT122, IFT140, IFT121, and IFT43 proteins all dis- 1998; Kozminski et al., 1995) or retrograde transport driven by played growth defects and wing notches or missing wing

Developmental Cell 34, 705–718, September 28, 2015 ª2015 Elsevier Inc. 707 Table 1. Secondary Screen Results: Analysis of Developmental Signaling Target Genes in the Third-Instar Wing Discs Hh Pathway Canonical Wnt Pathway N Pathway EGFR Pathway Gene Name Vertebrate Ortholog RNAi Line # Ptc Sens Wg Aos-lacZ Frizzled/Frizzled2 v43075;tJF01259 loss WT Notch v27228 loss loss Smoothened tJF02363 loss EGFR tJF01368 loss CG7735 BBS3, ARL6 v104462 WT loss loss WT CG13232, BBS4 BBS4 v100571 WT loss WT WT CG1126 BBS5 v18200 WT loss WT ND CG15666 BBS9 v40013 WT loss WT WT CG15923 MKS3 v37373;MS54 WT loss WT WT CG14870 B9D1 v107330 WT loss WT WT CG18631 CC2D2A, CEP76 v100302 WT WT WT WT CG15161 IFT46 d15161R-1 ND WT WT ND CG3259 IFT54 v46163 WT WT WT WT CG5142 IFT70, FLEER v22015 WT loss WT ND CG9333, Oseg5 IFT80 v100020 WT loss WT WT CG13809, osm-1 IFT172 v107157 WT loss WT WT CG7293, Klp68D KIF3B tJF03346 WT WT WT WT CG11759, Kap3 KIFAP3 v103548 WT loss WT WT CG2069, oseg4 IFT121 v109805 WT loss WT WT CG7161, oseg1 IFT122 v103598 WT loss WT WT CG11838, rempA IFT140 v31575 WT loss WT WT CG5780 IFT43 v106366 WT loss WT WT CG15148, btv DYNCH2H1 tJF03010 WT loss WT WT CG3769 DYNC2LI1 v40469 ND WT WT ND CG6998, ctp DYNLL1 v109084 WT loss increase WT CG11356 Arl13b v33812 WT WT WT ND CG1501, unc OFD1 tJF03403 WT WT WT ND CG9398, king-tubby TULP3 v29110 ND WT WT ND CG14367 CCDC104 v100799 ND WT WT ND CG17599 CLUAP1 v14682 WT WT WT WT CG4525 TTC26 v107708 ND WT WT ND CG3265, Eb1 EB1 v24451 WT loss WT WT CG6312, rfx RFX v10416 WT loss WT WT CG33957, cp309 Pericentrin v100969 WT WT WT ND CG15524, sas-6 SAS-6 v25073 WT WT WT increase CG6129, rootletin ROOTLETIN d6129R-1 WT WT WT WT CG6560, dnd ARL3 v104311 WT loss loss increase CG17286, Spd-2 CEP192 v36623 WT WT WT ND CG7186, Sak SAK/PLK4 v105102 WT loss WT ND CG10061, sas-4 SAS-4 v106051 WT WT WT WT CG14617, cp110 CP110 v101161 WT loss loss ND CG17081, cep135 CEP135 v14194 ND WT WT ND Transgenic RNAi strains were from the following sources: v, VDRC, Vienna, Austria; t, TRIP Collection, Bloomington; d, DGRC, Kyoto, Japan; MS, our lab at Mount Sinai. ND, not determined.

