Dishevelled Attenuates the Repelling Activity of Wnt Signaling During Neurite Outgrowth in Caenorhabditis Elegans

Home , Axon guidance, Dishevelled, Growth cone, Prickle (protein)

Dishevelled attenuates the repelling activity of Wnt signaling during neurite outgrowth in Caenorhabditis elegans

Chaogu Zheng (郑超固), Margarete Diaz-Cuadros, and Martin Chalfie1

Department of Biological Sciences, Columbia University, New York, NY 10027

Contributed by Martin Chalfie, September 21, 2015 (sent for review August 11, 2015; reviewed by Kang Shen and Yimin Zou)

Wnt proteins regulate axonal outgrowth along the anterior–pos- Here, using the morphologically well-defined PLM neurons in terior axis, but the intracellular mechanisms that modulate the C. elegans, we find that two Dsh proteins, DSH-1 and MIG-5, act strength of Wnt signaling in axon guidance are largely unknown. redundantly downstream of Fzd receptor to mediate the repelling Using the Caenorhabditis elegans mechanosensory PLM neurons, activity of Wnt signal, which guides the outgrowth of a long, an- we found that posteriorly enriched LIN-44/Wnt acts as a repellent teriorly directed neurite away from the cue. At the same time, to promote anteriorly directed neurite outgrowth through the LIN- DSH-1 also provides feedback inhibition to attenuate Fzd sig- 17/Frizzled receptor, instead of controlling neuronal polarity as naling. The net effect of these two actions is that the PLM neurons previously thought. Dishevelled (Dsh) proteins DSH-1 and MIG-5 can grow a posteriorly directed neurite against the Wnt gradients. redundantly mediate the repulsive activity of the Wnt signals to The dual functions of DSH-1 help establish the bipolar shape of induce anterior outgrowth, whereas DSH-1 also provides feedback the PLM neurons and other C. elegans posterior neurons. inhibition to attenuate the signaling to allow posterior outgrowth Results against the Wnt gradient. This inhibitory function of DSH-1, which requires its dishevelled, Egl-10, and pleckstrin (DEP) domain, acts LIN-44/Wnt Proteins Repel PLM Neurites Through LIN-17/Fzd. C. elegans by promoting LIN-17 phosphorylation and is antagonized by pla- has two pairs of embryonically derived touch receptor neurons (TRNs) (26). The anterior ALM neurons are unipolar, having a nar cell polarity signaling components Van Gogh (VANG-1) and single anteriorly directed neurite, whereas the posterior PLM Prickle (PRKL-1). Our results suggest that Dsh proteins both re- neurons are bipolar, with a long anterior neurite that extends to the spond to Wnt signals to shape neuronal projections and moderate center of the animal and a short posteriorly directed neurite that its activity to fine-tune neuronal morphology. extends to the tail (Fig. 1A). Previous studies showed that mutations in lin-44/Wnt, which is normally expressed from the tail epi- dishevelled | Wnt | axonal guidance | C. elegans | touch receptor neurons dermis, and lin-17/Fzd cause PLM neurons to have short anterior neurites and a long posteriorly oriented neurite that reaches the tip xonal navigation along the anterior–posterior (A-P) axis is of the tail, turns, and then extends anteriorly for a significant length Aoften guided by the gradients of morphogens, including (8) (Fig. 1B; this phenotype is hitherto referred to as the “Wnt those of the Wnt family of secreted glycoproteins (1). Wnt proteins phenotype”). Similarly, the double mutation of two other Wnt repel axons expressing the receptor tyrosine kinase Derailed/Ryk genes, cwn-1 and egl-20, reversed the orientation of ALM cells, (2, 3) or the Frizzled (Fzd, Fz) receptor (4–9), but also act as which then had long posterior neurites and little or no anterior attractive cues for neurite outgrowth and dendritic formation neurites (8, 27). The authors proposed that Wnt signaling estab- through the Fzd receptor (10–12). Moreover, Wnt proteins can lishes the A-P polarity of the TRNs. Although LIN-44 and LIN-17 promote axon outgrowth and at the same time induce repulsive regulate cellular polarity during asymmetric cell divisions (28, 29), turning in cultured cortical neurons (6). the TRN morphological defect is the only instance in which the Wnt signals evoke downstream activity mainly through three function of Wnt and Fzd receptors are thought to control the different intracellular pathways: the β-catenin-dependent canonical pathway, the planar cell polarity (PCP) pathway, and the Significance Wnt/calcium pathway (13, 14). Although the canonical pathway (7, 15) and the Wnt/calcium pathway (16) regulate axon guid- The growth of neuronal processes is guided not only by external ance in a few instances, Wnt-mediated axon pathfinding mainly molecules but also by the modulation of the intracellular re- uses the PCP pathway, which activates the small GTPases Rho, sponses to those extracellular guidance cues. The mechanism for Rac, and Cdc42, which regulate cytoskeletal dynamics (17, 18). such modulation is unclear. Using a pair of sensory neurons in the Studies from mice, Drosophila,andCaenorhabditis elegans collec- nematode Caenorhabditis elegans, we found that guidance mol- tively suggest that the core components of PCP signaling involved ecules activate two downstream molecules, which redundantly in axon guidance are the Wnt receptor Fzd, the phosphoprotein mediate the navigation of the main neurite away from the source Dishevelled (Dsh, Dvl), the transmembrane protein Van Gogh/ Strabismus (Vang/Stbm, Vangl) and its adaptor Prickle (Prkl, Pk), of the repulsive guidance cues. However, one of the two mole- and the atypical cadherin Flamingo/starry night (Fmi/Stan, Celsrs), cules also provides feedback inhibition to downregulate the sig- although the requirement for each component may depend on the naling activity, which allows the neurons to grow a second specific neuron type (17, 19). neurite against the gradient of the repulsive cues. This dual An important question is how these components interact with functionality of downstream signaling molecules thus fine-tunes each other to spatially and temporally control the neuronal re- the intracellular response to guidance molecules. sponse to the Wnt signal. One particularly intriguing aspect of this control is the relationship between Fzd and Dsh. Normally Author contributions: C.Z. and M.C. designed research; C.Z. and M.D.-C. performed re- Dsh is thought to act as a downstream effector of Fzd (20–22), search; C.Z., M.D.-C., and M.C. analyzed data; and C.Z. and M.C. wrote the paper. but Dsh can also promote the phosphorylation of Fzd, which Reviewers: K.S., Stanford University; and Y.Z., University of California, San Diego. attenuates Fzd activity and inhibits downstream PCP signaling The authors declare no conflict of interest. 1 (23–25). The physiological significance of this inhibition is un- To whom correspondence should be addressed. Email: [email protected]. BIOLOGY

