RESEARCH ARTICLE

JNK signaling coordinates with ecdysone signaling to promotepruning of Drosophila sensory neuron dendritesSijun Zhu1,2,*, Rui Chen1, Peter Soba2,3 and Yuh-Nung Jan2

ABSTRACTDevelopmental pruning of axons and dendrites is crucial for theformation of precise neuronal connections, but the mechanismsunderlying developmental pruning are not fully understood. Here, wehave investigated the function of JNKsignaling indendrite pruningusingDrosophila class IV dendritic arborization (c4da) neurons as a model.We find that lossof JNKor its canonical downstreameffectorsJunorFosled to dendrite-pruning defects in c4da neurons. Interestingly, our datashow that JNK activity in c4da neurons remains constant from larval topupal stages but the expression of Fos is specifically activated byecdysone receptor B1 (EcRB1) at early pupal stages, suggesting thatecdysone signaling provides temporal control of the regulation ofdendrite pruning by JNK signaling. Thus, our work not only identifies anovel pathway involved in dendrite pruning and a new downstreamtarget of EcRB1 in c4da neurons, but also reveals that JNK andEcdysone signaling coordinate to promote dendrite pruning.

KEY WORDS: Wnt5, JNK, Ecdysone, Dendrite pruning, Drosophila,Sensory neurons

INTRODUCTIONDuring the development of the nervous system, neurons often formexuberant axon branches and dendritic arbors and then selectivelyprune those that make incorrect connections. One major form ofdevelopmental pruning is stereotyped large-scale pruning, in which asignificant proportion of specific primary axons/dendrites and theirbranches are eliminated. Such large-scale pruning happens widelyduring the development of the nervous system, such as thedevelopment of subcortical projections and formation of retinotopicmaps, and is crucial for the formation of precise neuronal connections(Luo and O’Leary, 2005). However, molecular mechanisms thatcontrol large-scale pruning are not fully understood.In Drosophila, large-scale axon/dendrite pruning has been well

characterized in mushroom body (MB) γ neurons in the brain andclass IV dendritic arborization (c4da) sensory neurons in theperipheral nervous system (Yu and Schuldiner, 2014; Kanamoriet al., 2015). These neurons undergo drastic remodeling duringmetamorphosis. MB γ neurons prune both their larval axonsand dendrites, and then re-grow their axons and dendrites inadult-specific patterns (Lee et al., 1999), whereas c4da neurons onlyprune and re-grow their dendrites (Kuo et al., 2005). Studies in these

two types of neuron have begun to reveal cellular processes andmolecular mechanisms underlying axon/dendrite pruning. At thecellular level, both axon and dendrite pruning follow a similar seriesof cellular events, including breakdown of the microtubule and actincytoskeleton, blebbing and fragmentation of axons/dendrites, andclearance of axon/dendrite fragments by glial or epidermal cellsthrough phagocytosis (Watts et al., 2003; Lee et al., 2009; Awasakiet al., 2006; Han et al., 2014; Williams and Truman, 2005). Recentstudies also reveal that pruning of MB axons and da neurondendrites involves endocytosis (Zhang et al., 2014; Issman-Zecharya and Schuldiner, 2014) and a transient increase incalcium influx (Kanamori et al., 2013). At the molecular level,both axon and dendrite pruning require ecdysone receptor B1(EcRB1) and its co-receptor Ultraspiracle (Usp) (Lee et al., 2000;Kuo et al., 2005). In the MB, TGF-β signaling together with theimmunoglobulin protein Plum activates the expression of EcRB1 atearly pupal stages (Zheng et al., 2003; Yu et al., 2013). EcRB1promotes axon and dendrite pruning in part by activating Sox14(Kirilly et al., 2009). In addition, the ubiquitin-proteosome systemand caspases play crucial roles in both axon and dendrite pruning,and a few ubiquitylating enzymes and components of theproteasome involved in axon and/or dendrite pruning have beenidentified (Kuo et al., 2005; Watts et al., 2003; Kuo et al., 2006;Rumpf et al., 2011; Williams et al., 2006; Wong et al., 2013).Besides their shared mechanisms, MB axon pruning and da neurondendrite pruning also employ distinct mechanisms. For example,Ik2 kinase, the cytoskeleton regulators Katanin-60 and Mical, andthe ubiquitylating enzyme UbcD1 (Eff ) are required for da neurondendrite pruning but not for MB axon pruning (Lee et al., 2009;Kirilly et al., 2009; Kuo et al., 2006; Watts et al., 2003). However,despite the significant progress made over the past two decades, ourunderstanding about the molecular mechanisms regulating axon/dendrite pruning is far from complete.

The JNK signaling pathway plays diverse roles in both thedevelopment of the nervous system and neurological diseases.Studies in JNK knockout mice have shown that JNK is required forbrain morphogenesis, apoptosis, axon outgrowth/guidance anddendrite morphogenesis during the development. In addition, JNKalso triggers neuronal death in various neurodegenerative diseases(reviewed by Coffey, 2014). JNK performs these functions by eitherfunctioning in the nucleus or in the cytoplasm. In the nucleus, JNKregulates gene transcription by phosphorylating substrates such asJun or histone modifiers (Crocker et al., 2001; Tiwari et al., 2011).In the cytoplasm, JNK phosphorylates a variety of substrates,including microtubule-associate proteins, superior cervicalganglion 10 protein (SCG10), and postsynaptic density protein95 (PSD95) that are involved in neural development or degeneration(Bjorkblom et al., 2005; Shin et al., 2012; Kim et al., 2007).However, the function of JNK signaling in large-scale dendritepruning has not been investigated previously. In this study, wefound that JNK coordinates with EcRB1 signaling to promoteReceived 19 January 2018; Accepted 25 March 2019

1Department of Neuroscience and Physiology, State University of New YorkUpstateMedical University, Syracuse, NY 13210, USA. 2Department of Physiology, HowardHughes Medical Institute, University of California San Francisco, San Francisco,CA 20251, USA. 3Center for Molecular Neurobiology (ZMNH), University MedicalCenter Hamburg-Eppendorf, University of Hamburg, Hamburg, Germany.