margin bristles (Figures S2A–S2J; IFT144 KD did not show a thus suggested a loss or reduction of canonical Wg signaling phenotype). No KD of any of the IFT-A proteins induced de- (multiple cellular hairs and misoriented wing hair or thoracic fects in the adult thorax (Figures S2K–S2O). The phenotypes bristles observed in the Fz/Fz2 double KD are due to disrup- observed were comparable to Fz/Fz2 KDs (Figure S1C) and tion of the Wnt/Planar cell polarity pathway; Table S1 and

708 Developmental Cell 34, 705–718, September 28, 2015 ª2015 Elsevier Inc. Figure 2. Wg-Specific Markers Are Disrupted by KD of a Subset of IFT-A Components (A–A00) WT expression pattern of Sens (green, monochrome in A0) and Dll (red, monochrome in A00) in third-instar wing imaginal discs of en> flies. The en compartment (posterior) is marked in blue (A). Yellow rectangles in (A0) and (A00) indicate the areas used for quantifications of fluorescence intensity of Sens and Dll stainings; see (G) and (H) for graphs. (B–F) Sens staining is largely lost and Dll staining is reduced in en-driven RNAi-based KDs of IFT122IR (B–B00), IFT140IR (C–C00), IFT121IR (D–D00), and IFT43IR (E–E00) at 29C, whereas both remain WT in IFT144IR (F–F00)(nR 20 per genotype). Scale bar represents 25 mm. (G and H) Quantification of the fluorescence intensity ratio in the posterior compartment (PC) to the anterior compartment (AC) of Sens-stained (G) and Dll-stained (H) wing discs. The bars represent mean SD, n = 4 representative stainings per genotype, **p < 0.01 and ***p < 0.001, NS, nonsignificant; Student’s t test. (I and I0) WT pattern of Sens (marker for canonical Wg signaling, in red) and Wg (marker for N signaling, in blue, monochrome in I0) in a control wing disc expressing GFP under the control of enGal4. (J–N) Wg staining remains unaffected in all IFT-A KD (J0 –N0), whereas Sens is lost in the posterior compartment of IFT122IR (J), IFT140IR (K), IFT121IR (L), and IFT43IR (M) wing discs. See also Figure S2.

Developmental Cell 34, 705–718, September 28, 2015 ª2015 Elsevier Inc. 709 Figure S1C). For function within the cilium, it has been hypoth- fied by Sens expression (Figures S3E–S3F0). Again, Wg expres- esized that IFT121 and IFT43 are peripheral components of sion was not affected in IFT140/rempA21Ci clones (Figures 3D IFT-A, whereas IFT122, IFT140, and IFT144 form the core of and 3D0), in accordance with a role for IFT140 in canonical Wg the complex (Behal et al., 2012). Consistently, IFT122 and signaling downstream of Wg. In addition, IFT140 mutant clones IFT140 KD showed strong phenotypes, and IFT121 KD had sometimes induced apoptosis in mutant cells at the border of a generally weaker effect. Strikingly, KD of the peripheral pro- the clones (detected via cleaved-Caspase 3 staining; Figures tein IFT43 displayed a strong phenotype, and that of IFT144 S3D and S3D0), a feature that has been reported to occur at did not seem to disrupt any signaling pathway tested (Fig- sharp Wg signaling borders (Vincent et al., 2011) and thus is ure S2). To measure the extent of IFT144 KD, and to confirm consistent with a reduction in Wg signaling in IFT140/rempA21Ci that the lack of signaling phenotypes is not due to inefficient mutant cells. KD, we analyzed two GFP-tagged expression constructs: In parallel to analyzing existing mutant alleles of IFT-A compo- IFT144-GFP and, for comparison, IFT121-GFP (Avidor-Reiss nents, we generated IFT121/oseg4 mutants using the Clustered et al., 2004) (IFT121 KD served as the control, because it Regularly Interspaced Short Palindromic Repeat (CRISPR) tech- displayed a Wg-signaling defect-like phenotype). RNAi tar- nique (Figures S3G–S3J). Wings of adults that are homozygous geting of both genes reduced their protein expression to mutant for IFT121/oseg4 displayed wing margin defects with barely detectable levels, indicating that IFT144 was efficiently missing bristles (Figures S3H–S3J), reminiscent of hypomorphic silenced, similar to IFT121 (Figure S2S). Thus, these data sug- Wg signaling mutant effects and consistent with the weaker ef- gest that IFT-A has an alternative configuration, function, or fect of IFT121 in RNAi KD experiments (for example, Figures both during Wg signal transduction compared to its ciliary role. S2C and S2H as compared to Figures S2B and S2G). Consistent with the adult phenotypes, silencing of IFT122, Taken together, the analyses of mutant alleles of IFT-A com- IFT140, IFT121, and IFT43 (but again, not IFT144) markedly ponents confirmed the phenotypes observed in IFT-A KD back- reduced expression of Sens and Distalless (Dll, a lower threshold grounds, indicating that specific IFT-A components act in a cilia- target of the Wg pathway) (Figures 2A–2H), two molecularly well- independent context and are required for high levels of canonical defined Wg-signaling target genes. The reduction in expression Wg/Wnt signaling. was quantified by comparing ratios of posterior (experimental) to anterior (control) Sens and Dll expression