clear, but it suggests that Dsh proteins both promote and inhibit This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. DEVELOPMENTAL Wnt signaling in the same cellular context. 1073/pnas.1518686112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1518686112 PNAS | October 27, 2015 | vol. 112 | no. 43 | 13243–13248 Downloaded by guest on September 24, 2021 mutants (8). We found, however, that GFP::RAB-3 localized primarily to the positions where PLM neurites physically contacted the ventral cord. In the wild-type animals, this occurred when the anterior neurite sent a distal branch to synapse onto the ventral cord, whereas in the mutants, the GFP was distrib- uted along the ventral cord, where the anteriorly extending segment of the PLM posterior neurite, which made a U-turn at the tail, contacted the ventral cord (83%; n = 36; Fig. S1 A and B). GFP did accumulate in the tail region in the remaining 17% of PLM cells, as previously reported (8), but this distribution may be because these posterior neurites never made contact with the ventral cord. These results suggest that RAB-3 localization is more an indication of proximity to the ventral cord than a neurite- specific marker. We have, however, found a marker, the UNC-9 innexin gap junction protein (31), that localized to the anterior PLM neurite in both wild-type animals and lin-44 and lin-17 mutants. In wild- type animals, UNC-9::GFP was found in patches in the anterior neurite near the cell body. UNC-9::GFP was in the same position in lin-44 and lin-17 animals, even though the anterior neurite was much shorter (Fig. S1 C and D). The position of the label is where the PLM neurons form gap junctions with the downstream interneurons. Thus, synaptic proteins cannot be used to indicate polarity, undermining the main supporting evidence for the previous model. Moreover, the growth of a posterior neurite in PLM neurons requires the cell-intrinsic, Wnt-independent activity of the Hox protein EGL-5; mutations in egl-5 specifically suppressed posterior, but not anterior, outgrowth in otherwise wild-type animals Fig. 1. LIN-44/Wnt repels TRN neurites toward the anterior through Fzd (32). If PLM polarity is simply reversed in lin-44 or lin-17 ani- receptor LIN-17 and Dsh proteins DSH-1 and MIG-5. (A) TRN morphologies mals, the anterior neurite should act like the wild-type posterior visualized by RFP expression from the mec-17 promoter. (B) PLM in wild- neurite and be shortened by mutation of egl-5 in those animals. type, lin-44(n1792), and lin-17(n671) animals and those expressing lin-44(+) However, the loss of EGL-5 did not affect the growth of the and egl-20p::lin-44(+). (Scale bar, 20 μm.) (Right) Scheme of normal Wnt short anterior neurite in lin-44 or lin-17 animals (Fig. S2), which expression patterns adopted from ref. 30 and the diagrams of PLM shapes in further challenges the polarity theory. three different phenotypes: Wnt phenotype, posterior underextension, and For the above reasons and because all other neurons affected by posterior overextension. (C) The length of PLM posterior neurites in various the loss of Wnt signals only show outgrowth, and not polarity defects animals. (D) TRN morphology in dsh-1(u915) mig-5(u1030) double mutants. (7, 11, 12), we feel that the defects in the PLM neurons are best Arrows point to ALM and PLM neurons that showed the Wnt phenotype; described as alterations in outgrowth. Although we could not com- triangles point to the posteriorly directed neurites of the TRNs with morpho- pletely rule out the polarity model, the primary wild-type function of logical defects. (E) The penetrance of Wnt phenotypes in various mutants. Wnt and Fzd appears to be the stimulation of anterior neurite outgrowth and the inhibition of posterior neurite outgrowth. polarity of terminally differentiated neurons. Our results, however, challenge this interpretation and suggest that Wnt signaling plays Dsh Proteins, DSH-1 and MIG-5, Act Redundantly Downstream of Wnt its more expected role of providing guidance cues. Ligand and Fzd Receptor to Repel TRN Neurites. Dsh proteins link First, overexpression of LIN-44 changed the length of PLM Fzd receptors to all three Wnt signaling pathways (14). C. elegans has three Dsh paralogs: DSH-1, DSH-2, and MIG-5. Mutations posterior neurite, which is not expected if LIN-44 only provides in a single Dsh gene did not cause the Wnt phenotype, but positional information to establish “cellular polarity.” Wnt li- double mutations in dsh-1and mig-5 did (Fig. 1 D and E). Be- lin- gands are expressed in a specific pattern along the A-P axis. cause of their close linkage, we used CRISPR (clustered regu- 44 is expressed mainly posterior to the PLM cell body, whereas larly interspaced short palindromic repeats)/Cas9-mediated genome egl-20 is expressed exclusively anterior to the PLM cell body (Fig. editing (33) to introduce mutations in dsh-2 and mig-5 in animals 1B) (30). We found that overexpression of lin-44 from its own carrying the dsh-1(ok1445) allele. Loss-of-function dsh-2 and mig-5 promoter led to shortened PLM posterior neurites (Fig. 1B), alleles with small deletions that caused frameshifts were identified suggesting that Wnt signals act as repellents that suppress pos- by genotyping (Fig. S3B). PLM neurons in dsh-1 mig-5 animals terior outgrowth, rather than as a polarity signal. Misexpression were morphologically similar to those in lin-44 or lin-17 mutants, of lin-44 from the egl-20 promoter elongated the posterior neurite and ALM neurons in dsh-1 mig-5 double mutants resembled the but did not cause it to extend to the tail, turn, and then extend cells seen in cwn-1 egl-20 double mutants (Fig. 1D). ALM and anteriorly (Fig. 1 B and C). This posterior “overextension” phe- PLM neurons in dsh-1 dsh-2 double mutants did not have the Wnt notype also differed from the Wnt phenotype, in that the anterior phenotype (Fig. 1E); in fact, they were similar to the cells in dsh-1 neurite was not shortened. As a control, we misexpressed lin-44 animals described in the next paragraph. These results suggest that from the mom-2 promoter, which is expressed at a low level Dsh proteins DSH-1 and MIG-5 act redundantly downstream of around the PLM cell body, and found that PLM morphology did the Wnt ligand and Fzd receptor to propel TRN neurites toward not change significantly (Fig. 1C). These results suggest that the the anterior. repelling activities of the misexpressed LIN-44 countered the activities of the endogenous LIN-44 to allow the growth of a longer Mutations in Dsh Gene dsh-1 Caused the Shortening of the PLM Posterior PLM posterior neurite. Neurite. Given its role downstream of Wnt and Fzd, we were sur- One previous argument for polarity reversal in lin-44 and lin- prised that dsh-1 single mutants had significantly shorter PLM 17 mutants is the mistargeting of the synaptic vesicle protein posterior neurites compared with the wild-type (Fig. 2 A and B). RAB-3, which is transported toward the anterior in the wild-type We isolated three dsh-1 alleles (u915, u952,andu953)inagenetic animals but localized in the posterior neurite in lin-44 and lin-17 screen for mutants with TRN morphological defects (Fig. S3B). All