*Author for correspondence (zhus@upstate.edu)

S.Z., 0000-0003-2707-2031

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dendrite pruning and identified Fos as a novel downstream target ofEcRB1 in c4da neurons. Furthermore, we discovered that Wnt5signaling is also possibly required for dendrite pruning of c4daneurons and genetically interacts with JNK signaling.

RESULTSLoss of JNK leads to dendrite-pruning defectsof c4da neuronsIn each hemi-segment of Drosophila larvae, there are three c4daneurons, ddaC, v’ada and vdaB, from dorsal to ventral. All c4daneurons undergo similar dendrite pruning processes during earlymetamorphosis, although dendrite pruning of v’ada and vdaBneurons is slightly later than that of ddaCs. Furthermore, vdaBneurons do not survive to adulthood (Kuo et al., 2005). Therefore,we focused on ddaC neurons for phenotypic analyses of dendritepruning. During dendrite pruning, major dendritic branches,including primary and secondary branches of c4da neurons, arefirst severed from the soma at 5-10 h after puparium formation(h APF) (Fig. 1A-B). Severed dendrites are then cleared throughdegeneration and phagocytosis (Han et al., 2014; Williams andTruman, 2005). By 16-18 h APF, all dendrites are removed, leavingonly the soma and axons (Fig. 1C) (Kuo et al., 2005).To test whether the JNK signaling pathway is involved in dendrite

pruning of c4da neurons, we examined how loss of DrosophilaJNK, which is encoded by Basket (Bsk), would affect dendritepruning of c4da neurons. We first inhibited JNK activity in c4daneurons using ppk-Gal4 (Grueber et al., 2007) to drive theexpression of a dominant-negative form of JNK, JNKDN (Adachi-Yamada et al., 1999). We found that expressing JNKDN inhibiteddendrite pruning in a dose-dependent manner. When one copy ofUAS-JNKDN was expressed, about 11% of ddaC neurons still hadprimary dendritic branches attached to the soma and 24% of ddaCshad severed dendritic fragments at 16-18 h APF (Fig. 1D,E,H).When two copies ofUAS-JNKDNwere expressed, the percentages ofddaC neurons with attached dendritic branches or severed dendriticfragments were further increased to about 70% and 30%,respectively (Fig. 1H). In contrast, wild-type ddaC neurons onlyoccasionally had some severed dendritic fragments but no attacheddendritic branches at similar stages (Fig. 1C,H). The retention ofattached dendritic branches or severed dendritic fragments indicatesthat dendrite severing was blocked or delayed when JNK wasinhibited by its dominant-negative form. The dendrite-pruningdefects resulting from the expression of JNKDN is unlikely to be asecondary effect due to dendrite morphogenesis defects for thefollowing two reasons. First, no obvious dendrite morphogenesisdefects were observed in ddaC neurons expressing UAS-JNKDN orhomozygous mutant for bsk2 at 3rd instar larval stages (Fig. S1A-C). Second, similar dendrite-pruning defects were still observedwhen we used tub-GAL80ts to restrict the expression ofUAS-JNKDN

to 1 day before puparium formation (Fig. S1D).In order to confirm that dendrite-pruning defects resulting from

the expression of JNKDN were indeed caused by the loss of JNKfunction, we next examined whether knocking down JNK wouldlead to similar dendrite-pruning defects in c4da neurons. Tomaximize the RNAi knockdown efficiency, we raised animals at29°C after larval hatching and examined dendrite pruning at 11-13 hAPF, when dendrites are largely pruned in wild-type animals raisedat the same temperature. We found that 8% of JNK knockdownddaC neurons still had unsevered dendrites and 12% had severeddendritic fragments at 11-13 h APF, whereas in the wild type,severed dendritic fragments were only observed in fewer than 4% ofddaC neurons at the same stage (Fig. 1F,H).

To prove that the dendrite-pruning defects resulting fromthe expression of UAS-JNK RNAi were not off-target effects,we performed the following two experiments. First, we tried torescue the knockdown phenotypes by expressingUAS-JNK. Indeed,expressing UAS-JNK significantly rescued the dendrite-pruningdefects caused by JNK knockdown. Only about 1% of ddaCneurons had attached dendrites and 10% had severed dendriticfragments at 11-13 h APF when UAS-JNK was co-expressed withUAS-JNK RNAi (Fig. 1G,H). The rescue is unlikely due to dilutionof the GAL4 effect because similar dendrite-severing defects werestill observed in about 9% of ddaC neurons (13 out of 140 ddaCneurons examined) when one additional copy of UAS-mCD8-GFPwas included while knocking down JNK by RNAi (Fig. S1G).

Fig. 1. JNK is required for dendrite pruning in c4da neurons. ddaCneurons are labeled with mCD8-GFP driven by ppk-GAL4. (A-C) Wild-typeddaC neurons at 0, 10 and 16-18 h APF. Primary dendrites are severedfrom the soma at 10 h APF (B) and no dendrites remain at 16-18 h APF (C).(D,E) ddaC neurons expressing one copy of UAS-JNKDN still have severeddendritic fragments (D) or unsevered dendrites (E) at 16-18 h APF.(F) Knocking down Bsk (JNK) leads to retention of unsevered dendritesin c4da neurons at 11-13 h APF. Animals were kept at 29°C after larvalhatching for the knockdown of Bsk. (G) Expressing UAS-JNK rescuesthe dendrite-pruning defects resulting from JNK knockdown. Arrowsindicate the soma of ddaC neurons and arrowheads indicate the axons.(H) Quantification of ddaC neurons with attached or severed dendritesat 16-18 h APF (25°C) or 11-13 h APF (29°C) in animals with theindicated genotypes. Numbers in parentheses are the number of ddaCneurons examined. *P<0.05, **P<0.01.