levels; Figures 2Gand IFT-A Proteins Act Downstream of Fz2/Arr and 2H. Whereas the high-threshold-level target Sens was strongly Upstream of Armadillo reduced (Figure 2G), the expression of Dll showed a significant To begin to characterize the mechanistic involvement of IFT-A but milder reduction in the IFT-A KD compartment (Figure 2H). proteins in Wg signaling, we performed epistasis experiments These data are consistent with a role of these IFT-A proteins in in vivo. A Fz2-Arr chimeric protein, in which full-length Fz2 is Wg signal transduction. In contrast, Hh, N, and EGFR signaling fused to the cytoplasmic tail of Arr, constitutes an activated were not affected by KD of any IFT-A protein, as reflected form of the two receptors (Tolwinski et al., 2003). Expression of by wild-type (WT) expression patterns and levels of Ptc/Ci, Fz2-Arr under enGal4 (en > Fz2-myc-Arr) leads to induction of Wg/Notch-Reporter-Element (NRE)-GFP,andaos-lacZ stainings extra Sens-positive cells in wing discs and margin bristles in (Table 1 and Figures S2P–S2R). Importantly, none of the KDs of adult wings in the posterior compartment (Figures 4A and 4G, the IFT-A components affected Wg ligand expression (Figures S4A–S4A00, and S4G). Co-expression of RNAi against IFT122, 2I–2N and S2P), a target of N signaling, indicating that their func- IFT140, IFT121, and IFT43 suppressed this phenotype (for tion is linked to downstream signaling events during Wg-signal wing discs, see Figures S4B–S4E, and for adult wings, see Fig- transduction but not to Wg expression itself. ures 4A–4E, quantified in Figure 4G, and Figures S4H–S4K). Taken together, these data suggest that four out of five IFT-A IFT144 did not interact, as expected from earlier observations, proteins are specifically involved in Wg signal transduction in and served as a control (Figures 4F, S4F, and S4L). These data non-ciliated epithelial cell in Drosophila. indicate that the specific IFT-A proteins act downstream of the receptor complex. IFT-A Mutants Recapitulate RNAi-Mediated Phenotypes We next sought to establish whether IFT-A proteins act To confirm the RNAi-mediated phenotypes, we generated upstream of b-cat/Arm. A constitutively active form of Arm, loss-of-function clones for existing mutant alleles of two IFT-A ArmS10, which bears an N-terminal truncation inhibiting its components, IFT122/oseg1179 (Avidor-Reiss et al., 2004) and phosphorylation by the destruction complex, was selected (Pai IFT140/rempA21Ci (Lee et al., 2008), and analyzed these in et al., 1997). Expression of ArmS10 driven by C96Gal4 along third-instar wing discs. Consistent with the RNAi KD results, the developing wing margin induced extra margin bristles in Sens and Dll expression was reduced in IFT122 null cells (Figures adult wings (Figures 4H and S4M; other drivers could not be 3A–3B000 and S3A–S3A000). Ptc and Wg expression was again not used for ArmS10 expression, because they caused early affected in IFT122/oseg1179 clones (Figures 3A00 and S3B– lethality). None of the IFT-A component KDs had a detectable S3C00), indicating that IFT122 specifically modulates canonical effect on the ArmS10 phenotype (Figures 4H–4L and quantified Wg signaling downstream of Wg with no effect on other develop- in Figure 4N; see also Figures S4N–S4R). Nonetheless, as a con- mental pathways. Similarly, mutants in the IFT-A component trol, known downstream components can suppress the ArmS10 IFT140/rempA21Ci caused a reduction or loss of Sens expression phenotype, for example, a dominant-negative isoform of the (Figures 3C–3C000; compare Sens levels in mutant cells to neigh- transcription factor TCF (Figures 4M, 4N, and S4S). These data boring WT tissue), and such clones consistently caused partial are consistent with a role for the IFT-A proteins upstream of nu- loss of margin bristles in adult wings, because these are speci- clear b-cat/Arm transcriptional activation.

710 Developmental Cell 34, 705–718, September 28, 2015 ª2015 Elsevier Inc. Figure 3. IFT122 and IFT140 Mutant Clones Disrupt Wg Signaling (A and B) Expression of Wg-signaling targets Sens and Dll is reduced in IFT122/oseg1179 mutant cells, whereas the N pathway target Wg remains unaffected. (A) IFT122/oseg1179 cells (marked by the absence of GFP in a minute background) display reduced levels of nuclear Sens (red, monochrome in A0), whereas Wg staining is WT (blue, monochrome in A00). (B) IFT122/oseg1179 cells (marked by the absence of GFP) show reduced levels of Dll (red, monochrome in B0); this is quantified in B00 (along the white line in B). A.U., arbitrary units. (C–C00) IFT140/rempA21Ci mutant cells (marked by the absence of GFP) display reduction or loss of Sens expression (red, monochrome in C0–C000). High magnification of two areas (boxed in C) is shown as monochrome in C00 (with reduced Sens expression) and C000 (with loss of Sens expression). (D and D0) Wg expression is not affected in IFT140/ rempA21Ci mutant cells (magenta, monochrome in D0). See also Figure S3.