13244 | www.pnas.org/cgi/doi/10.1073/pnas.1518686112 Zheng et al. Downloaded by guest on September 24, 2021 three (presumably loss-of-function) alleles and the deletion allele presumably through mig-5, which led to defects in posteriorly ok1445 resulted in a similar shortening of the PLM posterior directed outgrowth. The length of PLM anterior neurites was not neurites (Fig. 2B). DSH-1 is expressed and functions cell auton- affected by either lin-44 overexpression or dsh-1 mutations, omously in the TRNs, as a dsh-1 promoter reporter sIs12076 suggesting that outgrowth toward the anterior is not sensitive to [C34F11.9a::gfp] wasexpressedinbothALMandPLMneurons the hyperactivity of Wnt signaling. Because DSH-1::GFP was rel- (Fig. S3A), and expression of a wild-type copy of the a isoform of atively enriched in the posterior neurite compared with the anterior dsh-1 (www.wormbase.org) from the TRN-specific mec-17 pro- neurite (Fig. 2 D and E), we propose that high Wnt concentration moter rescued the PLM morphological defects (Fig. 2B). These in the tail region leads to the accumulation of DSH-1 in the pos- results suggest DSH-1 normally promotes posteriorly directed terior neurites, which triggers the down-regulation of the Wnt sig- neurite outgrowth. This function of DSH-1 depended on Wnt signaling. This DSH-1-mediated negative feedback is not evoked in nals because dsh-1 lin-44 and dsh-1 lin-17 double mutants had the the anterior neurite, presumably because of the low LIN-44 con- samedefectsasthelin-44 and lin-17 animals. In both the single and centration surrounding it. double mutants, about 80% of PLM neurons showed the Wnt phenotype (Fig. 1E), and the other 20% had a slightly elongated The Dishevelled, Egl-10, and Pleckstrin Domain of DSH-1 Is Required for posterior neurite (Fig. S4), suggesting lin-44 and lin-17 are epistatic the Attenuation of Wnt Signaling and the Growth of PLM Posterior to dsh-1. These results support the conclusion that Wnt proteins act Neurites. DSH-1-mediated down-regulation of the repulsive Wnt as guidance molecules for the PLM neurons. activities resembles the inhibition of Wnt5a-stimulated growth of Among the three Dsh genes, the effect on the growth of the commissural axons in mouse embryos by Dvl1, which induces PLM posterior neurite is specific to dsh-1; mutations in dsh-2 phosphorylation of Fzd3 (24). Biochemical studies in Xenopus and mig-5 did not result in the shortening of PLM posterior found that the dishevelled, Egl-10, and pleckstrin (DEP) domain neurite (Fig. 2B). Therefore, in addition to promoting the out- of Dsh proteins was needed for Dsh-dependent Fzd3 phos- growth of anterior neurites away from the Wnt signal DSH-1, but phorylation (25). The DEP domain of DSH-1 is essential for not the other two Dsh paralogs, also promoted posterior neurite attenuating the repelling activity of Wnt signaling that allows the outgrowth against the Wnt gradients. This second, inhibitory posterior neurite to grow to a normal length. The expression of function of DSH-1 was further confirmed by the observation that DEP-less DSH-1 or DSH-1 N terminus in dsh-1 animals failed to dsh-1 overexpression from a mec-17 promoter produced the Wnt restore the PLM posterior neurite in dsh-1 mutants, whereas the phenotype in wild-type PLM neurons (Fig. 2C), which suggests expression of the DEP domain-containing C terminus did (Fig. 3 that an excess amount of DSH-1 can completely shut down Wnt A and B). Moreover, the missense mutation in dsh-1(u952), signaling. Consistent with the absence of this second function in which alters G512 in the DEP domain (Fig. S3B), gave the same DSH-2 and MIG-5, overexpression of dsh-2 and mig-5 had little shortened PLM posterior neurites as the null alleles (Fig. S3C). effect on PLM neurites (Fig. 2C). Overexpression of the full-length DSH-1 protein and the DEP The observation that overexpression of lin-44 from its own domain-containing C terminus caused the Wnt phenotype, al- promoter caused a similar shortening of the PLM posterior though at a lower penetrance than in lin-17 or lin-44 mutants neurite, as seen in dsh-1 mutants (Fig. 1B), is consistent with (Fig. 3C). In contrast, overexpression of DSH-1 N terminus and DSH-1 having a negative effect on Wnt signaling. Misexpression DSH-1ΔDEP had little effect (Fig. 3C). These results suggest of lin-44 from the egl-20 promoter (i.e., from a more anterior that DSH-1 provides inhibitory feedback to the Wnt signaling site) partially rescued dsh-1 mutants (Fig. 2B). These results pathway through its DEP domain. suggest that the loss of dsh-1 increased the Wnt repelling activity, The DEP domains of DSH-1 and MIG-5 have 85% sequence similarity, and swapping the most divergent region of the domains (Lys-Leu-Lys-Tyr-Ile-Ala in DSH-1 and Ala-Ala-Gly-Leu-Ile-Arg in the MIG-5) did not affect the inhibitory function of DSH-1 (Fig. S5). Therefore, the functional differences between DSH-1 and MIG-5 cannot be attributed to the DEP domain alone.