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Second, we examined whether removing one wild-type copy of thebsk gene would enhance the RNAi knockdown phenotypes. To thisend, we performed the knockdown experiments at 25°C to reducethe efficiency of JNK knockdown. When JNK was knocked downin the wild-type background at 25°C, ∼3% of ddaC neurons hadunsevered dendrites and 8% had severed dendritic fragments at16-18 h APF. However, when JNK was knocked down in a bsk2

heterozygous mutant background, the percentage of ddaC neuronswith attached dendrites was increased to about 7% and the other14% had severed fragments. The rescue of the knockdownphenotypes by UAS-JNK and the enhancement of the phenotypesin a bsk2 heterozygous mutant background indicate that thedendrite-pruning defects resulting from the RNAi knockdownwere not off-target effects. Consistent with these results, weobserved unsevered dendrites in one out of 10 bsk2 mutant ddaCclones and two out of 23 bskflp147Emutant ddaC clones (Fig. S1E-F).Taken together, the dendrite-pruning defects observed in ddaCneurons expressing JNKDN or JNK RNAi and bsk mutant clonesdemonstrate that JNK is indeed required for dendrite severing ofc4da neurons.

JNK regulates dendrite pruning, likely by promotingmicrotubule disassemblyLocal disassembly of microtubule structures in the dendrites is one ofthe earliest visible signs during dendrite pruning and occurs beforethinning and severing of membrane dendrites (Fig. 2A-C′) (Williamsand Truman, 2005). Previous studies have shown that loss of themicrotubule-severing protein Katanin p60-like 1 (Kat-60L1) or theTau kinase PAR-1 blocks dendrite pruning (Herzmann et al., 2017;Lee et al., 2009). In addition, JNK is known to regulate microtubuledynamics by phosphorylating microtubule-associated proteins,

including Tau (Bjorkblom et al., 2005; Buée-Scherrer and Goedert,2002). In order to understand how JNK regulates dendrite pruning,therefore, we next examined how microtubule structures in thedendrites might be affected when JNK was inhibited by JNKDN. Wefound that microtubule structures remained intact in unsevereddendrites even at 11 h APF when JNKDN was expressed in c4daneurons (Fig. 2D-F′), whereas in the wild-type c4da neurons,microtubules have already broken down around 5 hAPF (Fig. 2A-C′).These results indicate that JNK is required for disassembly ofmicrotubules during dendrite pruning in c4da neurons.

JNK activity does not show dynamic changes during thetransition from larval to pupal stagesDendrite pruning of c4da neurons occurs only at early pupal stages.Therefore, we wondered whether JNK is only activated at earlypupal stages and whether JNK acts through the canonical nuclearpathway in c4da neurons. To address these issues, we examined theexpression of the JNK reporter puckered ( puc)-lacZ in c4da neuronsfrom larval to early pupal stages. puc encodes a JNK phosphataseand is a transcriptional target of JNK signaling (Martin-Blancoet al., 1998). Activation of JNK signaling upregulates pucexpression, which in turn provides negative feedback to JNK bydephosphorylation (Martin-Blanco et al., 1998). puc-lacZ lines( pucA251.1F3 or pucE69) are lacZ enhancer trap lines (Dobens et al.,2001) that have been widely used as reporters of nuclear JNKactivity (Neisch et al., 2010; Willsey et al., 2016). Using thepuc-lacZ ( pucA251.1F3) as a reporter, we observed that the lacZexpression could be detected in c4da neurons at both larval andearly pupal stages, and its expression levels remained similar(Fig. 3A-D). These data suggest that JNK is constantly activated inc4da neurons from larval and pupal stages, and its activity does notshow obvious dynamic changes. Furthermore, the expression ofpuc-lacZ also indicates that JNK likely acts through the canonicalnuclear pathway.

The expression of the Drosophila Fos in c4da neurons isactivated by EcRB1 at early pupal stagesIf JNK is activated at both larval and pupal stages, then why doesJNK promote dendrite pruning only at early pupal stages? Could itbe that JNK downstream effectors are only expressed/activated atearly pupal stages? JNK controls various cellular processes byregulating the activity of a number of substrates throughphosphorylation. As puc-lacZ is expressed in c4da neurons, it ispossible that JNK acts through the canonical pathway in the nucleusto phosphorylate and activate the AP-1 complex, which iscomposed of Drosophila Jun and Drosophila Fos. Studies in bothvertebrates and invertebrates show that Fos expression isdynamically regulated by various stimuli (Greenberg and Ziff,1984; Weiser et al., 1993; Rusak et al., 1990; Morgan et al., 1987).Therefore we wondered whether the expression of Drosophila Fos,which is encoded by kayak (kay), in ddaC neurons shows dynamicchanges during the transition from larval to early pupal stages.Immunostaining of Fos in ddaC neurons showed that this is indeedthe case. At the 3rd instar larval stages, no obvious Fos expressioncould be detected (Fig. 4A,E). At the white pupal stage (0 h APF),we observed relatively low levels of Fos expression (Fig. 4B,E). Theexpression of Fos further increased at 2-3 h APF (Fig. 4C,E). Thesedata demonstrate that Fos expression in ddaC neurons is specificallyactivated at early pupal stages.

Given that ecdysone signaling plays a key role in regulating axon/dendrite pruning in Drosophila (Lee et al., 2000; Kuo et al., 2005),we then asked whether the expression of fos was activated by

Fig. 2. JNK regulates dendrite pruning likely in part by promotingmicrotubule disassembly in the dendrites. (A-C′) In a wild-type ddaCneuron at 5 h APF, the membrane (labeled with mCD8-GFP) of a primarydendrite remains unsevered (A,C), while the microtubule has disassembled,as indicated by MAP1B staining (B,C). (A′-C′) Enlarged views of the regionshighlighted by dotted boxes in A-C. (D-F′) A primary dendrite is still unsevered(green in D,F) and the microtubule structure (red in B,C) remains intact inthe unsevered dendrite at 11 h APF in a ddaC neuron expressingUAS-JNKDN.(D′-F′) Enlarged views of the areas highlighted by dotted boxes in D-F.Arrows indicate the soma of ddaC neurons. ddaC neurons are labeled withmCD8-GFP driven by ppk-GAL4.