marked by GFP, and single-mutant axnE77 patches can be identified by high Sens and Dll levels; see Figures S5F and S5F0 for axn clones positively labeled with GFP). Together, these analyses sug- gest that IFT-A proteins function down- stream of or in parallel to the destruction complex and upstream of Arm nuclear function, suggesting a role in Arm stabili- zation in the prior to its nuclear transcriptional activity. In Drosophila imaginal discs, like all epithelial tissues, b-cat/Arm localizes both to apical adherens junctions (via interaction with E-cadherin [E-cad]) and in the cytoplasm, with the cytoplasmic b-cat/Arm pool being used in Wg signaling. Within the cytoplasm, b-cat/ IFT-A Proteins Modify Phosphorylation Levels of Arm is present in two forms: the unphosphorylated ‘‘active’’ Armadillo form, which can translocate to the nucleus to promote target Next, we addressed whether IFT-A proteins acted upstream or gene activation, and the phosphorylated (‘‘inactive’’) form, which downstream of the destruction complex. The en-driven KD of is targeted for degradation by the proteasome. To remove junc- Axin, a key component of the destruction complex, leads to tional Arm and allow specific analysis of the cytoplasmic fraction, constitutive activation of Wg signaling in almost every cell in we used concanavalin-A-coupled beads, which bind to cadher- the posterior compartment of the wing pouch (Figure 5A), ins and thus sequester molecules associated with E-cad, i.e., causing pupal lethality. Co-KD of IFT122, IFT140, IFT121, and Arm (Peifer, 1993). In WT third-instar wing discs, equal amounts IFT43, together with Axin, suppressed the overactivation of the of the active and the inactive forms of cytoplasmic Arm were Wg pathway and associated Sens expression (Figures 5B–5F present in the fraction of Arm not bound to E-cad. When the and quantified in Figure 5G; IFT144 served as the control). destruction complex was inhibited in nubGal4-driven Axin KD Viability was also restored, and adult flies were recovered (Fig- wing discs, this balance changed to 80% of active Arm (Figures ures S5A–S5D). Importantly, double-mutant clones for IFT140/ 6A and 6B), leading to activated Wg signaling and causing the in- rempA21Ci; axnE77 also suppressed ectopic activation of Sens duction of Sens expression throughout the wing pouch (Figures and Dll normally observed in axnE77 single-mutant clones (Fig- S6A and S6B; nubGal4-driven Axin KD does not completely ures 5H–5H00, S5E, S5E0, and S5G–S5G000; only the double- disrupt the destruction complex, with basal phosphorylation mutant clones lack GFP, because both FRT are of Arm still observed). In this background, we tested whether

Developmental Cell 34, 705–718, September 28, 2015 ª2015 Elsevier Inc. 711 Figure 4. In Vivo Epistasis Analyses Reveal IFT-A Proteins Function Downstream of the Fz2-Arr Receptor Complex and Upstream of b-Cat/Arm (A–G) High-magnification view of extra-margin bristle phenotype (examples indicated by white arrowheads) observed near margins of adult wings between L3 and L4 from flies expressing activated Fz2-Arr chimeric receptors (A) and Fz2-Arr combinations with IFT-A protein RNAi KD (B–F; genotypes are indicated in panels) under the control of enGal4 (image field as indicated in the overview in Figure S4G). (G) Quantification of the number of extra bristles per wing area shown, as induced by expression of Fz2-Arr (A) or Fz2-Arr and IFT-A KD factors (B–F) as indicated. All IFT-A KDs, except IFT144, suppress the extra-bristle phenotype. Bars represent mean SD; n R 10 fields per genotype; **p < 0.01; ***p < 0.001; NS, nonsignificant (Student’s t test). (H–N) High-magnification view of the extra-margin bristle phenotype near the margin of adult wings in C96 > ArmS10 flies (image field as indicated in Figure S4M). (I–L) Co-expression of ArmS10 and RNAi against IFT-A components does not affect the C96 > ArmS10 phenotype (see also Figures S4N–S4S for whole adult wing images). (M) Co-expression of TCFDN suppresses the C96 > ArmS10 phenotype. (N) Quantification of the number of extra bristles per wing area in the respective IFT-A protein KD backgrounds. Bars represent mean SD; n R 10 fields per genotype; ***p < 0.001; NS, nonsignificant (Student’s t test). See also Figure S4.