Protein Kinase C Phosphorylation Sites in LIN-17/Fzd Cytoplasmic Tail Mediate the DSH-1 Functions in Attenuating Wnt Signaling. We next confirmed that phosphorylation of the Fzd receptor LIN-17 was required for DSH-1-mediated attenuation of Wnt signaling activity. Although the lin-17 gene produces several isoforms varying in their C terminus, we found that the expression of the cDNA of the lin-17a isoform (www.wormbase.org) from a TRN-specific mec-17 promoter fully rescued the morphological defects of lin- 17 mutants (Fig. 4D). Thus, LIN-17a responds to Wnt ligands cell-autonomously in PLM neurons. The cytoplasmic tail of Fz1 can be phosphorylated and then inhibited by protein kinase C ζ (PKCζ)inDrosophila eyes (23), and multiple phosphorylation sites have been identified in mouse Fzd3 and Xenopus Fz3 (24, 25). Using the online tools of the Group-based Prediction System (34), we identified four potential PKC phosphorylation sites (S523, T546, S551, and S555) in the C-terminal cytoplasmic region of LIN-17a and created LIN-17a mutants with nonphosphorylatable (5A) and phosphomimetic Fig. 2. DSH-1 promotes posteriorly directed neurite outgrowth by attenu- (5E) amino acids at those sites plus the adjacent T524 (Fig. 4A). ating Wnt signals. (A) PLM posterior neurites in wild-type and dsh-1 animals. (B) The length of PLM posterior neurites in various mutants and transgenic Expression of either LIN-17a(5A) or LIN-17a(5E) in the TRNs animals. (C) The percentage of PLM neurons showing the Wnt phenotype in rescued the PLM neurite Wnt phenotype in lin-17 null mutants animals overexpressing Dsh proteins from mec-17 promoter. Constructs were (Fig. 4B). However, the morphologically restored PLM neurons injected at the concentration of 15 ng/μL. (D) The localization of DSH-1::GFP that express the nonphosphorylatable LIN-17a(5A) had signifi- in the anterior (AN) and posterior (PN) neurites of PLM neurons. The dashed cantly shorter posterior neurites, whereas cells that express LIN-

boxes, which are enlarged in the lower panels, represent 35 μm in neurite 17a(5E) carrying the phosphomimetic mutations had longer BIOLOGY

length; arrows point to the fluorescent puncta. (E) The average number of posterior neurites compared with cells rescued by the wild-type DEVELOPMENTAL fluorescent DSH-1::GFP puncta/35 μm in ALM and PLM neurites. LIN-17a (Fig. 4 C and D). These results suggest that the potential

Zheng et al. PNAS | October 27, 2015 | vol. 112 | no. 43 | 13245 Downloaded by guest on September 24, 2021 three core components of PCP signaling in axonal guidance (17) in PLM outgrowth. Although their loss did not produce the Wnt phenotype seen in lin-44 and lin-17 mutants, mutations in vang-1 and prkl-1 resulted in the abnormal elongation of the PLM posterior neurite (Fig. 6A). This posterior overextension phenotype, although less severe than the Wnt phenotype, suggests a weakening of the repulsive Wnt signal and a role, albeit a weak one, for VANG-1 and PRKL-1 in Wnt signaling activity in the PLM neurons. This conclusion is supported by the observation that mutations in vang-1 and prkl-1 partially rescued the dsh-1 phenotype by increasing the length of PLM posterior neurite. Mutations in fmi-1 also rescued the PLM posterior outgrowth defects in dsh-1 mutants, but elongated the posterior neurite only slightly in the wild- type background (Fig. 6A). Thus, these three positive regulators of PCP signaling antagonize the DSH-1-mediated feedback inhibition. Mutations in unc-44/Diego-like and lin-18/Derailed/Ryk did not affect PLM outgrowth (SI Results). Expression of nonphosphorylatable LIN-17a(5A) suppressed Fig. 3. The DEP domain of DSH-1 mediates its inhibitory function. (A) The the elongation of the PLM posterior neurite in vang-1 and prkl-1 schemes representing the domain organization of DSH-1 protein and its mutants (Fig. 6A). These results suggest that VANG-1 and PRKL-1 variants. (B) The length of PLM posterior neurites in dsh-1 animals expressing normally act to reduce LIN-17 phosphorylation and, thus, coun- various transgenes. Constructs were injected at the concentration of 3 ng/μL. (C) Penetrance of the Wnt phenotype in PLM neurons from animals over- teract the increase in LIN-17 phosphorylation caused by DSH-1. expressing DSH-1 variants from mec-17 promoter. Constructs were injected These data are consistent with a previous report that mouse Vangl2 at 15 ng/μL. promotes Wnt5a-stimulated outgrowth of commissural axons by suppressing Fzd3 hyperphosphorylation (24).

