© 2020. Published by The Company of Biologists Ltd | Development (2020) 147, dev179168. doi:10.1242/dev.179168

RESEARCH ARTICLE Caenorhabditis elegans Flamingo FMI-1 controls self-avoidance through F-actin assembly Hao-Wei Hsu*, Chien-Po Liao*, Yueh-Chen Chiang*, Ru-Ting Syu and Chun-Liang Pan‡

ABSTRACT protease KPC-1 (Salzberg et al., 2014) and the Wnt-secretory Self-avoidance is a conserved mechanism that prevents crossover factor Wntless (Liao et al., 2018). The signaling pathways through between sister from the same , ensuring proper which these molecules control self-avoidance are incompletely functioning of the neuronal circuits. Several adhesion molecules are defined. known to be important for dendrite self-avoidance, but the underlying A crucial observation made by live imaging in the nematode molecular mechanisms are incompletely defined. Here, we show that Caenorhabditis elegans indicates that dendrite self-avoidance FMI-1/Flamingo, an atypical cadherin, is required autonomously for occurs through contact-dependent dendrite retraction (Smith et al., self-avoidance in the multidendritic PVD neuron of Caenorhabditis 2012). This finding is consistent with the fact that the majority of elegans. The fmi-1 mutant shows increased crossover between sister self-avoidance molecules are cell-membrane proteins and several of PVD dendrites. Our genetic analysis suggests that FMI-1 promotes these display homophilic interactions (Goodman et al., 2016; transient F-actin assembly at the tips of contacting sister dendrites to Rubinstein et al., 2015; Wojtowicz et al., 2007). It is less clear what facilitate their efficient retraction during self-avoidance events, drives subsequent dendrite retraction after the initial transient probably by interacting with WSP-1/N-WASP. Mutations of vang-1, contact between sister dendrite branches. Recently, F-actin which encodes the planar cell polarity protein Vangl2 previously assembly has emerged as a potential cytoskeletal platform that shown to inhibit F-actin assembly, suppress self-avoidance defects of integrates signaling from the plasma membrane to promote self- the fmi-1 mutant. FMI-1 downregulates VANG-1 levels probably avoidance. In C. elegans, UNC-6/Netrin signaling and Wntless through forming protein complexes. Our study identifies molecular engage F-actin assembly to promote dendrite self-avoidance (Liao links between Flamingo and the F-actin cytoskeleton that facilitate et al., 2018; Sundararajan et al., 2019). The Netrin receptor UNC- efficient dendrite self-avoidance. 40/DCC (deleted in colorectal cancer) contains a conserved motif for direct interaction with the WAVE regulatory complex (WRC), KEY WORDS: Actin, C. elegans, Dendrite, Flamingo, Planar polarity, providing a molecular link between Netrin signaling and F-actin Self-avoidance assembly (Chen et al., 2014). Wntless genetically interacts with neural Wiskott–Aldrich syndrome protein (N-WASP), a conserved INTRODUCTION actin regulator (Derivery and Gautreau, 2010), further strengthening Dendrite self-avoidance prevents crossover between sister dendrites the idea that F-actin dynamics play an important role in mediating from the same neuron, maximizing coverage of the sensory territory contact-dependent dendrite repulsion (Liao et al., 2018). of individual dendrites with minimal overlapping (Zipursky and Flamingo is an atypical cadherin that has seven transmembrane Grueber, 2013). In mammals, defects in dendrite self-avoidance are domains, making it structurally reminiscent of a G protein-coupled associated with abnormal gait patterns or impaired directional receptor (GPCR) (Langenhan et al., 2016). In Drosophila, Flamingo sensitivity in vision, suggesting that self-avoidance is essential for promotes the self-avoidance of sister dendrites from the same larval the proper functioning of the neural circuitries (Gibson et al., 2014; class IV dendrite arborization (da) neuron (Matsubara et al., 2011). Kostadinov and Sanes, 2015). Many molecules have been identified Flamingo binds the LIM domain protein Espinas and also genetically as important regulators for dendrite self-avoidance in diverse interacts with the small GTPase RhoA and Van Gogh, molecules that species, including Down’s syndrome cell adhesion molecules govern planar cell polarity (PCP) across epithelial tissues (Matsubara (Hughes et al., 2007; Matthews et al., 2007; Soba et al., 2007), et al., 2011). As RhoA is a well-studied regulator of F-actin dynamics, γ-protocadherins (Lefebvre et al., 2012), integrins (Han et al., 2012; it is tempting to speculate that Flamingo promotes dendrite self- Kim et al., 2012), the atypical cadherin Flamingo and the avoidance by engaging F-actin assembly. In this study, we provide transmembrane polarity protein Van Gogh (Matsubara et al., experimental evidence that FMI-1, the C. elegans Flamingo, 2011), the immunoglobulin superfamily protein Turtle (Long genetically interacts with WSP-1/N-WASP and probably regulates et al., 2009), the secreted proteins Netrin and Slit (Gibson et al., dendrite self-avoidance by orchestrating spatially and temporally 2014; Smith et al., 2012; Sundararajan et al., 2019), the furin-like defined F-actin activity at the dendrite tips. Our data further suggest that FMI-1 antagonizes, rather than collaborates with, VANG-1/Van

Institute of Molecular Medicine and Center of Precision Medicine, College of Gogh in dendrite repulsion. These findings provide insights into the Medicine, National Taiwan University, Taipei 10002, Taiwan. molecular mechanisms by which Flamingo shapes the fine *These authors contributed equally to this work architecture of dendrite arborization. ‡Author for correspondence ([email protected]) RESULTS H.-W.H., 0000-0002-9941-3256; R.-T.S., 0000-0001-8828-4885; C.-L.P., 0000- C. elegans FMI-1/Flamingo regulates dendrite self-avoidance 0003-0108-3138 To understand how dendrite self-avoidance is regulated at the Handling Editor: Susan Strome molecular and cellular level, we focused on PVD, a bilaterally

Received 8 April 2019; Accepted 29 June 2020 symmetric multidendritic nociceptive neuron in C. elegans that has DEVELOPMENT