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ecdysone signaling. To address this issue, we inhibited ecdysonesignaling in c4da neurons by expressing a dominant-negative form ofecdysone receptor B1 (EcRB1DN) (Cherbas et al., 2003). ExpressingEcRB1DN completely inhibits dendrite pruning of c4da neurons (datanot shown) (Kuo et al., 2005). Interestingly, expressing EcRB1DN

abolished the expression of Fos in ddaC neurons at early pupal stages(Fig. 3D-E). These results suggest that the expression of Fos in c4daneurons is activated by ecdysone signaling.Previous studies have shown that EcRB1 regulates dendrite

pruning by activating the expression of the transcription factorSox14 (Kirilly et al., 2009). Therefore, we wondered whether theremight be a cross-regulatory relationship between Fos and Sox14 fortheir expression in c4da neurons. To address this issue, we examinedhow loss of Sox14 would affect the expression of Fos and viceversa. We expressed UAS-sox14 RNAi to knockdown Sox14 orexpressed a dominant-negative form of Fos (FosDN) to inhibit theactivity of Fos in c4da neurons. Knocking down Sox14 by RNAicompletely inhibited dendrite pruning like the expression ofEcRB1DN (Fig. S2E-F,H) as reported previously (Kirilly et al.,2009). However, Fos expression in c4da neurons at 3 h APFremained comparable with that in wild-type animals when Sox14was knocked down by RNAi (Fig. S2A-B′). Likewise, expressingFosDN did not lead to obvious changes of the expression of Sox14 inc4da neurons at 3 h APF either (Fig. S2C-D′). These data suggestthat, although the expression of both Fos and Sox14 is activated byEcRB1, Fos and Sox14 do not depend on each other for their

expression and likely act in two independent pathways downstreamof EcRB1. In support of this notion, expressing UAS-Fos couldnot rescue the dendrite-pruning defects resulting from Sox14knockdown (Fig. S2G,H).

JNK downstream effectors Jun and Fos are required fordendrite pruning in c4da neuronsWe next wanted to determine whether Jun and/or Fos functiondownstream of JNK to promote dendrite pruning of ddaC neurons.We first examined how loss of Jun, encoded by Jra (Jun-relatedantigen), or Fos would affect dendrite pruning of ddaC neurons. Forthe loss-of-function phenotypic analyses of Jun, we either generatedjunmutant clones or knocked down Jun using RNAi. We found thatover 60% of ddaC neurons homozygous mutant for jun1, a nonsensemutant allele that produces a truncated protein lacking the basicregion and leucine zipper (Kockel et al., 1997), still had unsevereddendrites at 16-18 h APF (Fig. 5A,B,F). Similarly, knockdown ofJun led to retention of unsevered dendrites in about 40% of ddaCneurons (Fig. 5C,F). The retention of unsevered dendrites in jun1

mutant and Jun knockdown ddaC neurons demonstrates that Jun isrequired for dendrite pruning of c4da neurons.

Next, we tried to determine whether Fos is similarly required fordendrite pruning of c4da neurons. To this end, we inhibited Fosactivity by expressing a dominant-negative mutant form of Fos(FosDN), which is a truncated version of Fos carrying the bZIPdomain only (FosbZIP) (Eresh et al., 1997). The isolated bZIPdomain can still dimerize with Jun but lacks transcriptional

Fig. 3. The expression of the JNK reporter puc-lacZ in c4da neuronsdoes not show dynamic changes from larval to early pupal stages.(A-C′) puc-lacZ (pucA251.1F3/+) expression in ddaC neurons at the 3rd instarlarval stage (A,A′), 0 h (B,B′) and 2 h (C,C′) APF. Arrows indicate the nucleiof ddaC neurons. Only the soma regions of ddaC neurons are shown.(D) Quantification of relative β-gal staining intensities in ddaC neurons atdifferent developmental stages. The staining intensities of β-gal in the nucleiwere measured using Adobe Photoshop. The mean staining intensity ofβ-gal in ddaC neurons at the 3rd instar larval stage is set as 100%. Data aremean±s.d. Numbers in parentheses on top of each bar are sample sizes.ddaC neurons are labeled with mCD8-GFP driven by ppk-GAL4.

Fig. 4. Expression of Fos in c4da neurons is activated by EcRB1 at earlypupal stages. (A-C′) Fos staining in wild-type ddaC neurons at the 3rd instarlarval stage (A,A′), 0 h (B,B′) and 3 h (C,C′) APF. (D,D′) No obvious Fosstaining is detected in ddaC neurons expressing EcRB1DN at 3 h APF. Thecell body regions are outlined by dashed lines and the nuclei are outlinedby dashed circles. (E) Quantification of relative Fos staining intensities inwild-type ddaC neurons from the 3rd instar larval stage to 3 h APF or in ddaCneurons expressing EcRB1DN at 3 h APF. The staining intensities of Fos inthe nuclei were measured using Adobe Photoshop. The average stainingintensity of Fos in the wild-type ddaCs at 3 h APF is set as 100%. Data aremean±s.d. Numbers on top of each bar represent sample sizes. ddaCneurons are labeled with mCD8-GFP driven by ppk-GAL4.

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activation activity. FosDN has been widely used for inhibiting Fosfunction (Collins et al., 2006; West et al., 2015). Our results showedthat inhibiting Fos activity by FosDN led to dendrite-severing defectsin ddaC neurons in a dose-dependent manner. When one copy ofUAS-d-FosDN was expressed, ∼23% of ddaC neurons hadunsevered dendrites and other 10% of ddaCs had severeddendritic fragments at 16-18 h APF (Fig. 5D,F). When two copiesofUAS-FosDNwere expressed, the percentage of ddaC neurons withattached dendrites was increased to nearly 50% and another 20%had severed dendritic fragments (Fig. 5E,F). Dendrite-pruningdefects resulting from the expression of UAS-d-FosDN suggest that,like Jun, Fos is also required for dendrite pruning of c4da neurons.Taken together, the dendrite-pruning defects observed in junmutantand FosDN-expressing ddaC neurons suggest that JNK promotesdendrite pruning likely by regulating the activity of the AP1complex in C4da neurons.