IFT-A protein KD could affect Arm phosphorylation levels. In clones for IFT140/rempA21Ci; axnE77 suppressed the elevated wing discs with combined nub > IFT-A and Axin KDs, the ratio expression of Sens and Dll seen in their neighboring axnE77 sin- of active to inactive Arm is significantly decreased (Figures 6A gle-mutant clones (Figures 5H, 5H0, S5E, and S5E0), we exam- and 6B; i.e., inactive or phosphorylated Arm levels are increased ined the level of cytoplasmic Arm staining in these clones relative to Axin KD alone), which is consistent with Sens expres- compared to either the axnE77 single-mutant clones or the sur- sion patterns reverting similar to the WT (Figures S6A–S6D). To rounding tissue. Cytoplasmic Arm staining was elevated in dou- address this further, we tested whether endogenous (cyto- ble-mutant clones, similar to in neighboring axnE77 clones, when plasmic) Arm levels are affected in IFT-A component KDs or mu- compared to the surrounding tissue (Figure 6G; surrounding tis- tants. Generally, the levels of Arm were reduced in such mutant sue is heterozygous and so phenotypically WT). The elevated scenarios. For example, in enGal4-driven KD of IFT43 or IFT140 levels of Arm detected by immunofluorescence comprise both in the posterior compartment (Figures 6D–6E0; compared to the phosphorylated inactive and unphosphorylated active forms. control in Figures 6C and 6C0 and quantified in Figures S6E– Based on western blot data and ratiometric analysis, we infer S6G0) or in mutant clones of IFT122 (Oseg1179;inFigures 6F that the elevated Arm staining in the double-mutant clones rep- and 6F0, compare Arm levels to surrounding WT cells), the intra- resents more of the phosphorylated inactive form and less cellular, cytoplasmic b-cat/Arm levels were reduced. These active Arm, thus explaining the reduction in Arm transcriptional results are in agreement with the increased proportion of phos- activity (as shown previously in Figures 5H, 5H0, S5E, and phorylated Arm in axin, IFT-A double KDs, which is targeted S5E0). Taken with the epistasis analyses, these data suggest for degradation (Figures 6A and 6B). Because double-mutant that IFT-A components are necessary for proper stabilization

712 Developmental Cell 34, 705–718, September 28, 2015 ª2015 Elsevier Inc. Figure 5. IFT-A Proteins Act Downstream or in Parallel to the Destruction Complex (A and A0) Axin RNAi KD in the posterior compartment of the wing disc induces a strong increase in Sens-positive cells at 29C (red, monochrome in A0). (B–E) Co-expression of RNAi against Axin and IFT122 (B), IFT140 (C), IFT121 (D), or IFT43 (E) suppresses extra Sens-positive cells in the posterior compartment (PC). n > 20 wings per genotype. (F) KD of Axin and IFT144 resembles the Sens pattern in Axin KD alone. (G) Quantification of the ratio of PC to the anterior compartment (AC) of fluorescence intensity in A0–F0 in third-instar wing discs of Axin KD alone and in com- bination with IFT-A RNAi. Bars represent mean SD; n = 4 representative stainings per genotype; ***p < 0.001; NS, nonsignificant (Student’s t test). Scale bar in (A) represents 25 mm. (H) Dll and Sens expression in axnE77 and IFT140/rempA21Ci double-mutant clones. Within the GFP-negative cells (double mutant), Sens (blue, monochrome in H0) and Dll (red, monochrome in Figure S5E0) are not highly induced compared to axnE77 clones (visible as high levels of Sens in margin proximal regions, see also Figure S5F), which is consistent with the RNAi data above (A–E0). See also Figure S5. of unphosphorylated b-cat/Arm in the cytoplasm to allow its roles for several cilia-associated proteins in regulating N, Wg, translocation to the nucleus, downstream or in parallel to the and EGFR signaling (Table 1), demonstrating cilia-independent destruction complex. functions in vivo in Drosophila. We show that ciliary proteins To analyze the localization of IFT-A proteins in Drosophila wing have no effect on Hh signaling in non-ciliated cells. cells and test for potential co-localization with Arm, we ex- Components of the Hh/Shh pathways are highly conserved pressed myc-tagged IFT122 and IFT43 using tubGal4, which is between Drosophila and vertebrates, with