1 RESEARCH ARTICLE Development (2020) 147, dev179168. doi:10.1242/dev.179168

Fig. 1. fmi-1 regulates dendrite self-avoidance in the C. elegans PVD neuron. (A) Diagram of the dendrite arborization of the C. elegans PVD neuron. The degree values indicate the order of the dendritic branches from the PVD cell body. (B) Schematic of the structure of the FMI-1 protein and fmi-1 mutant alleles used in this study. (C) PVD dendrite morphology as revealed by wdIs52[F49H12.4::GFP]. Red arrowheads, self-avoidance defects; yellow arrowheads, normal self-avoidance between sister 3° dendrites. Details of 3° dendrite morphology in the boxed regions are highlighted to the right. (D) Quantification of dendrite self-avoidance defects. Data are mean±s.e.m. The numbers of PVD scored are indicated. **P<0.01; ***P<0.001, Mann–Whitney U test followed by Bonferroni’s multiple comparison. n.s., not significant. (E) Snapshots of time-lapse imaging during self-avoidance events. Images were taken from dendritic arbors anterior to the PVD soma in the control and the fmi-1 mutant. t=0 indicates the time of dendrite contact. Yellow and red arrowheads mark gaps and sustained contact or continuity between sister tertiary dendrites, respectively. (F) Quantification of the duration of dendrite contact. The numbers at the top of the graph represent the sample size of the dendrite contact events examined. Scale bars: 10 µm (C); 5 µm (E). extensive dendrite arborization (Fig. 1A). PVD elaborates phenotypes (Fig. 1B,D), suggesting that self-avoidance defects in peripheral branches orthogonal to the more proximal dendrites, these mutants are probably caused by loss of fmi-1 gene activity. forming a stereotyped, menorah-like dendrite morphology (Albeg Trans-heterozygotes between fmi-1(rh308) and nDf42, a deficiency et al., 2011; Oren-Suissa et al., 2010; Smith et al., 2010). The chromosome that deletes the entire fmi-1 locus, showed self-avoidance horizontal 3° branches display robust self-avoidance, leaving gaps defects similar to those of the fmi-1(rh308) homozygotes (Fig. 1D), of variable length with minimal contact or crossover between sister suggesting that rh308 is a null allele of fmi-1. Gross PVD dendrite 3° dendrites (Smith et al., 2012). The C. elegans gene fmi-1 encodes morphology of the fmi-1(rh308) mutant was indistinguishable from Flamingo, an atypical cadherin with enormous extracellular that of the control (Fig. S1). As rh308 is a putative null mutation, we domains, seven transmembrane domains and a short cytoplasmic focused the rest of our investigation on this allele. tail (Fig. 1B) (Steimel et al., 2010). The fmi-1(rh308) allele, a Previous studies suggest that growing PVD 3° dendrites briefly nonsense mutation predicted to truncate most of the FMI-1 protein contact each other and promptly retract, typically within 3 to 5 min (Steimel et al., 2010), displayed increased self-avoidance defects after the initial contact (Liao et al., 2018; Smith et al., 2012). To gain compared with those of the control, with 3° dendrites in contact with insight into the cellular basis of self-avoidance defects in the fmi-1 each other and missing gaps between them (Fig. 1B,C). Another mutant, we performed live imaging by spinning-disk confocal nonsense allele, hd121 (Steimel et al., 2010), showed similar microscopy in third-stage larvae (L3) when dendrite self-avoidance DEVELOPMENT

2 RESEARCH ARTICLE Development (2020) 147, dev179168. doi:10.1242/dev.179168 is being established (Smith et al., 2012). Consistent with the development, as revealed by a high-dose translational FMI-1:: published literature, we found that contact of 3° dendrites was GFP reporter that contained the 2615 bp fmi-1 promoter and the resolved by dendrite retraction within 3 min in more than 60% of the genomic fmi-1 sequence injected at a DNA concentration of 30 ng/ contact events, with more than 90% of contact events resolved in µl (twnEx458; Fig. 2A). FMI-1::GFP was enriched in the nerve ring 10 min (Fig. 1E,F, Movie 1). By contrast, in the fmi-1 mutant, 3° and the ventral nerve cord (Fig. 2A), highlighting the role of FMI-1 dendrites remained in contact for 10 min or longer in more than 60% in and synapse development. These findings do not exclude of the events (Fig. 1E,F, Movie 2). As defects in self-avoidance the possibility that fmi-1 expression levels in non-neural tissues scored in late L4 fmi-1 animals are considerably less penetrant than were lower than the detection threshold of our transgene. FMI-1:: dendrites in contact in our live-imaging experiments, it is possible GFP showed membrane enrichment in the cell body of PVD that some contacting events are resolved at later time points beyond (Fig. 2B). Punctate FMI-1::GFP could also be found in the 1° our live-imaging sessions. Possible interference of dendrite dendrites and occasionally in the 2° or 3° dendrites (Fig. 2C). The development might arise from worm manipulation, immobilization low signal-to-noise ratio of FMI-1::GFP in peripheral PVD or phototoxicity during live imaging. Low concentrations of branches precluded verification of possible FMI-1 localization in levamisole and microbeads were used to minimize toxicity possibly the tips of growing 3° dendrites. Even at such marginal signal caused by immobilization, and we tried to reduce phototoxicity by intensity, this transgene caused self-avoidance defects in an using spinning-disk confocal microscopy, which markedly shortened otherwise wild-type genetic background [control, median=2.9% image acquisition time. Taken together, these data suggest that fmi-1 (2.3-7.9%), n=11; twnEx458, median=8.2% (0-21.7%), n=12, regulates self-avoidance by promoting efficient retraction after P<0.05, Mann–Whitney U test]. Another Pfmi-1::FMI-1::GFP transient contact of the 3° dendrites. transgene that was expressed at a lower dosage (10 ng/µl of DNA Consistent with previous reports (Huarcaya Najarro and Ackley, injected; twnEx423) fully rescued self-avoidance defects in the 2013; Steimel et al., 2010), we found that fmi-1 was expressed fmi-1 mutant (Fig. 2D), and it did not cause self-avoidance defects throughout the C. elegans nervous system at all stages of in the control background. Because the 3° dendrites of PVD are not

Fig. 2. fmi-1 expression patterns and rescue of dendrite self-avoidance defects. (A) Epifluorescent photographs of a strain expressing twnEx458[Pfmi-1::FMI-1::GFP] at embryonic (Em), L1 and L3 larval stages. GFP was nearly exclusively observed in the nervous system at larval stages. (B,C) Single optical section confocal image of Pfmi-1::FMI- 1::GFP expression in the PVD soma (B) and confocal projection image of 3° dendrites (C). FMI-1:GFP puncta in 3° dendrites are marked by arrowheads. The dotted line indicates the ventral segment of the PVD axon. The asterisk marks the PVD soma. (D,E) Quantification of dendrite self- avoidance defects. ‘−’ in D indicates progeny from the fmi-1; Pfmi-1::FMI-1:: GFP animals that lose the Pfmi-1::FMI- 1::GFP transgene. These animals serve as controls for their transgenic siblings. Data are mean±s.e.m. The numbers at the top of the graph represent the neuron sample size. *P<0.05, **P<0.01, ***P<0.001; n.s., not significant, Mann–Whitney U test followed by Bonferroni’s correction. Scale bars: 10 µm (Em and L1), 50 µm (L3) (A); 5 µm (B,C). DEVELOPMENT