Wnt5 genetically interacts with JNK in regulating dendritepruning of c4da neuronsJNK signaling is involved in many cellular processes and can beactivated by a variety of extracellular stimuli (Behrens et al., 2000;Leppä and Bohmann, 1999). We next asked what extracellularstimuli activated JNK signaling during dendrite pruning. Weinvestigated some candidates that could potentially activate JNKsignaling by examining dendrite-pruning defects in ddaC neuronsexpressing UAS-RNAi transgenes or homozygous mutant forcandidate genes. These candidates include TNFα (Eiger) and itsreceptor (Wengen) as well as TNF receptor-associated factors,receptors for growth factors (including EGFR, VEGFR andTGFβR) and Wnt5 (Table S1). We found that knockdown of theActivin receptor Baboon (Babo) (Fig. S3A-B) and mutations of

wnt5 (Fig. 6A-C,G) led to dendrite-pruning defects in c4da neurons.It has been demonstrated previously that Babo regulates axonpruning by functioning through Smad to activate EcRB1 expressionin MB neurons (Zheng et al., 2003). Consistently, knockdown ofSmad also resulted in dendrite-pruning defects in ddaC neurons(Fig. S3C), suggesting that Babo regulates dendrite pruning of c4daneurons similarly by activating Smad in ddaC neurons. Therefore,we focused on Wnt5 in our studies.

Wnt5 is not essential for viability, making it possible to examinedendrite pruning in wnt5 homozygous mutant animals. We used twownt5 mutant alleles, wnt5400 and wnt5D7, both of which are loss-of-function alleles generated by imprecise excision from the P-elementinsertion line P{GT1} wnt5BG00642 (Fradkin et al., 2004; Yoshikawaet al., 2003). We found that ∼7-10% of ddaC neurons in wnt5400 orwnt5D7 mutant animals still had unsevered dendrites and another 10-12% had severed dendritic fragments at 16-18 h APF, when wild-type c4da neurons have already pruned their dendrites (Fig. 6A-C,G).Similar dendrite-pruning defects were also observed in wnt5400/wnt5D7 transheterozygous mutant animals (Fig. 6D,G), indicatingthat the dendrite-pruning defects in wnt5mutants were not caused bybackground mutations. These results demonstrate that Wnt5 isrequired for dendrite pruning in c4da neurons.

To determine whether Wnt5 regulates dendrite pruning byactivating JNK signaling in c4da neurons, we then performed thefollowing three experiments. First, we carried out dominant geneticinteraction tests to determine whether Wnt5 and JNK act in the samepathway. To this end, we examined whether removing one wild-typecopy of wnt5 would enhance the dendrite-pruning defects resultingfrom the expression of one copy ofUAS-JNKDN in c4da neurons. Wereasoned that if Wnt5 activates JNK in c4da neurons, reducing theexpression of Wnt5 would lead to reduced JNK activity and enhancethe dendrite-pruning defects resulting from the expression of UAS-JNKDN. Indeed, dendrite-pruning defects induced by one copy ofUAS-JNKDN were significantly enhanced by the reduction of Wnt5expression. The percentage of c4da neuronswith attached dendrites at16-18 h APF increased from 12% to ∼25% when one copy of UAS-JNKDNwas expressed in animals heterozygous mutant for wnt5D7 orwnt5400, whereas wnt5D7 or wnt5400 heterozygous mutants did notshow obvious dendrite-pruning defects at the same stage (Fig. 6H).The enhancement of JNKDN-induced dendrite-pruning defectsappears to be specific for wnt5 because no similar enhancementwas observed in animals heterozygousmutant forwntDKO1,wntDKO2,wg1-8,wnt2I andwnt4EMS23 (Fig. 6H and data not shown). These datasuggest that Wnt5 and JNK genetically interact and possibly functionin the same pathway to promote dendrite pruning.

Second, we examined whether the dendrite-pruning defects inwnt5D7 mutants could be partially rescued by enhancing JNKsignaling. We enhanced JNK activity by reducing the expression ofPuc, which provides inhibitory feedback on JNK (Martin-Blancoet al., 1998). Interestingly, the dendrite-pruning defects in wnt5D7

mutant animals were significantly rescued by removing one wild-typecopy of puc. The percentage of wnt5D7 mutant ddaC neurons withattached dendrites was reduced from 10% to only 1.5% at 16-18 hAPF in pucA251.1F3/+ heterozygous mutant background, whereaspucA251.1F3/+ heterozygotes did not show any dendrite-pruning defects(Fig. 6E-G). The partial rescue of the dendrite-pruning defects inwnt5D7 mutants by increasing JNK activity and the enhancement ofthe JNKDN-induced dendrite-pruning defects by removing onewild-type copy of wnt5 strongly suggest that Wnt5 likely regulatesdendrite pruning of c4da neurons by activating JNK signaling.

Last, we examined JNK activity in wnt5 mutants using puc-lacZas a reporter to see whether JNK activity might be reduced in wnt5

Fig. 5. Jun and Fos are required for dendrite pruning of c4da neurons.(A) Dendrites of a wild-type ddaC neuron are pruned at 16-18 h APF.(B,C) ddaC neurons homozygous mutant for jun1 (B) or expressing UAS-JunRNAi (C) still have unsevered primary dendrites at 16-18 h APF. (D,E) ddaCneurons expressing one (D) or two (E) copies of UAS-FosDN retainunsevered primary dendrites at 16-18 h APF. (F) Quantification of thepercentages of ddaC neurons with attached or severed dendrites at 16-18 hAPF. Numbers on top of each bar represent the total number of neuronsexamined. Arrows indicate the somaof ddaC neurons. ddaC neurons are labeledwith mCD8-GFP driven by ppk-GAL4. **P<0.01 compared with the wild type.

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mutants. However, we did not observe dramatic reduction inpuc-lacZ expression in wnt5D7 hemizygous mutant animals at 0 hAPF compared with that in wild-type animals (Fig. S4A-C),indicating that Wnt5 might not be solely responsible for theactivation of JNK signaling in c4da neurons.