the main difference within the physiological range of their endogenous expression being that Shh/Hh signaling takes place in the cilium in verte- levels. Both IFT122-myc and IFT43-myc localize in punctae brate cells (Goetz and Anderson, 2010). The lack of a require- below the apical junctions (Figures 6H–6I00). Importantly, these ment for conserved ciliary proteins in Hh signaling in Drosophila punctae are often co-labeled with Arm: approximately 40%– epithelial cells thus suggests that the cilium provides a structural 50% of the pool of cytoplasmic Arm punctae co-stain with the compartment for transducing Hh signals, rather than that spe- IFT-A components (Figures 6H–6J and S6H–S6I00; in particular, cific cilia-associated proteins are required within the pathway. the brightest Arm spots overlap with IFT-A staining). Consistent with this, it was recently demonstrated that Hh- Taken together, our data argue for a role for IFT-A proteins in signaling components are localized within the cilium in ciliated Wg/Wnt signaling in Drosophila via an effect on the stabilization Drosophila neuronal cells (Kuzhandaivel et al., 2014). The IFT-B or localization of active, unphosphorylated Arm upstream of protein IFT25, which has no effect on cilia assembly but its nuclear translocation and downstream or in parallel to the is required for Hh signaling in vertebrates, is the exception. destruction complex. However, IFT25 is not conserved in Caenorhabditis elegans or Drosophila (Keady et al., 2012). The importance of IFT25 DISCUSSION suggests that moving Shh-signaling components along the (in and out of the cilium) is also critical for Shh signaling We have performed a systematic screen to test for potential roles in vertebrates, which is again consistent with the notion that Shh of ciliary proteins in non-ciliated epithelial cells and develop- signaling needs to take place inside the cilium when cilia are pre- mental signaling pathway contexts. This approach identified sent. Evolutionary aspects of the Hh/Shh pathway components,

Developmental Cell 34, 705–718, September 28, 2015 ª2015 Elsevier Inc. 713 Figure 6. IFT-A Proteins Regulate Wg Signaling by Affecting the Stability of Excess Arm Resulting from Destruction Complex Inhibition (A and B) Western blot analysis of cytoplasmic levels of Arm in WT, Axin-IR, and Axin-IR combined with IFT122-IR or IFT121-IR wing disc extracts (black and red arrowheads indicate the upper inactive or phosphorylated Arm band and the lower active or stable Arm band, respectively (n = 300 wing discs per genotype). (B) Quantification of three independent experiments of the ratio of lower band (stable Arm, red arrowhead) compared to the total cytoplasmic Arm levels. Bars represent mean SD; *p < 0.05, **p < 0.01, and ***p < 0.001; Student’s t test. (C–E) Cytoplasmic levels of Arm are reduced in cells of IFT43IR (magenta in D, monochrome in D0) and IFT140IR (magenta in E, monochrome in E0) compared to the WT control levels (magenta in C, monochrome in C0). See also the line scan quantification of protein staining in Figures S6E–S6G. (F and F0) Cytoplasmic Arm levels are reduced in IFT122/oseg1179 clones (marked by GFP absence below the junctional level; red in F, monochrome in F0). (G and G0) Cytoplasmic Arm levels are elevated in axnE77; IFT140/rempA21Ci double-mutant clones (GFP-negative cells; magenta in G, monochrome in G0) and indistinguishable from neighboring axnE77 single-mutant clones (GFP-positive cells with high levels of cytoplasmic Arm). (H–J) IFT122 and IFT43 partially colocalize with Arm. The tub-promoter-driven IFT122-myc (green in H, monochrome in H0) and IFT43-myc (green in I, mono- chrome in I0) show partial co-localization (in punctate structures) with Arm (scan is just below the apical junctional region; see also apical staining of Arm and IFT in Figures S6H and S6I). (J) Quantification of the number of punctae containing Arm alone (magenta) or Arm with IFT122 or IFT43 (white; n R 200 punctae per genotype, from four different wing discs). See also the line scan quantification of respective co-localization in Figures S6H00–S6I00. Scale bar represents 15 mmin C–E and 3 mm in H and I. See also Figure S6.