3 RESEARCH ARTICLE Development (2020) 147, dev179168. doi:10.1242/dev.179168 associated with other neuronal processes (Dong et al., 2013), and By contrast, fmi-1 overexpression from the fmi-1 promoter failed to fmi-1 is specifically expressed in neurons, including PVD, we rescue the wsp-1 mutant. These results indicate that wsp-1 acts cell- speculate that FMI-1 acts autonomously to promote PVD dendrite autonomously in PVD and probably downstream of fmi-1.Wenext self-avoidance. As a definitive test, we expressed the fmi-1a tested how FMI-1 interacts with WSP-1. Diffuse cytosolic signal of transcript in PVD, and confirmed that the dendrite self-avoidance an mCherry::WSP-1 transgene expressed in PVD made it difficult defects of the fmi-1 mutant were completely rescued (Fig. 2E). to conclude whether mCherry::WSP-1 colocalizes with FMI-1:: These data suggest that fmi-1 acts cell-autonomously in PVD to GFP. We were unable to detect WSP-1 protein distribution in PVD, regulate dendrite self-avoidance. possibly due to a low expression level, in a strain in which the endogenous wsp-1 gene was tagged with GFP using the CRISPR/ FMI-1 is required for local F-actin assembly during the Cas9 technique (Zhu et al., 2016). establishment of dendrite self-avoidance Our model suggests that the inhibition of F-actin disassembly Recent reports show that contact and retraction of PVD 3° dendrites might offset the deleterious effects of fmi-1 or wsp-1 mutations on during self-avoidance is associated with local F-actin assembly at F-actin dynamics. The C. elegans gene unc-60 encodes cofilin, a dendrite tips (Liao et al., 2018; Sundararajan et al., 2019). This protein that promotes F-actin disassembly (Ono and Benian, 1998). spatially and temporally defined F-actin activity is significantly An unc-60 mutation suppressed self-avoidance defects in the fmi-1 diminished in mutants that display self-avoidance defects, such as mutant but did not cause dendrite defects in an otherwise control mig-14/Wntless and wsp-1/N-WASP (Liao et al., 2018). Cell- background (Fig. 4B). Taken together, our data indicate that FMI-1 adhesion molecules and surface receptors engage the actin leverages F-actin dynamics to promote dendrite self-avoidance. cytoskeleton to regulate axon branching and synapse formation To further understand whether fmi-1 regulates dendrite self- (Chen et al., 2014; Chia et al., 2014). Therefore, we tested whether avoidance by interacting with known signaling pathways, we made FMI-1 promotes dendrite self-avoidance through F-actin assembly. double mutants that contained fmi-1(rh308) and either the unc-40 or To monitor F-actin activity in live animals, we expressed EGFP- mig-14 mutations. We showed in previous work that mig-14 and tagged LifeAct, a small peptide that binds F-actin and reports unc-40 act independently to regulate dendrite self-avoidance in F-actin dynamics (Riedl et al., 2008) in the PVD neuron, and PVD (Liao et al., 2018). We found that self-avoidance defects were performed time-lapse spinning-disk confocal microscopy. We more severe in the unc-40; fmi-1 and the mig-14; fmi-1 double identified dendrite contacts by quantifying mCherry fluorescent mutants, compared with those of the unc-40, mig-14 and fmi-1 signals that labeled PVD dendrites, as reported previously (Fig. 3A, single mutants (Fig. S3A,B). These observations suggest that fmi-1 Fig. S2; see also Materials and Methods) (Liao et al., 2018). Briefly, acts in a genetic pathway distinct from that of mig-14 and unc-40 to a5μm region centering at the dendrite contact point was defined as regulate dendrite self-avoidance. the contact site, and another 5 μm region along the same 3° dendrite but away from the contact point was selected as the control non- FMI-1 antagonizes the polarity protein VANG-1/Vangl2 to contact site (Fig. 3B). Using this method, we quantified LifeAct:: regulate dendrite self-avoidance EGFP fluorescent signal intensity and confirmed that F-actin Flamingo is a component of the PCP molecular cascade, which activity increased at the contact sites that lasted for the duration of specifies planar tissue polarity in Drosophila and mammals dendrite contact and diminished before dendrite retraction (Fig. 3C,D, (Devenport, 2014). VANG-1, the C. elegans homolog of the PCP Movie 3). No such F-actin activity was observed at the non-contact component Vangl2/Strabismus/Van Gogh, regulates several aspects sites (Fig. 3C,D), suggesting that dendrite self-avoidance is of neural development, including neuronal migration and neurite associated with temporally and spatially defined F-actin dynamics. branching (Chen et al., 2017; He et al., 2018; Mentink et al., 2014; In the fmi-1 mutant, F-actin assembly at the contact sites during self- Sanchez-Alvarez et al., 2011). We recently showed that VANG-1 avoidance events was markedly decreased, whereas F-actin dynamics acts in the Wnt- pathway to specify neurite branching sites at non-contact sites were comparable with those in the control in the PLM mechanosensory neuron by restricting F-actin dynamics (Fig. 3A,C,D, Movie 4). These observations indicate that FMI-1 (Chen et al., 2017). In the vang-1 mutant, F-actin assembly regulates dendrite self-avoidance by promoting F-actin assembly at increased and was distributed to ectopic sites. This observation dendrite tips that coincides with the self-avoidance event. raises the intriguing possibility that vang-1 mutations might suppress dendrite self-avoidance defects of the fmi-1 mutant by FMI-1 promotes F-actin dynamics through WSP-1/N-WASP upregulating F-actin assembly. To explore how FMI-1 regulates F-actin dynamics, we next tested To test this hypothesis, we examined two vang-1 mutants wsp-1, which encodes the C. elegans homolog of N-WASP known (Fig. 5A,B). In addition to the commonly used tm1422 deletion as a crucial regulator of F-actin assembly (Derivery and Gautreau, allele, we generated a new vang-1 allele, twn2, using CRISPR/Cas9 2010). We previously showed that wsp-1 promotes PVD dendrite gene editing. twn2 contains a premature stop codon predicted to self-avoidance probably through facilitating local F-actin assembly truncate the majority of the VANG-1 protein and thus represents a at dendrite tips (Liao et al., 2018). gm324 is a deletion allele that putative null mutation (Fig. 5A). Dendrite self-avoidance in the produces no detectable wsp-1 transcripts or WSP-1 protein, and is vang-1(tm1422) and vang-1(twn2) mutants was indistinguishable thus a putative null wsp-1 allele (Withee et al., 2004). Defects of from the control (Fig. 5B). Interestingly, both vang-1 mutations self-avoidance were comparable in the fmi-1 and wsp-1 single significantly suppressed self-avoidance defects of the fmi-1 mutant mutants and were not further increased in the wsp-1; fmi-1 double (Fig. 5B). Expression of vang-1 specifically in PVD of the fmi-1; mutant, suggesting that these two genes act in a common pathway vang-1 double mutant restored self-avoidance defects to the level (Fig. 4A). PVD-specific expression of wsp-1 rescued self-avoidance observed in the fmi-1 mutant, indicating that vang-1 acts cell- defects in the wsp-1 and fmi-1 mutants (Fig. 4A), and it did not autonomously (Fig. 5C). Of note, PVD-specific vang-1 expression cause self-avoidance defects in the wild-type background [control, did not cause self-avoidance defects in the wild-type background median=2.9% (2.3-7.9%), n=11; Pser-2.3::mCherry::WSP-1, [control, median=2.9% (2.3-7.9%), n=11; Pser-2.3::mCherry:: median=5.15% (0-9.7%), n=12, P=0.51, Mann–Whitney U test]. VANG-1, median=2.65% (0-7.3%), n=14, P=0.23, Mann– DEVELOPMENT