Derailed and Derailed-2 are possibly involved in dendritepruning of c4da neuronsIn Drosophila, Wnt5 binds to the RYK (related-to-tyrosine-kinase)receptor Derailed (Drl) to regulate axon guidance and salivarygland development (Yoshikawa et al., 2003; Harris andBeckendorf, 2007). A recent study also demonstrated that bothDrl and a closely related RYK receptor, Derailed-2 (Drl-2), have apartially redundant function in c4da neurons to mediate Wnt5signaling to specify the dendritic territory of adult c4da neurons(Yasunaga et al., 2015). To investigate whether Wnt5 similarlyfunctions through Drl and/or Drl-2 to regulate dendrite pruning ofc4da neurons, we first examined whether Drl was expressed inlarval c4da neurons. Antibody staining revealed that Drl was indeedexpressed in c4da neurons at both the 3rd instar larval stage andearly pupal stages (Fig. 7A-B′). Consistently, UAS-mCD8-GFPdriven by drl-GAL4, a GAL4 enhancer trap line in drl that reflectsthe endogenous expression pattern of Drl (Moreau-Fauvarqueet al., 1998), was also specifically expressed in c4da neurons on thelarval body surface but not in other peripheral sensory neurons(Fig. 7C). The expression pattern of drl-GAL4 in larval c4daneurons is consistent with its expression in adult c4da neurons(Yasunaga et al., 2015). Therefore, Drl is endogenously expressedin c4da neurons throughout development and could potentiallymediate Wnt5 signaling.

We next investigated whether Drl and Drl-2 are required fordendrite pruning of c4da neurons. We used three viable mutantalleles, drl2, drlP1 and drlRed2 (Dura et al., 1995; Bonkowsky et al.,1999), for examining the involvement of Drl in dendrite pruning.We observed consistent weak dendrite-pruning defects in animalhomozygous mutant for drl2, drlP1 or drlRed2. About 6-11% of ddaCneurons still had attached dendrites and the other 8-22% had severeddendritic fragments in these mutant animals at 16-18 h APF(Fig. 7D-G,I). To determine whether the pruning defects in drlmutants were indeed caused by loss of Drl rather than backgroundmutations and whether Drl function cell autonomously in c4daneurons, we next examined whether similar dendrite-pruningdefects could still be observed in drl2/drlP1 transheterozygousmutants and whether expressing Drl in c4da neurons could rescuethe pruning defects. We found that dendrite-pruning defects stillexisted but were much weaker in drl2/drlP1 transheterozygousmutants. Only 2% of ddaC neurons that still had unsevered dendritesand 5% had severed fragments in drl2/drlP1 animals at 16-18 h APF(Fig. 7H,L). However, expressing UAS-drl-myc in c4da neuronscould not rescue the dendrite-pruning defects in drlP1 or drlRed2

mutants (Fig. 7L). Given that similar dendrite-pruning defects wereobserved in pupae homozygous mutant for three independent drlalleles, as well as in drl2/drlP1 transheterozygotes, the pruningdefects are likely caused by the loss of Drl. However, failure of therescue of the pruning defect by expressing UAS-drl-myc indicatesthat Drl may actually function non-cell-autonomously to regulatedendrite pruning in spite of the expression of Drl in c4da neurons.Alternatively, the pruning defects observed in drl mutants mayresult from other subtle developmental defects that were not visibleat the gross morphology level.

Fig. 6. Wnt5 promotes dendrite pruning of c4da neurons likely by activating JNK. (A) Dendrites are pruned in a wild-type ddaC neuron at 16-18 h APF.(B-D) ddaC neurons homozygous mutant for wnt5400 (B) or wnt5D7 (C), or transheterozygous mutant for wnt5400/wnt5D7 (D), have attached primary dendrites at16-18 h APF. (E) wnt5D7 mutant ddaC neurons largely prune their dendrites at 16-18 h APF in the pucA251.1F3 heterozygous mutant background. (F) Dendritesof a pucA251.1F3 heterozygous mutant ddaC neuron are pruned normally at 16-18 h APF. Arrows indicate the soma of ddaC neurons. (G) Quantification of wnt5mutant ddaC neurons with attached primary dendrites or severed dendritic fragments at 16-18 h APF with or without the pucA251.1F3 heterozygous mutantbackground. (H) Quantification of ddaC neurons with attached dendrites at 16-18 h APF in wnt5 or wntD heterozygous mutant animals with or without theexpression of one copy of UAS-JNKDN. Numbers on top of each bar in F and G are the total number of ddaC neurons examined. ddaC neurons are labeled withmCD8-GFP driven by ppk-GAL4. *P<0.05, **P<0.01.

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For assessing the potential role of Drl-2 in dendrite pruning, weexamined dendrite pruning in drl2E124 (Inaki et al., 2007) anddrl2DG38805 (Bellen et al., 2004) homozygous mutant animals at16-18 h APF. Our results showed that, in drl-2E124mutants,∼14% ofddaC neurons still had attached dendrites and 27% had severeddendritic fragments at 16-18 h APF (Fig. 7I,L). In drl-2DG38805

mutants, about 12% of ddaC neurons still had attached dendrites andmany higher-order branches were also retained in these neurons at16-18 h APF. In addition, over 70% of drl-2DG38805 mutant ddaCneurons had a large amount of severed dendritic fragments at 16-18 hAPF (Fig. 7J,L), similar to wild-type ddaC neurons at around 10 hAPF (Fig. 1B), indicating that severing of primary dendrites had beensignificantly delayed in most drl-2DG38805 mutant ddaC neurons.Similarly, in drl-2E124/drl-2DG38805 transheterozygotes, about 8% and12% of ddaC neurons still had unsevered dendrites or severeddendritic fragments, respectively, at 16-18 h APF. However, thedendrite-pruning defects in drl-2 mutants were only observed inanimals with the 3rd chromosome homozygous but not heterozygousfor UAS-mCD8-GFP, ppk-GAL4, although animals homozygous forUAS-mCD8-GFP, ppk-GAL4 alone on the 3rd chromosome did nothave any obvious dendrite-pruning defects in c4da neurons. Theseresults suggest that Drl-2 could also be potentially involved in dendritepruning inC4daneurons, but itmay not playamajor role given that thepruning defects were only observed in specific genetic backgrounds.