PKC phosphorylation sites in the cytoplasmic tail of LIN-17/Fzd DAAM-1 and JNK-1 Act Downstream of PCP Signaling. The PCP sig- are important for the regulation of neurite outgrowth by Wnt naling pathway activates Rac and its downstream kinase JNK, as signaling, although we could not rule out the phosphorylation of well as the small GTPase Rho and its effector ROCK, through the these sites by other kinases. Formin family protein Daam1 (37). We found that mutations in Unlike the overexpression of lin-44/Wnt, overexpression of lin- jnk-1, the sole homolog of JNK, caused the elongation of PLM 17(+) did not cause the premature termination of the PLM posterior neurites, but not the Wnt phenotype, and mutations in posterior neurite (Fig. 4D). This result could occur if Wnt, and daam-1 had much weaker effects (Fig. 6C). Mutations in both not its receptor, is limiting, as seems likely. In contrast, over- genes significantly increased the length of PLM posterior neurites expression of the LIN-17a(5A) in wild-type animals did result in in dsh-1 background (Fig. 6C). jnk-1 daam-1 double mutants had a short PLM posterior neurite (Fig. 4D), possibly through com- slightly longer posteriorly directed neurites than jnk-1 animals petition with endogenous LIN-17a. We also found that the ex- (Fig. 6 B and C). These results suggest that both Rac/JNK and pression of LIN-17a(5E), but not LIN-17a(+) or LIN-17a(5A), was sufficient to rescue the defects in posteriorly directed outgrowth in dsh-1 mutants (Fig. 4 D and E). These results suggest that DSH-1 negatively regulates Wnt signaling activity by af- fecting the phosphorylation state of LIN-17.

The Canonical Pathway Contributes Weakly to the Wnt Activities in Neurite Guidance. We tested whether both the canonical and non- canonical Wnt pathways are needed for Wnt repellent activity in PLM neurons. In the canonical pathway, the binding of Wnt ligands to Fzd receptor leads to the disassembly of the GSK-3/Axin/ APC destruction complex, which leads to the stabilization and nuclear translocation of β-catenin and activation of downstream genes (35). This signaling cascade requires the C-terminal cytoplasmic Lys-Thr-X-X-X-Trp motif in Fzd receptors (36). We found that the expression of LIN-17a lacking amino acids 497–502 (Lys- Thr-Val-His-Ala-Trp) rescued the Wnt phenotype in lin-17 mutants (Fig. 5A), suggesting the canonical pathway is not required for Wnt activities. In addition, mutations in either pry-1/Axin,acom- ponent of the destruction complex, or any of the four β-catenin genes (bar-1, hmp-2, wrm-1,andsys-1) did not significantly change the length of PLM posterior neurites in wild-type animals (Fig. 5B). Loss of bar-1 and overexpression of LIN-17a (ΔLys-Thr-Val-His- Ala-Trp) could, however, partially restore the PLM posterior neurite defect in dsh-1 mutants where the strength of Wnt signaling was elevated (Fig. 5B). These results suggest that the β-catenin- Fig. 4. Phosphorylation sites in the cytoplasmic tail of LIN-17 are needed for DSH-1-mediated inhibition of Wnt signaling. (A) The sequence of the dependent canonical pathway indeed contributes to the guidance C-terminal cytoplasmic region of LIN-17a proteins. Predicted PKC phos- function of Wnt proteins, as had been previously found for the phorylation sites are labeled in red. Five serine/threonine sites were mu- anteroposterior guidance of D-type axons in C. elegans (7). tated to alanine in LIN-17a(5A) and to glutamic acid in LIN-17a(5E) mutants. (B) Percentage of the Wnt phenotype in PLM neurons of lin-17 animals PCP Signaling Components Vang, Prkl, and Fmi Antagonize the Function expressing wild-type or mutant LIN-17a proteins in TRNs. (C–E) PLM poste- of DSH-1 in Promoting Outgrowth. We also tested the role of Van rior neurites in wild-type, lin-17, and dsh-1 animals carrying transgenes that Gogh (VANG-1)/Vang, Prickle (PRKL-1)/Prkl, and FMI-1/Fmi, express LIN-17a mutants.