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Fig. 3. fmi-1 regulates F-actin dynamics at dendrite tips. (A) Time-lapse imaging of F-actin assembly during self-avoidance events in the control and fmi-1 mutants. F-actin and PVD dendrite signals are represented by LifeAct::EGFP and mCherry, respectively. Imaging speed was 30 s/frame. t=0 represents the moment when visually defined contact between sister tertiary dendrites, marked by arrowheads, was first observed. For dendrite contact events in the fmi-1 mutant shown here, no resolution of dendrite contact was observed during the imaging experiment. (B) Schematic of dendrite contact and non-contact sites. (C) Quantification of LifeAct::EGFP signals as a percentage change from the baseline (1 min before visually defined dendrite contact) at the contact or non-contact sites. t=0 represents visually specified dendrite contact. Data are mean±s.e.m. *P<0.05, **P<0.01, two-tailed, unpaired multiple t-test. (D) Heat map representations of percentage change of F-actin signal for individual self- avoidance events at the contact or non- contact sites. Individual LifeAct::EGFP signal stripes are aligned at the dendrite contact time point (time zero, arrows, dotted lines). The vertical bars (asterisks) in individual events indicate dendrite separation, distinguishing the last image frame of dendrite contact (left of the bar) from the first image frame of dendrite retraction (right of the bar).

Whitney U test]. We failed to detect vang-1 expression in PVD subcellular localization of VANG-1 by expressing VANG-1 tagged using the transgene syIs202, which contains 3 kb of the vang-1 with GFP or mCherry at the N-terminus in PVD. Punctate GFP:: promoter and part of the vang-1-coding sequence to drive YFP VANG-1 or mCherry::VANG-1 signals were found in PVD soma expression (Green et al., 2008). However, PVD-specific and infrequently in dendrites (Fig. 5D-F). We found that some transcriptional profiling by mRNA tagging and sequencing had GFP::VANG-1 puncta in peripheral PVD dendrites showed detected vang-1 expression in PVD (Smith et al., 2013). colocalization with mCherry-fused COR-1/coronin, an F-actin- We hypothesize that fmi-1 antagonizes vang-1 to ensure efficient binding protein (Fig. 5E), although we were not able to identify dendrite self-avoidance. To test this model, we first investigated the VANG-1 signals at the tips of growing 3° dendrites with confidence. DEVELOPMENT

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Fig. 4. fmi-1 regulates dendrite self-avoidance through F-actin cytoskeleton. (A,B) Quantification of dendrite self-avoidance defects in strains containing the wsp-1 (A) or unc-60 (B) mutations. The numbers at the top of the graphs in A and B represent the neuron sample size. Data are mean±s.e.m. ***P<0.001; n.s., not significant, Mann–Whitney U test followed by Bonferroni’s correction.