DISCUSSIONIn this study, we found that JNK signaling is required for dendritepruning of c4da neurons. Our data suggest that JNK regulatesdendrite pruning likely by acting through its canonical pathway topromote microtubule disassembly. Remarkably, the temporalcontrol of dendrite pruning by JNK at early pupal stages seems tobe achieved not through changes in JNK activity but rather by theavailability of its substrate Fos. We show that the expression of Fosis specifically activated at early pupal stages by EcRB1, a majorregulator of axon/dendrite pruning (Kuo et al., 2005; Lee et al.,2000). Thus, our work not only identified a novel signaling pathwayinvolved in dendrite pruning and a new downstream effector ofEcRB1 in c4da neurons, but also revealed that JNK and ecdysonesignaling coordinate to regulate dendrite pruning (Fig. S5).

Dendrite pruning shares many common cellular and molecularmechanisms with pathological axon degeneration as well asdevelopmental axon pruning (Yu and Schuldiner, 2014; Luo andO’Leary, 2005). It has been well documented that JNK and otherMAPKs are central to axon degeneration resulting from injury orneurodegenerative diseases (reviewed by Geden and Deshmukh,2016; Coffey, 2014). A recent study reported that JNK is alsorequired for developmental pruning of mushroom body axons indeveloping Drosophila brains (Bornstein et al., 2015). However,mechanisms of JNK-mediated dendrite pruning of c4da neurons

Fig. 7. Drl and Drl2 are likely requiredfor dendrite pruning of c4daneurons. (A-B′) Drl staining in c4daneurons at the 3rd instar larval stage(A,A′) and 3 h APF (B,B′). The cellbodies are outlined with dashed lines.(C) UAS-mCD8-GFP driven by drl-GAL4 is expressed in c4da neuronsat the 3rd instar larval stage. (D) Awild-type ddaC neuron does not haveremaining dendrites at 16-18 h APF.(E-H) Unsevered primary dendritesare observed in drl2 (E), drlP1 (F),drlRed2 (G) or drl2/drlP1 (H) mutantddaC neurons at 16-18 h APF.(I-K) Dendrite-severing defects areobserved in drl-2E124 (I), drl-2DG38805

(J) and drl-2E124/drl-2DG38805 (K)mutant ddaC neurons at 16-18 h APF.The dendrite-severing defects in drl-2mutants were observed only inanimals homozygous for ppk-GAL4,UAS-mCD8-GFP on the 3rdchromosome. Arrows indicate the somaof ddaC neurons. (L) Quantification ofdrl or drl-2 mutant ddaC neurons withattached or severed dendrites at16-18 h APF. Quantification of dendrite-pruning defects in drl-2 mutants werefrom animals homozygous for ppk-GAL4, UAS-mCD8-GFP on the 3rdchromosome. Numbers on top ofeach bar represent the total numberof ddaC neurons examined. ddaCneurons are labeled with mCD8-GFPdriven by ppk-GAL4. *P<0.05,**P<0.01 compared with the wild type.

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are likely different from those of JNK-mediated developmentalaxon pruning or injury-induced axon degeneration. In injury-induced axon degeneration or developmental axon pruning, JNKmainly functions locally in the axon to promote axon degenerationinstead of acting through the canonical pathway to regulate geneexpression by activating the AP-1 complex in the nucleus. Ininjury-induced axon degeneration, JNK acts locally in the axon topromote the degradation of the axon survival factors NMNAT2 andSCG10, and the subsequent depletion of NAD+ and ATP in severedaxons (Walker et al., 2017; Shin et al., 2012; Yang et al., 2015). Inaxon pruning of MB neurons, JNK destabilizes the cell-adhesionmolecules Fasciclin (FasII) in axons (Bornstein et al., 2015). Thenuclear reporter of JNK, puc-lacZ, is not expressed in MB neurons,nor does loss of Jra or Kay function lead to axon-pruning defects inMB neurons (Bornstein et al., 2015). In contrast, in c4da neurons,our results demonstrate that JNK likely functions through thecanonical pathway to promote dendrite pruning. First, puc-lacZ isexpressed in the nuclei of c4da neurons. Second, loss of either Junor Fos results in dendrite-pruning defects. It will be interesting toidentify target genes that are activated by the AP-1 complex duringdendrite pruning of c4da neurons, which may provide mechanisticdetails of developmental dendrite pruning. Interestingly, eventhough JNK is required for dendrite pruning of da neurons and axonpruning of MB neurons, loss of JNK does not block dendritepruning in MB neurons (Bornstein et al., 2015). Therefore, evendendrites in different neuronal types could employ distinctmechanisms for regulating their development pruning.Although JNK is required for the dendrite pruning of da neurons,

our work shows that the temporal control of the JNK-mediateddendrite pruning is achieved not through dynamic changes of theJNK activity but rather through activation of Fos expressionspecifically at early pupal stages. The expression of the JNKreporter puc-lacZ in c4da neurons remains constant from larval toearly pupal stages, whereas the expression of Fos can only bedetected at early pupal stages. Therefore, JNK may performdifferent functions in c4da neurons at different developmentalstages, depending on the availability of distinct substrates. It hasbeen well documented that the expression of Fos is dynamicallyregulated and can be induced by various physiological andpathological stimuli, such as growth factors, neuronal activity andinjury (Greenberg and Ziff, 1984; Morgan et al., 1987; Rusak et al.,1990; Weiser et al., 1993). In this study, we found that expression ofFos in c4da neurons was activated by EcRB1 at early pupal stages.Therefore, JNK signaling coordinates with ecdysone signaling toregulate dendrite pruning of da neurons. Ecdysone signalingactivates the expression of Fos either directly or indirectly,whereas JNK regulates the activity of the AP-1 complexcomposed of Fos and Jun through phosphorylation.In addition to Fos, a previous study has shown that EcRB1 also