13246 | www.pnas.org/cgi/doi/10.1073/pnas.1518686112 Zheng et al. Downloaded by guest on September 24, 2021 only displayed a loss of the inhibitory Dsh function, so this activity could be studied in isolation. A dual role for Dsh proteins provides explanations to several previously puzzling observations. For example, the axons of posterior D-type motor neurons in C. elegans are repelled by LIN-44/Wnt; however, mutations in Dsh genes mig-5 and dsh-1 resulted in the underextension phenotype, opposite to the overextension phenotype observed in lin-44 mutants (7). Results from our study suggest that in this scenario, MIG-5 and DSH-1 (or the three Dsh proteins) may act redundantly to mediate the repulsive activities of LIN-44, but both also carry the inhibitory functions. A dual function could explain the opposing phenotypes between Dsh and Wnt mutants. When Wnt proteins act as attractive cues, Dsh proteins also play both positive and negative Fig. 5. The canonical pathway contributes weakly to the activity of Wnt roles, depending on the cellular contexts. The mushroom body signaling. (A) The penetrance of the PLM Wnt phenotype in lin-17 animals axons and embryonic sensory axons in Drosophila require Dsh expressing LIN-17a that lacks the transmembrane motif Lys-Thr-Val-His-Ala- for Wnt-stimulated outgrowth (20, 21), whereas mammalian Trp (aa 497–502). (B) The length of PLM posterior neurites in various strains. Dvl1 inhibits Wnt5a-stimulated growth of commissural axons (24). Although our model is consistent with the regulatory function of Dvl1 in providing feedback inhibition of Fzd3, we do Daam1/Rho/ROCK pathways contribute to the repulsive Wnt not know whether Dvl1 also positively contributes to Wnt sig- effect in the PLM neurons. naling. A more recent study found that Dvl2 positively promotes bar-1 jnk-1 daam-1 triple mutants did not show the Wnt Wnt/PCP signaling in the commissural axons and antagonizes phenotype either, although the PLM posterior neurites were the Dvl1-mediated inhibition of Fzd3 (38), which suggests that the most extended of any of the mutant combinations (Fig. 6C). dual function of Dsh proteins could also be executed separately Thus, both the canonical and the PCP pathways contribute to by distinct Dsh paralogs. Wnt signaling in the PLM neurons. In contrast, the Wnt/calcium Because the shortened PLM posterior neurite in dsh-1 mu- pathway did not appear to be important (Fig. S6 and SI Results). tants represents a state of hyperactive Wnt signaling, these animals provided a sensitized background to identify genes that DSH-1-Mediated Attenuation of Wnt Signaling Generally Enables positively contribute to the actions of Wnt proteins. We found Posteriorly Directed Neurite Outgrowth in Tail Neurons. The tail that the PCP core components VANG-1, PRKL-1, and FMI-1 region of C. elegans contains the cell bodies of more than a dozen promoted the repulsion of neurites, but the relatively weak ef- neurons in addition to the PLM neurons. Two pairs of these fects of their loss compared with Wnt and Fzd suggest they play neurons, ALNL/R and PLNL/R, are bipolar, similar to the PLM modulatory, instead of essential, roles in Wnt signaling. This neurons (Fig. S7). We found that the posterior ALN and PLN modulation involves antagonizing the inhibitory function of DSH-1 neurites were also repelled by LIN-44/Wnt signals, and DSH-1 and reducing LIN-17/Fzd phosphorylation. Consistent with our promoted posteriorly directed outgrowth against the Wnt gra- results, a previous study reported that Vangl2 in mouse com- dients. In lin-44, lin-17, and dsh-1 mig-2 mutants, the extent of missural axons blocked the Dvl1-mediated inhibition of Fzd3 the two neurites of the ALN and PLN neurons were reversed: (24). Because Vang physically interacts with Prkl and recruits it the anterior neurite was shorter and the posterior neurites grew to the membrane (39), our studies further establish that the to the end of the tail, turned, and extended anteriorly for a – Vang/Prkl complex positively promotes Wnt/PCP signaling and considerable length (Fig. S7 A D). In the dsh-1 single mutants, axon guidance by Wnt proteins. Furthermore, Fmi, a cadherin- the posterior neurites of the ALN and PLN neurons were sig- like membrane protein that serves as coreceptors for Fzd and nificantly shorter than in the wild-type (Fig. S7E). These results Vang (40), promotes the function of both in neurite guidance. suggest that DSH-1-mediated feedback inhibition may generally Our results suggest that the effectors downstream of Wnt sig- balance the repulsive activities of Wnt signals and enable the naling are most probably highly redundant. First, in the saturated growth of posterior neurites in tail bipolar neurons. screen that searched for mutants with TRN morphological defects and identified dsh-1 alleles, we isolated four lin-44 alleles and four Discussion lin-17 alleles but did not find mutants of any other genes that In this study, we found that LIN-44/Wnt and LIN-17/Fzd act to showed a similar PLM Wnt phenotype. Second, the bar-1 jnk-1 repel PLM neurites toward the anterior instead of controlling daam-1 triple mutants, in which both the canonical pathway and neuronal polarity, as previously thought. In this reinterpretation, PCP signaling pathway are blocked, still failed to reproduce the Wnt proteins function in their conventional role as guidance lin-44 phenotype, although an intermediate phenotype with a molecules during PLM morphogenesis. We propose that the default outgrowth pattern in PLM neurons is determined by the intrinsic propensity to grow toward the posterior in the absence of the guidance cues. The Wnt signal released from the posterior side of the cell body likely determines the length of the two neurites by regulating neurite extension. The anterior neurite grows down the repulsive Wnt gradient, whereas the posterior neurite grows against it. Importantly, we found that the outgrowth of both neurites requires DSH-1, which has two seemingly paradoxical functions in both promoting and inhibiting Wnt activity. Two Dsh paralogs, DSH-1 and MIG-5, redundantly mediate the intracellular response to the repulsive activities of Wnt proteins. This repulsive activity promotes the production of a long, anteriorly directed neurite in PLM and other tail neurons. At the same time, DSH-1 Fig. 6. The PCP signaling pathway is downstream of the Wnt activities in

acts specifically in the posterior neurite to attenuate the strength neurite guidance. (A–C) The length of PLM posterior neurites in various BIOLOGY

of Wnt signaling, allowing it to grow against the repelling gra- strains. In A, lin-17a(5A) was expressed from the mec-17 promoter and re- DEVELOPMENTAL dients. The dsh-1 mig-5 redundancy meant that dsh-1 mutants pressed the phenotype of vang-1 and prkl-1.