In the fmi-1 mutant, signal intensity of mCherry::VANG-1 in the navigated anteriorly to terminate at or beyond the vulval region PVD soma was significantly upregulated (Fig. 5F,G). The (Fig. 6A,B). In the fmi-1 mutant, PVD displayed either frequency of mCherry::VANG-1 puncta in peripheral PVD premature truncation or posterior misrouting, or both, in more than dendrites was also dramatically increased in the fmi-1 mutant 90% of the animals (Fig. 6A,B, Table 1). Genomic fmi-1 sequences compared with the control background (Fig. 5H,I). Overexpression expressed from the fmi-1 promoter significantly rescued PVD axon of FMI-1 did not further decrease mCherry::VANG-1 level or defects when expression in PVD could be confirmed with a cell- frequency, suggesting that the VANG-1 level in PVD was minimal specific marker, but not in mosaic transgenic animals that lost fmi-1 in the control (Fig. 5F-I). Using a transgenic C. elegans strain that expression from PVD (Fig. 6C, Table 1). These observations expresses FMI-1::GFP and HA::VANG-1 broadly in the nervous indicate that fmi-1 activity in PVD is necessary to facilitate PVD system (from the fmi-1 and rgef-1 promoter, respectively), we axon navigation in the VNC, but do not rule out the possibility that showed that FMI-1 co-immunoprecipitated VANG-1 (Fig. 5J, Fig. non-autonomous fmi-1 activity is also required. In contrast to its S4). Taking these data together, we conclude that FMI-1 forms rescue of self-avoidance defects, PVD-specific expression of fmi-1a protein complexes with VANG-1 in C. elegans neurons, and it failed to rescue axon phenotypes. As the PVD axons fasciculate decreases VANG-1 levels in PVD. with other VNC axons, this result suggests that fmi-1 needs to be present in both PVD and its fasciculating partners for anterior FMI-1 regulates PVD axon extension both autonomously and projection, although it might also be possible that other fmi-1 non-autonomously isoforms are necessary for complete functional complement of fmi-1 In addition to dendrite development, FMI-1 is important for the loss. extension and guidance of certain axons in the C. elegans ventral We noted that some fmi-1 animals still displayed PVD axon nerve cord (VNC) that require pioneer axons (Steimel et al., 2010). defects even with the Pfmi-1::FMI-1::GFP transgene expressed in The AVG axon is the first nerve process that establishes the right PVD (Fig. 6C, Table 1). This raises the possibility that fmi-1 bundle of the embryonic VNC (Durbin, 1987). The PVP axons expression outside PVD is also important for PVD axon navigation. subsequently join the embryonic VNC and serve as pioneers or PVD axons form chemical synapses with several command scaffolds for the following PVQ axons, which enter the embryonic interneurons that express the GLR-1 , including VNC later (Durbin, 1987; Wadsworth and Hedgecock, 1996; White AVA, AVB and AVD (Goodman, 2006), which also express fmi-1. et al., 1986). Previous studies show that the navigation of follower fmi-1 might function in one or several of these neurons and signal in PVQ axons requires both cell-autonomous and non-autonomous a non-autonomous fashion to regulate PVD axon guidance. To test (from PVP pioneers) fmi-1 activities (Steimel et al., 2010). this, we achieved cell-specific fmi-1 RNAi by expressing a fmi-1 Although axons of the HSN motor neurons, which join the VNC RNA duplex in glr-1(+) neurons (Fig. S5A,B). Pglr-1::fmi- at L3, show navigation and guidance errors in the fmi-1 mutant, the 1(RNAi) significantly reduced the intensity of FMI-1::GFP in the site of action for fmi-1 in postembryonic VNC axon navigation nerve ring, which contains axons of AVA, AVB and AVD, remains incompletely understood. The axon of PVD extends and suggesting that the Pglr-1::fmi-1(RNAi) transgene effectively enters the VNC at late L2 to L3 stages, making it a good model for decreases fmi-1 expression in these neurons (Fig. S5B,C). studying the autonomous requirement of fmi-1 in postembryonic Dimmer signal intensity and susceptibility to photobleaching axon development. precluded the use of FMI-1::GFP signals in the VNC The axons of left and right PVDs fasciculate after entering the for evaluating the efficiency of fmi-1 RNAi. Silencing fmi-1 in right bundle of the VNC (Fig. 6A,B). In the control, PVD axons glr-1(+) interneurons triggered PVD axon navigation defects in DEVELOPMENT

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Fig. 5. See next page for legend.

∼20% of the wild-type animals (Fig. 6D, Table 1), implying DISCUSSION that, in addition to its major, cell-autonomous function, fmi-1 In this study, we present evidence indicating that the C. elegans can also regulate PVD axon development in a non-autonomous Flamingo FMI-1 promotes dendrite self-avoidance by orchestrating manner. spatially and temporally defined F-actin assembly at the dendrite DEVELOPMENT