714 Developmental Cell 34, 705–718, September 28, 2015 ª2015 Elsevier Inc. studied in planarians, revealed that Hh signaling might have other IFT-A components), suggesting that some aspects of originally been organized by the cilium, with cilia serving as a IFT-A are nonetheless preserved between the cilia and the cyto- signaling compartment (Rink et al., 2009). The evolutionary plasmic locations. loss of IFTs in planaria does not affect Hh signaling (Rink et al., Can these observations be related to disease aspects of the 2009), consistent with the notion that the cilium serves as a spectrum of ciliopathies? Mutations in human IFT-A components Hh/Shh signaling hub but that specific components of ciliogene- cause a specific subcategory of ciliopathies, called skeletal cili- sis are not required for Hh signaling per se. opathy, which are characterized by limb morphogenesis defects, Our screen for developmental signaling requirements of ciliary as well as extra-skeletal abnormalities, including retinal or renal proteins in non-ciliated cells identified several such factors as defects (Huber and Cormier-Daire, 2012). IFT-A mutations often being required for canonical Wg/Wnt signaling. Previous studies lead to Shh/Hh pathway disruption (Liem et al., 2012; Qin et al., have proposed conflicting effects of the loss of cilia on canonical 2011), which in turn induces skeletal morphogenesis problems Wnt signaling in vertebrates, ranging from reduction or loss similar to the ones observed in skeletal ciliopathies (Ashe et al., of signaling to overactivation of the Wnt pathway (reviewed 2012; Cortellino et al., 2009; Mill et al., 2011). Importantly, the ca- in Oh and Katsanis, 2013). These different, or even opposing, nonical Wnt-signaling pathway has been proposed to act down- outcomes were possibly caused by pathway crosstalks, stream of Hh and bone morphogenetic protein signaling in bone context-specific interactions, or both. For example, Shh morphogenesis (Baron et al., 2006), and it is therefore possible signaling and Wnt signaling often mutually affect each other that defects observed in these syndromes are due to impaired (Borday et al., 2012; Rink et al., 2009; van den Brink et al., Hh and Wnt signaling. Retinal and renal defects can also be 2004; Yanai et al., 2008), and when cilium biogenesis is impaired, induced by defective Wnt signaling levels (Kawakami et al., Shh signaling is reduced or abolished. Thus, the potential posi- 2013; Lad et al., 2009). Our work supports this concept and tive effect of impaired ciliary function on Wnt signaling could adds insight into the function of ciliary proteins with regards to be secondary to the loss of Shh signaling, leading to an overac- Wnt signaling. tivation of Wnt signaling in ciliary mutants. In particular, Lancas- It remains unclear whether the cytoplasmic IFT-A proteins (in ter et al. (2011) showed in vitro and in vivo that mutations in a their possibly altered configuration) associate with microtubular dynein subunit of the retrograde transport complex (Dync2h1) structures and whether such an association is required for func- display cell-type-specific effects on cilium integrity and canoni- tion in Wnt/Wg signaling. Interestingly, a role for kinesin II in Wg cal Wnt signaling. The dync2h1 mouse embryonic fibroblasts signaling and the transport of b-cat/Arm was recently reported (MEFs) and regions of dync2h1 mutant embryos that exhibit (Vuong et al., 2014), consistent with a link to microtubules. One loss of cilia, and thus loss of Shh signaling, induce overactivation hypothesis is that the IFT-A proteins associate with microtubules of Wnt signaling. However, in dync2h1 mutant embryos or small and play a similar role to Costal-2 (Cos-2)/Kif7 in Hh signaling. In interfering RNA in MEFs that disrupt retrograde transport but the absence of Hh signals, Cos-2 binds to the Hh/Shh effector leave the cilium intact (and thus do not disrupt Shh signaling) Ci/Gli and tethers it to microtubules, together with Fused, Sup- display reduced canonical signaling levels (Lancaster et al., pressor of Fused, and several kinases (He et al., 2014; Robbins 2011). et al., 1997). Hh activation reverses this binding and frees Ci Here we demonstrate that a subset of IFT-A components, from the complex (Goetz and Anderson, 2010). Cos-2 is a nega- as well as other ciliary proteins (Table 1), act positively in canon- tive regulator of Hh signaling, and our data suggest that IFT-A ical Wnt/Wg