Zheng et al. PNAS | October 27, 2015 | vol. 112 | no. 43 | 13247 Downloaded by guest on September 24, 2021 markedly elongated PLM posterior neurite was observed. These CATTTATGTGG-3′ in exon 1 of dsh-2, respectively, were injected into results suggest that a strong genetic redundancy and/or com- dsh-1(u915) mutants, using a previously described method (33). Most of the pensating mechanism exist downstream of the Wnt/Fzd/Dvl constructs were made using the Gateway cloning system (Life Technologies). complex, which ensures a robust output of Wnt signaling. Q5 Site-Directed Mutagenesis Kit (New England Biolabs) was used to create At least six types of neurons [PLM, ALN, PLN (this study), constructs that express dsh-1a and lin-17a mutants. Transgenes uIs31[mec- DD6, VD12, and VD13 (7)] require Dsh proteins to grow toward 17p::GFP] III, uIs115[mec-17p::RFP] IV, and uIs134[mec-17p::RFP] Vwereused the posterior and against the gradient of repulsive LIN-44 con- to visualize TRN morphology. Transgenes jsIs821[Pmec-7::GFP::rab-3] and centration. Given the specific expression of LIN-44 from the tail uIs178[mec-17p::unc-9::GFP; mec-17p::RFP] were used to label presynaptic hypodermis (8, 29), we propose that Dsh-mediated attenuation vesicles and gap junctions, respectively. The relative length of PLM neurites of the Wnt signaling is a common mechanism used by the pos- was calculated by dividing the length of the neurite by the diameter of PLM teriorly directed neurite to navigate against the Wnt gradients. cell body along the A-P axis. To test transgenic animals, DNA constructs were This region-specific activity suggests that fine-tuning the strength injected at a concentration of 5 ng/μL (unless otherwise indicated) into the of Wnt activity by intracellular mechanisms may be essential for animals to establish stable lines carrying the extrachromosomal array. For the local shaping of the posterior neuroanatomy. statistical analysis, ANOVA and a post hoc Tukey–Kramer method were used to identify significant difference between the sample means in multiple Materials and Methods comparisons. Single and double asterisks indicated P < 0.05 and P < 0.01, C. elegans wild-type (N2) and mutant strains were maintained at 20 °C, as respectively. More details are provided in SI Materials and Methods. previously described (41). Most strains were provided by the Caenorhabditis Genetics Center, which is funded by NIH Office of Research Infrastructure ACKNOWLEDGMENTS. We thank Dan Dickinson for sharing materials and Programs (P40 OD010440). To create dsh-1 mig-5 and dsh-1 dsh-2 double mu- reagents. We also thank Oliver Hobert, Richard Mann, and the members of tants, constructs that express CRISPR/Cas9 with sgRNAs targeting 5′-GAGA- our laboratory for stimulating discussions and comments. This work was AGGAGTAGCGACGCTTGG-3′ in exon 2 of mig-5 and 5′-GATGTTTCTAA- supported by NIH Grant GM30997 (to M.C.).