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Fig. 5. fmi-1 regulates dendrite self-avoidance by antagonizing vang-1. hexapeptide motif in the cytosolic tail of cell-membrane receptors (A) Schematic of the vang-1(twn2) mutation generated by CRISPR/Cas9 gene that directly bind WRC (Chen et al., 2014). We therefore suspect editing. Boxes and lines represent exons and introns, respectively. that the interaction between FMI-1 and the actin regulatory (B,C) Quantification of dendrite self-avoidance defects. Self-avoidance defects of vang-1(tm1422) and vang-1(twn2) were similar and were not significantly machinery is indirect. The identification of FMI-1-interacting different from the control (P>0.05). The vang-1(tm1422) allele was used in proteins will shed light on the molecular link between Flamingo, C. The numbers at the top of the graph represent the neuron sample size. N-WASP, WRC, Arp2/3 and other actin regulators. (D) Confocal projection images of mCherry:VANG-1 (arrowheads) in the 3° PVD dendrites. (E) Fluorescent confocal projection images of the PVD Antagonism between Flamingo and Van Gogh in dendrite dendrites expressing Pser-2.3::GFP::VANG-1 and Pser-2.3::COR-1:: self-avoidance mCherry, which label F-actin. Arrowheads indicate colocalization of the GFP and mCherry signals. (F) Representative confocal images of mCherry::VANG- The core PCP component Van Gogh acts with Flamingo in a number 1 in the PVD soma under different FMI-1 levels. FMI-1(+++) indicates Pfmi-1:: of neurodevelopmental processes (Tissir and Goffinet, 2013). The FMI-1::GFP expression. (G) Quantification of mCherry::VANG-1 intensity in antagonism between VANG-1/Van Gogh and FMI-1 during dendrite the PVD soma. The numbers at the top of the graph represent the neuron self-avoidance was thus unexpected and highlighted the complex sample size. (H) Confocal projection images of mCherry::VANG-1 in PVD interaction between different PCP molecules. FMI-1 forms protein dendrites and soma outlined by dotted lines. Arrow indicates mCherry::VANG- complexes with VANG-1 and lowers the level of VANG-1 in neurons, 1 puncta in the dendrites. (I) Percentage of animals with detectable mCherry:: which raises the possibility that the retention of VANG-1 by FMI-1 in VANG-1 signals in PVD dendrites under different FMI-1 levels. The numbers in each bar represent the PVD neuron sample size. (J) Co-immunoprecipitation the protein complex facilitates VANG-1 turnover. Our previous work of FMI-1::GFP and HA::VANG-1 from the lysate of C. elegans expressing on neurite branching revealed that VANG-1 restricts F-actin assembly Pfmi-1::FMI-1::GFP and Prgef-1::HA::VANG-1. N2, and worms expressing in the C. elegans PLM mechanosensory neuron (Chen et al., 2017). either Pfmi-1::FMI-1::GFP or Prgef-1::HA::VANG-1 served as controls. Consistent with this, we find that vang-1 mutations suppressed self- Data are mean±s.e.m. *P<0.05, **P<0.01, ***P<0.001, n.s., not significant, avoidance defects of the fmi-1 mutant, probably through restoring F- – χ2 ’ Mann Whitney U test (B,C,G) or test (I) followed by Bonferroni s correction. actin formation in the dendrites. It remains unclear how VANG-1 Scale bars: 10 μm (D,E); 5 μm (F,H). inhibits F-actin assembly, although some VANG-1 proteins appear to colocalize with F-actin in PVD neurons. A recent study in the rat testis tips. This intricate regulation of F-actin dynamics by FMI-1 is found that Vangl2, the mammalian Van Gogh, bound actin and probably mediated through two molecules (Fig. 7A). First, FMI-1 modulated F-actin configuration (Chen et al., 2016). Loss or facilitates F-actin assembly by genetically interacting with N- overexpression of Vangl2 changed F-actin organization and WASP, which is well-known for promoting F-actin polymerization subcellular distribution in Sertoli cells, with knockdown of Vangl2 through Arp2/3 (Derivery and Gautreau, 2010). Second, FMI-1 enhancing F-actin and N-cadherin levels. The elucidation of the antagonizes VANG-1/Van Gogh, which has recently been found to protein structure of Van Gogh, which remains largely unexplored, is restrict F-actin assembly in C. elegans neurites (Chen et al., 2017). necessary to understand how Van Gogh modulates F-actin These two molecules might act in similar or distinct genetic organization, as well as how Flamingo antagonizes Van Gogh. pathways to ensure precise F-actin assembly at the tips of contacting sister dendrites for efficient retraction. Together with recent studies F-actin assembly and dendrite retraction on the effects of MIG-14/Wntless and Netrin signaling in PVD Previous studies using LifeAct::EGFP as a reporter for F-actin dendrite morphogenesis, our work reinforces the notion that F-actin dynamics suggest that F-actin assembly at dendrite tips increases is a central cytoskeletal component for dendrite self-avoidance in around the time of contact and retraction of sister dendrites in PVD C. elegans. Validation in other systems, such as insects and (Liao et al., 2018; Sundararajan et al., 2019). The spatial resolution mammals, is important to establish this as a conserved mechanism of time-lapse confocal microscopy does not allow us to distinguish that sculpts non-overlapping dendrite morphology. whether F-actin assembly occurs after physical contact of dendritic membrane, or whether it occurs when sister dendrites are close but FMI-1 is required for contact-dependent dendrite retraction still separate. Dendrite extension occurs normally in the fmi-1 and Cadherins play important roles in different aspects of neural wsp-1 mutants but their retraction is impaired, together with development, such as neurite extension, axon fasciculation and diminished F-actin assembly. These observations imply that F-actin synapse formation (Takeichi, 2007). The role of Flamingo, an assembly is required for dendrite repulsion, a notion further atypical cadherin, in dendrite self-avoidance was first discovered in supported by the observation that unc-60/cofilin mutations suppress Drosophila (Matsubara et al., 2011). Given its multiple extracellular dendrite self-avoidance defects. How does F-actin assembly drive domains characteristic of an adhesion molecule, such as cadherin dendrite retraction? Semaphorin 3A has been shown to collapse the repeats and laminin G domains, it was somewhat unexpected that axon growth cone and induce axon retraction in cultured chicken the absence of Flamingo activity resulted in ectopic dendrite dorsal root ganglion neurons in an F-actin-dependent manner (Gallo, contact. Matsubara et al. (2011) showed that Flamingo genetically 2006). The addition of semaphorin 3A diminishes F-actin in the interacts with the LIM-domain protein Espinas through the growth cone but increases F-actin assembly in an axonal segment juxtamembrane domain A (JM-A). LIM domain proteins, such as behind the growth cone. Inhibition of RhoA or Rho-dependent kinase C. elegans UNC-115, regulate cytoskeletal elements, including (ROCK) decreases intra-axonal F-actin and significantly suppresses actins (Gitai et al., 2003; Smith et al., 2014). We performed amino semaphorin 3A-induced axon retraction. It is thus hypothesized that acid sequence analysis of FMI-1 and Drosophila Flamingo, but we intra-axonal F-actin drives axon retraction in the presence of repulsive did not find FMI-1 sequences homologous to the Drosophila signals, probably through engaging myosin II (Gallo, 2006). Flamingo JM-A. Flamingo also interacts with RhoA, a well- Interestingly, a recent study found that mutations in the C. elegans established F-actin regulator. These observations are consistent with gene nmy-1, which encodes non-muscle myosin II, resulted in PVD our findings that F-actin assembly mediates the effects of Flamingo dendrite self-avoidance defects (Sundararajan et al., 2019). One on dendrite self-avoidance. FMI-1 lacks the conserved WIRS possibility is that the activation of membrane receptors, such as

(WAVE regulatory complex interacting receptor sequence), a Flamingo, Wntless or DCC, leads to a redistribution of F-actin DEVELOPMENT

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Fig. 6. fmi-1 regulates axon development of the PVD neuron. (A) Schematics of PVD axon defects in the fmi-1 mutant. (B) Representative epifluorescent images of PVD axon projection in the control and the fmi-1 mutant. Yellow and white arrowheads mark the anteriorly and posteriorly directed PVD axons, respectively. The vulva is indicated by dotted lines. Anterior is to the left. (C,D) Quantification of PVD axon defects. (PVD−) indicates mosaic transgenic fmi- 1(rh308); Pfmi-1::FMI-1::GFP animals that do not have FMI-1::GFP expression in PVD. The numbers in each bar represent the PVD neuron sample size. *P<0.05, ***P<0.001, Fisher’s exact test with Bonferroni’s correction. Scale bar: 20 µm. assembly towards dendrite tips. Retrograde actin flow driven by self-avoidance defects in the fmi-1 mutant suggest that other myosin II, along with enhanced membrane retrieval, leads to axon pathways act in parallel to ensure the robustness of dendrite retraction (Gallo, 2006; Yang et al., 2012). The termination of repulsion. By contrast, most fmi-1 mutant animals show PVD axon receptor activation following dendrite separation from the initial guidance defects, implicating FMI-1 as an essential factor in PVD contact decreases F-actin assembly and stops dendrite repulsion, axon development. FMI-1 is required for the development of which might explain the transient nature of dendrite retraction during multiple C. elegans neuronal classes whose axons travel in the self-avoidance. F-actin polymerization may also facilitate membrane VNC, including PVP, PVQ, HSN and glr-1(+) command retrieval at distal dendrites, which contributes to dendrite retraction. interneurons (Steimel et al., 2010). The axon phenotypes are We speculate that F-actin dynamics orchestrate concerted cytoskeletal complex in the fmi-1 mutant and seem to depend on the neuronal shortening and retrieval of the dendrite membrane, resolving sister type. Axons of the left and right PVP travel in the right and left dendrites in transient contact during self-avoidance. However, spatial fascicles of the VNC, respectively. Axons of the left and right and temporal resolution of fluorescent imaging in this study was not PVQ use the PVP axons as a guidepost and follow them in the sufficient for documenting fine F-actin features in the dynamic VNC. In the absence of fmi-1, the pioneer PVPR axon often dendritic branches during self-avoidance. Super-resolution defasciculates from the left VNC fascicle and crosses to the microscopy that offers temporal resolution at the time scale of a contralateral right VNC fascicle. As a result, the follower PVQ hundred milliseconds, and an F-actin reporter with improved signal- axons display penetrant crossover defects and join inappropriate to-noise ratio, should help to clarify the correlation between F-actin VNC fascicles in the fmi-1 mutant. fmi-1 expression in the pioneer dynamics and the distinct steps in dendrite self-avoidance. PVP axons rescues crossover defects in both PVP and the follower PVQ, whereas fmi-1 expression in PVQ rescues defects of the Autonomous and non-autonomous FMI-1 functions in axon PVQ but not those of the pioneer PVP axons. These observations guidance suggest that fmi-1 controls PVQ axon development both cell- In addition to dendrite self-avoidance, we found that FMI-1 is also autonomously and non-autonomously. By contrast, premature required for navigation of the PVD axon in the VNC. The modest truncation of PVQ axons in the fmi-1 mutant could only be