signaling in a cilium-independent manner. This proteins act antagonistically to the destruction complex by pro- is consistent with the preceding data, in particular the effects moting b-cat/Arm stabilization. Sequestering Arm away from the of the dync2h1 mutants that leave cilia intact. Importantly, our destruction complex to prevent its phosphorylation and allow data provide insight into the role of cilia-associated proteins activation of the pathway makes this an attractive hypothesis outside of the ciliary structure. We have focused our mechanistic for future study. studies in Drosophila and on the IFT-A proteins, and we demon- strate that a subset of these is required for stabilization, localiza- EXPERIMENTAL PROCEDURES tion, or both of b-cat/Arm prior to its activity in the nucleus. Taken with the Lancaster et al. (2011) observations, this non-ciliary Fly Stocks Drosophila strains were raised at 25C on standard medium unless otherwise function is likely conserved in vertebrates. Due to the omnipres- indicated. The following lines were used: axin-IR (Bloomington Stock Center, ence of primary cilia in vertebrate cells and the associated 31705); UAS-IFT121-GFP, UAS-IFT144-GFP, and oseg1179/TM6b (gifts from difficulty of uncoupling ciliary and non-ciliary functions of IFT-A T. Avidor-Reiss); rempAl(2)21Ci-1 (gift from M. Kernan); UAS-Fz2-myc-Arr (gift proteins, Drosophila provides a unique and ideal model system from M. Wehrli); UAS-ArmS10 (Pai et al., 1996); UAS-TCFDN (Bloomington to dissect their non-ciliary function. Our data also suggest that Stock Center, 4784); and FRT82B,axnE77/TM6b and aoslacZ/TM6b (gifts IFT-A functions in a different configuration in ciliary versus non- from J. Treisman). ciliary contexts, because IFT144 (although efficiently knocked The following lines were used to induce and analyze mutant clones: down; Figure S2S) did not affect any detectable aspects of y, w, hsFlp; armlacZ, FRT40 developmental signaling pathways tested. IFT144 is a core y, w, hsFlp; ; P(w+), min55, UbiGFP, FRT80B structural component of IFT-A in cilia, thus suggesting a different y, w, hsFlp; ; UbiGFP(nls), FRT80B composition of IFT-A outside of the ciliary compartment. How- y, w, UbxFlp; UbiGFP(nls), FRT40; UbiGFP(nls), FRT82B/SM5:TM6b ever, IFT43 and IFT121, which are described as peripheral within y, w, hsFlp, tubGal4, UAS-GFP; ; FRT82B, tubGal80, CD21/TM6b IFT-A in the cilium, show effects (although the IFT121 effects are See Supplemental Experimental Procedures for generation of UAS-IFT122- generally weaker, both in KDs and in mutants, as compared to myc, UAS-IFT43-myc, and oseg1 CRISPR alleles.

Developmental Cell 34, 705–718, September 28, 2015 ª2015 Elsevier Inc. 715 The Gal4/UAS system (Brand and Perrimon, 1993) was used for gene ACKNOWLEDGMENTS expression studies, with the following Gal4 drivers used: We thank the Bloomington Stock Center, Kyoto DGRC Stock Center, UAS-dcr2; enGal4/CyoGFP VDRC, DGRC (supported by NIH 2P40OD010949-10A1), and Develop- UAS-dcr2; enGal4, UAS-GFP mental Studies Hybridoma Bank (DSHB, created by the NICHD of the UAS-dcr2; nubGal4 NIH and maintained at the University of Iowa) for fly strains and antibodies. apGal4; UAS-GFP/S-T We are grateful to U. Weber for help with the generation of CRISPR allele. UAS-dcr2; neurGal4/S-T We thank H. Bellen for the Sens antibody, J. Treisman and K. Legent for fly tubGal4/TM3 strains and discussions, T. Avidor-Reiss for the IFT-A-GFP and oseg1179 fly UAS-dcr2; C96Gal4 strains, M. Wehrli for the Fz2-myc-Arr fly strains, M. Kernan for the enGal4, UAS-myrRFP, NRE-GFP rempA21Ci fly strain, and J.R. Huynh, B. Mollereau, A. Benmerah, A. Gui- chet, and A. Audibert for discussions. We thank all M.M. and C.I. lab mem- Our 62 screen candidates were identified using the NCBI protein-blast bers for help and discussions and A. Humphries and N. Founounou for (http://blast.ncbi.nlm.nih.gov/Blast.cgi), and expression levels were verified helpful comments on the manuscript. Confocal microscopy was performed on FlyBase (http://flybase.org). RNAi lines used for the screen were ordered at the Icahn School of Medicine at the Mount Sinai Microscopy Shared from the Vienna Drosophila RNAi Center (VDRC), Kyoto Drosophila Genomics Resource Facility. This work was supported by NIH/NIGMS GM102811 Resource Center (DGRC) Kyoto, or Bloomington stock centers (Table S1). and HD066319 to M.M. and NIH/NIGMS EY022639 to C.I.

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