1. Schnorrer F, Dickson BJ (2004) Axon guidance: Morphogens show the way. Curr Biol 24. Shafer B, Onishi K, Lo C, Colakoglu G, Zou Y (2011) Vangl2 promotes Wnt/planar cell 14(1):R19–R21. polarity-like signaling by antagonizing Dvl1-mediated feedback inhibition in growth 2. Yoshikawa S, McKinnon RD, Kokel M, Thomas JB (2003) Wnt-mediated axon guidance cone guidance. Dev Cell 20(2):177–191. via the Drosophila Derailed receptor. Nature 422(6932):583–588. 25. Yanfeng WA, Tan C, Fagan RJ, Klein PS (2006) Phosphorylation of frizzled-3. J Biol 3. Liu Y, et al. (2005) Ryk-mediated Wnt repulsion regulates posterior-directed growth Chem 281(17):11603–11609. of corticospinal tract. Nat Neurosci 8(9):1151–1159. 26. Chalfie M, Sulston J (1981) Developmental genetics of the mechanosensory neurons 4. Fenstermaker AG, et al. (2010) Wnt/planar cell polarity signaling controls the anterior- of Caenorhabditis elegans. Dev Biol 82(2):358–370. posterior organization of monoaminergic axons in the brainstem. J Neurosci 30(47): 27. Prasad BC, Clark SG (2006) Wnt signaling establishes anteroposterior neuronal po- 16053–16064. larity and requires retromer in C. elegans. Development 133(9):1757–1766. 5. Blakely BD, et al. (2011) Wnt5a regulates midbrain dopaminergic axon growth and 28. Sternberg PW, Horvitz HR (1988) lin-17 mutations of Caenorhabditis elegans disrupt guidance. PLoS One 6(3):e18373. certain asymmetric cell divisions. Dev Biol 130(1):67–73. 6. Li L, Hutchins BI, Kalil K (2010) Wnt5a induces simultaneous cortical axon outgrowth 29. Herman MA, Vassilieva LL, Horvitz HR, Shaw JE, Herman RK (1995) The C. elegans and repulsive turning through distinct signaling mechanisms. Sci Signal 3(147):pt2. gene lin-44, which controls the polarity of certain asymmetric cell divisions, encodes a 7. Maro GS, Klassen MP, Shen K (2009) A beta-catenin-dependent Wnt pathway mediates Wnt protein and acts cell nonautonomously. Cell 83(1):101–110. anteroposterior axon guidance in C. elegans motor neurons. PLoS One 4(3):e4690. 30. Harterink M, et al. (2011) Neuroblast migration along the anteroposterior axis of 8. Hilliard MA, Bargmann CI (2006) Wnt signals and frizzled activity orient anterior- C. elegans is controlled by opposing gradients of Wnts and a secreted Frizzled-related – posterior axon outgrowth in C. elegans. Dev Cell 10(3):379 390. protein. Development 138(14):2915–2924. 9. Pan CL, et al. (2006) Multiple Wnts and frizzled receptors regulate anteriorly directed 31. Liu Q, Chen B, Gaier E, Joshi J, Wang ZW (2006) Low conductance gap junctions cell and growth cone migrations in Caenorhabditis elegans. Dev Cell 10(3):367–377. mediate specific electrical coupling in body-wall muscle cells of Caenorhabditis ele- 10. Lyuksyutova AI, et al. (2003) Anterior-posterior guidance of commissural axons by gans. J Biol Chem 281(12):7881–7889. Wnt-frizzled signaling. Science 302(5652):1984–1988. 32. Zheng C, Diaz-Cuadros M, Chalfie M (2015) Hox genes promote neuronal subtype di- 11. Kirszenblat L, Pattabiraman D, Hilliard MA (2011) LIN-44/Wnt directs dendrite out- versification through posterior induction in Caenorhabditis elegans. Neuron, in press. growth through LIN-17/Frizzled in C. elegans Neurons. PLoS Biol 9(9):e1001157. 33. Dickinson DJ, Ward JD, Reiner DJ, Goldstein B (2013) Engineering the Caenorhabditis 12. Song S, et al. (2010) A Wnt-Frz/Ror-Dsh pathway regulates neurite outgrowth in elegans genome using Cas9-triggered homologous recombination. Nat Methods Caenorhabditis elegans. PLoS Genet 6(8):e1001056. 10(10):1028–1034. 13. Zou Y (2004) Wnt signaling in axon guidance. Trends Neurosci 27(9):528–532. 34. Xue Y, et al. (2008) GPS 2.0, a tool to predict kinase-specific phosphorylation sites in 14. Gordon MD, Nusse R (2006) Wnt signaling: Multiple pathways, multiple receptors, hierarchy. Mol Cell Proteomics 7(9):1598–1608. and multiple transcription factors. J Biol Chem 281(32):22429–22433. 35. Logan CY, Nusse R (2004) The Wnt signaling pathway in development and disease. 15. Ciani L, Krylova O, Smalley MJ, Dale TC, Salinas PC (2004) A divergent canonical WNT- – signaling pathway regulates microtubule dynamics: Dishevelled signals locally to Annu Rev Cell Dev Biol 20:781 810. stabilize microtubules. J Cell Biol 164(2):243–253. 36. Umbhauer M, et al. (2000) The C-terminal cytoplasmic Lys-thr-X-X-X-Trp motif in – 16. Hutchins BI, Li L, Kalil K (2011) Wnt/calcium signaling mediates axon growth and frizzled receptors mediates Wnt/beta-catenin signalling. EMBO J 19(18):4944 4954. guidance in the developing corpus callosum. Dev Neurobiol 71(4):269–283. 37. Endo Y, Rubin JS (2007) Wnt signaling and neurite outgrowth: Insights and questions. – 17. Zou Y (2012) Does planar cell polarity signaling steer growth cones? Curr Top Dev Biol Cancer Sci 98(9):1311 1317. 101:141–160. 38. Onishi K, et al. (2013) Antagonistic functions of Dishevelleds regulate Frizzled3 en- 18. Schlessinger K, Hall A, Tolwinski N (2009) Wnt signaling pathways meet Rho GTPases. docytosis via filopodia tips in Wnt-mediated growth cone guidance. J Neurosci 33(49): – Genes Dev 23(3):265–277. 19071 19085. 19. Ackley BD (2014) Wnt-signaling and planar cell polarity genes regulate axon guidance 39. Jenny A, Darken RS, Wilson PA, Mlodzik M (2003) Prickle and Strabismus form a along the anteroposterior axis in C. elegans. Dev Neurobiol 74(8):781–796. functional complex to generate a correct axis during planar cell polarity signaling. 20. Mrkusich EM, Flanagan DJ, Whitington PM (2011) The core planar cell polarity gene EMBO J 22(17):4409–4420. prickle interacts with flamingo to promote sensory axon advance in the Drosophila 40. Wu J, Mlodzik M (2009) A quest for the mechanism regulating global planar cell embryo. Dev Biol 358(1):224–230. polarity of tissues. Trends Cell Biol 19(7):295–305. 21. Shimizu K, Sato M, Tabata T (2011) The Wnt5/planar cell polarity pathway regulates 41. Brenner S (1974) The genetics of Caenorhabditis elegans. Genetics 77(1):71–94. axonal development of the Drosophila mushroom body neuron. J Neurosci 31(13): 42. Kühl M (2004) The WNT/calcium pathway: Biochemical mediators, tools and future 4944–4954. requirements. Front Biosci 9:967–974. 22. Rosso SB, Sussman D, Wynshaw-Boris A, Salinas PC (2005) Wnt signaling through 43. Thompson O, et al. (2013) The million mutation project: A new approach to genetics Dishevelled, Rac and JNK regulates dendritic development. Nat Neurosci 8(1):34–42. in Caenorhabditis elegans. Genome Res 23(10):1749–1762. 23. Djiane A, Yogev S, Mlodzik M (2005) The apical determinants aPKC and dPatj regulate 44. Dupuy D, et al. (2007) Genome-scale analysis of in vivo spatiotemporal promoter Frizzled-dependent planar cell polarity in the Drosophila eye. Cell 121(4):621–631. activity in Caenorhabditis elegans. Nat Biotechnol 25(6):663–668.

13248 | www.pnas.org/cgi/doi/10.1073/pnas.1518686112 Zheng et al. Downloaded by guest on September 24, 2021