Table 1. Classification of PVD axon defects Genotypes Bifurcation Premature truncation Posterior misrouting Bifurcation+truncation Defasciculation N Control 0 0 0 0 0 22 fmi-1(rh308) 2.9 35.3 29.4 26.5 0 34 fmi-1(rh308)Pser2.3::fmi-1a 16.7 16.7 16.7 38.9 0 18 fmi-1(rh308)Pfmi-1::fmi-1 (+++) 0 0 8.3 8.3 4.2 24 fmi-1(rh308)Pfmi-1::fmi-1 (−) 0 34.4 34.4 25 0 32 fmi-1(rh308)Pfmi-1::fmi-1 (PVD−) 6.3 31.3 25 25 0 16 Pglr-1::fmi-1 RNAi 13.3 3.3 0 3.3 0 30 DEVELOPMENT

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Fig. 7. Model of the FMI-1 pathways that regulate PVD development. (A) FMI-1 engages the F-actin cytoskeleton and VANG-1 to regulate dendrite self- avoidance. (B) Cell-autonomous and non-autonomous functions of FMI-1 to control PVD axon navigation. rescued when fmi-1 is expressed in PVQ, suggesting a cell- Plasmid construction and germline transformation autonomous requirement of fmi-1 in axon extension. Analysis of Constructs used in this study were generated by standard molecular fmi-1 mutant animals mosaic for PVD-specific fmi-1 rescue, using biological techniques. Constructs used to generate the twnEx series of the Pfmi-1::FMI-1::GFP transgene, suggests that fmi-1 activity in transgenes were in the pPD95.77 vector, except for Pfmi-1::FMI-1::GFP, PVD is essential for axon development. However, expression of which was kindly provided by Harald Hutter (Simon Fraser University, Burnaby, Canada). The fmi-1a cDNA was a generous gift from Georgia fmi-1a in PVD fails to rescue axon defects in the fmi-1 Rapti and Shai Shaham (The Rockefeller University, New York, NY, USA). mutant. Moreover, reducing fmi-1 activity in glr-1(+) command Germline transformation by microinjection was performed as described interneurons, with which PVD axons make synapses, results in previously (Mello et al., 1991). defective PVD axon navigation. These data are also consistent with the model that FMI-1 acts both autonomously and non- CRISPR/Cas9 genome editing autonomously to control PVD axon development. It is also CRISPR/Cas9 mutagenesis was performed by germline transformation possible that more than one fmi-1 isoform is necessary to fully using pDD162(Peft-3::Cas9, Addgene #47549) inserted with vang-1 rescue the axon defects. Crossover defects in axons of command sgRNA (GACACGAGGAGTTGCGTT). An unc-22 sgRNA construct interneurons are found in 10-20% of the fmi-1 mutant animals was co-injected as a co-CRISPR marker to select F1 animals whose (Steimel et al., 2010), implying that PVD axon defects in some fmi-1 genomes were successfully edited at the unc-22 locus to indicate possible animals are probably the consequence of disruption of the editing at the vang-1 locus. Confirmation of editing of vang-1 was also development of interneuron axons. This also raises the possibility sought by performing T7-endonuclease I digestion of the PCR products of target vang-1 sequence from F1 (Mashal et al., 1995; Shen et al., 2014), and of heterophilic FMI-1 interaction with other membrane-tethered or was verified by DNA sequencing. diffusible ligands. Candidates include molecules with cadherin repeats, EGF or laminin domains. The identification of FMI-1 Scoring of PVD axon projection defects ligands will advance our understanding of how Flamingo signaling PVD axon defects were scored with the transgene wdIs52, which expresses controls axon extension and navigation to sculpt the connectivity of soluble GFP from the regulatory sequence of the C. elegans gene F49H12.4 neuronal circuitries. in PVD, AQR and a cell in the tail (Smith et al., 2010). For characterizing PVD axon projection, L4 animals were collected and immobilized by 1% MATERIALS AND METHODS sodium azide and were manually oriented with the ventral side up. PVD C. elegans strains axons were imaged using the 40× objective of the Zeiss AxioImager M2 All strains were cultured and maintained as described previously (Brenner, system. Axons that projected anteriorly beyond the vulva were defined as 1974). The alleles and integrated transgenes used in this study were: LG I, unc- ‘normal’. Axons that projected both anteriorly and posteriorly were defined 40(n324); LG II, mig-14(ga62); LG III, unc-119(ed3); LG IV, wsp-1(gm324), as ‘axon bifurcation’. Axons that projected anteriorly but failed to reach the cas723[gfp::wsp-1a knock-in]; LG V, fmi-1(rh308), fmi-1(hd121), nDf42, midpoint between the vulva and the PVD soma were classified as ‘premature unc-60(su158), syIs202(Pvang-1::YFP, Pmyo-2::DsRed); LGX, vang- truncation’. Axons that projected posteriorly were classified as ‘posterior 1(tm1422), vang-1(twn2), wdIs52[F49H12.4::GFP, unc-119(+)]. misrouting’. Unbundling of the left and right PVD axons was defined as Extrachromosomal arrays used were: twnEx382[Pser-2.3::LifeAct::EGFP, ‘defasciculation’. Pser-2.3::mCherry, Pgcy-8::GFP]; twnEx422[Pfmi-1::FMI-1::GFP (30 ng/ μl), Pser-2.3::mCherry, Pgcy-8::mCherry]; twnEx423[Pfmi-1::FMI-1::GFP Quantification of dendrite self-avoidance defects (10 ng/μl), Pser-2.3::mCherry, Pgcy-8::mCherry]; twnEx444[Pegl-17:: Quantification of PVD dendrite self-avoidance was described previously GFP::VANG-1, Pser-2.3::mCherry, Pgcy-8::mCherry]; twnEx445[Pser-2.3:: (Liao et al., 2018; Smith et al., 2010). Briefly, the number of estimated gaps mCherry::WSP-1, Pttx-3::GFP]: twnEx446[Pglr-1::fmi-1(RNAi: sense/anti- (G) in the entire PVD dendritic arbor was defined as sense), Pglr-1::mCherry, Pgcy-8::mCherry]; twnEx450[Pser-2.3::mCherry:: VANG-1, Pgcy-8::mCherry]; twnEx451[Pfmi-1::FMI-1::GFP, Pser-2.3:: ¼ð Þþð Þ mCherry::VANG-1, Pgcy-8::mCherry]; twnEx452[Pser-2.3::GFP::VANG-1, G K 1 N 1 , Pser-2.3::COR-1::mCherry, Pgcy-8::mCherry]; twnEx458[Pfmi-1::FMI-1:: in which K and N are the numbers of dorsal and ventral secondary branches, GFP (30 ng/μl), Pgcy-8::mCherry] (‘high-dose FMI-1::GFP’); twnEx483 respectively. The percentage of PVD dendrite self-avoidance defect was [Pglr-1::mCherry]; twnEx488[Prgef-1::HA::VANG-1, Pfmi-1::FMI-1:: defined as: GFP(30 ng/μl), unc-119(+)]; twnEx571[Pser-2.3::FMI-1a::gfp, Pgcy-8:: mCherry];andtwnEx572[Prgef-1::HA::VANG-1]. Self-avoidance defectð% Þ¼Mðmissing gapsÞ=G 100% : DEVELOPMENT

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Time-lapse imaging AffiniPure goat anti-rabbit IgG (1:5000, Jackson ImmunoResearch, 111- For time-lapse imaging of dendrite self-avoidance, laid embryos were 035-003) and HRP goat anti-mouse IgG (1:5000, BioLegend, 405306). See cultivated at 20°C for ∼30 h to obtain L3 larvae. Animals were immobilized supplementary Materials and Methods for further details regarding antibody in 2 μl of 1 mM levamisole with 2 μl of polystyrene beads (0.1 μm, validation. Polysciences) on 10% agar pads. Images were taken using the 40× objective of the Carl Zeiss Cell Observer SD equipped with a Yokogawa CSU-X1 Acknowledgements spinning disk and EMCCD Qimaging Rolera EM-C2 at 2 frames/min for at We thank Gian Garriga, Harald Hutter, Georgia Rapti, Shai Shaham and the least 30 min per animal. For F-actin imaging during dendrite contact, L3 C. elegans Genetics Center (CGC) for the provision of worm strains and plasmids. larvae expressing LifeAct::EGFP were collected and immobilized in 1 mM We thank Chun-Hao Chen for the vang-1(twn2) allele; Chun-Wei He and Ya-Wen Liu levamisole and polystyrene beads (as described earlier) on 10% agar pads. for advice on biochemistry; and Hwa-Man Hsu for assistance with confocal imaging experiments. We thank Chun-Wei He for comments on the manuscript. CGC is The imaging apparatus and condition were the same as those for time-lapse funded by the National Institutes of Health Office of Research Infrastructure imaging of dendrite self-avoidance. Programs (P40 OD010440).

Fluorescence confocal microscopy Competing interests For analyzing dendrite self-avoidance defects and protein localization, L4 The authors declare no competing or financial interests. hermaphrodites were anesthetized in 1% sodium azide and mounted on 5% agar pads. To assess fmi-1 RNAi knockdown efficiency, FMI::GFP intensity Author contributions in the nerve ring of L2 hermaphrodites was quantified. C. elegans protein Conceptualization: H.-W.H., C.-P.L., Y.-C.C., C.-L.P.; Methodology: H.-W.H., C.-P.L., Y.-C.C., R.-T.S., C.-L.P.; Validation: H.-W.H., C.-L.P.; Formal analysis: expression pattern and neuronal morphology were imaged using a Zeiss H.-W.H., C.-P.L., Y.-C.C., R.-T.S., C.-L.P.; Investigation: H.-W.H., C.-P.L., Y.-C.C., LSM700 or LSM880 Airyscan Imaging system. R.-T.S., C.-L.P.; Data curation: C.-L.P.; Writing - original draft: H.-W.H., C.-L.P.; Writing - review & editing: C.-L.P.; Supervision: C.-L.P.; Project administration: Quantification of fluorescent signal intensity C.-L.P.; Funding acquisition: C.-L.P. To evaluate fmi-1 RNAi knockdown efficiency, z-stack projection images of the nerve ring were acquired using a Zeiss LSM700 Confocal Imaging Funding System and FMI-1::GFP signals were quantified using ImageJ. For This study was supported by the Center of Precision Medicine from The Featured μ Areas Research Center Program within the framework of the Higher Education analyzing LifeAct::EGFP signals in PVD dendrites, two 5 m regions Sprout Project administered by the Ministry of Education (NTU-109L901402A to (contact site and non-contact site) were selected and quantified using ZEN C.-L.P.) and the Ministry of Science and Technology, Taiwan (MOST 106-2320-B-002- software (2011 blue edition). For analyzing mCherry::VANG-1 signals in 051-MY3, MOST 108-3017-F-002-004 and MOST 109-2634-F-002-043 to C.-L.P.). PVD soma, single optical sections of a z-stack series, obtained using a Zeiss LSM700 Confocal Imaging System, were quantified using ImageJ and then Supplementary information summed to derive mean signal intensity, which is total mCherry pixel Supplementary information available online at intensity divided by the total area of the PVD soma. To quantify mCherry:: https://dev.biologists.org/lookup/doi/10.1242/dev.179168.supplemental VANG-1 puncta in PVD dendrites, z-stack projection images were obtained using a Zeiss LSM700 Confocal Imaging System. Each single mCherry:: References VANG-1 punctum in PVD dendrites was first quantified using Zen software Albeg, A., Smith, C. J., Chatzigeorgiou, M., Feitelson, D. G., Hall, D. H., Schafer, (2010 black edition). We defined discrete mCherry::VANG-1 puncta with W. R., Miller, D. M., III and Treinin, M. (2011). 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