activates the expression of Sox14 to regulate dendrite pruning ofc4da neurons. Sox14 in turn activates the expression of thecytoskeleton regulator Mical (Kirilly et al., 2009). However, Fosand Sox14 likely function in parallel instead of linearly because ourresults show that the expression of Fos and Sox14 does not seem todepend on each other, nor could expression of UAS-Fos rescue thedendrite-pruning defects resulting from Sox14 knockdown.Therefore, EcRB1 promotes dendrite pruning possibly by activatingthe expression of multiple target genes, including Sox14, Fos andpossibly other unknown targets.How is JNK activated in c4da neurons? JNKs are components of

the canonical MAPK pathway and can be activated by a variety ofstimuli, such as cytokines, growth factors and cellular stress (Leppä

and Bohmann, 1999; Behrens et al., 2000). In this study, we showedthat loss of Wnt5 led to similar dendrite-pruning defects that couldbe significantly rescued by reducing the expression of JNK inhibitorPuc, whereas removing one wild-type copy of wnt5 significantlyenhanced the dendrite-pruning defects caused by the expression ofJNKDN. These genetic interaction data suggest that Wnt5 regulatesdendrite pruning possibly by activating JNK signaling in c4daneurons, but Wnt5 might be only partially responsible for theactivation of JNK in c4da neurons because there was no dramaticreduction in the expression of the JNK reporter puc-lacZ in wnt5D7

hemizygous mutants. Other unknown signals might also beinvolved in the activation of JNK in c4da neurons. Alternatively,Wnt5 and JNK signaling could act independently in parallel toregulate dendrite pruning of c4da neurons. However, we do not ruleout the possibility that the dendrite-pruning defects observed inwnt5 as well as drl mutants (see discussion below) might besecondary effects due to other subtle developmental defects that arenot obviously visible at the gross morphological level. It will beinteresting in the future to investigate whether other unknownsignals that activate JNK signaling regulate dendrite pruning inc4da neurons.

Studies in various model systems have shown that Wnt5 can bindto different receptors, including Frizzled, Ror2, CD146 and RYK, toactivate JNK (Kumawat and Gosens, 2016; Fradkin et al., 2010;Nishita et al., 2010). A recent article reported that, in adult c4daneurons, Wnt5 acts through Drl and Drl2 to restrict the growth ofdendrites within specific dendritic territories (Yasunaga et al.,2015). We show in this study that there are consistent dendrite-pruning defects in different drl or drl-2 mutant alleles, suggestingthat Drl and Drl-2 may also be involved in dendrite pruning in c4daneurons. However, the failure of UAS-drl-myc to rescue the pruningdefects observed in drl mutants indicates that the dendrite-pruningdefects in drl mutants are likely non-cell-autonomous, or may besecondary to other subtle developmental defects in drlmutants. Thefact that the dendrite-pruning defects in drl-2 mutants were onlyobserved in a genetic background with homozygous UAS-mCD8-GFP, ppk-GAL4 on the 3rd chromosome suggests that Drl-2 mayonly have a minor role in mediating dendrite pruning. Therefore,further investigations are needed in the future to determine whichreceptor(s) mediate(s) Wnt5 function in dendrite pruning ifWnt5 indeed activates JNK signaling in c4da neurons to promotedendrite pruning.

MATERIALS AND METHODSFly geneticsFor dominant-negative inhibition, the following transgenic lines were used:UAS-JNKDN [6409, Bloomington Drosophila Stock Center (BDSC)], UAS-d-FosDN (also named UAS-Fra.Fbz, BDSC 7214 and BDSC 7215), andUAS-EcRB1DN (BDSC 6872). The following lines were used for RNAiknockdown:UAS-djun RNAi [fly stocks of the National Institute of Genetics(NIG), Japan, 2275R-1], UAS-sox14 RNAi (BDSC 34794), UAS-bsk RNAi[BDSC 31323, targeting to a 483 bp sequence (from +1028 bps to +1051bps downstream of the transcription start site) within the coding region],UAS-babo RNAi [Vienna Drosophila Resource Center (VDRC), Austria,853] and UAS-dSmad2 RNAi (NIG 2262-R-1). bsk2, FRT40A/Cyo,bskflp147E, FRT40A/Cyo, FRTG13, jun1/Cyo, wnt5D7, wnt5400, drl2, drlP1,drlRed2, drl2E124 and drl-2DG38805 were used for mutant phenotypicanalyses. elav-GAL4, hs-FLPase, UAS-mCD8-GFP; tubP-GAL80,FRT40A/Cyo and elav-GAL4, hs-FLPase, UAS-mCD8-GFP; FRTG13,tubP-GAL80/Cyo fly lines were used for generating bsk and jun mutantclones of c4da neurons with the mosaic analysis with repressible cell marker(MARCM) technology (Lee et al., 1999). ppk-GAL4 lines on the 2nd or 3rdchromosome (BDSC 32078 and BDSC 32079) were used to drive theexpression of transgenes in c4da neurons.

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Mosaic analyses, RNAi knockdown and dominant-negativeinhibitionMARCM clones of c4da neurons were induced by 1 h heat shock at 38°C at3 h after egg laying. Animals were then raised at 25°C until 3rd instar larvalstage for identifying larvae with c4da neuron clones with live confocalimaging. Larvae with c4da neuron clones were further kept at 25°C until16-18 h APF for examining dendrite-pruning defects. For Jun RNAiknockdown and dominant-negative inhibition of JNK and Fos, animals werealso maintained at 25°C and dendrite-pruning defects were examined at16-18 h APF. RNAi knockdown of JNK was performed at 25°C or 29°C,and dendrite pruning was examined at either 16-18 h APF or 11-13 h APF,respectively, as indicated in the text. UAS-Dcr2 (BGSC #24650) wasco-expressed in ddaC neurons to boost RNAi knockdown efficiency.

Immunohistochemistry and confocal microscopyFor examining the expression of puc-lacZ, Fos, Sox14 or Drl in c4daneurons, animals at 3rd instar larval or early pupal stages were dissected,fixed and stained with the following primary antibodies: rat anti-mCD8(Life Technologies, 1:100, 14-0081-82), rabbit anti-galactosidase (MPBiomedicals, 1:1000, 55976), rabbit anti-Fos (FabGennix International,1:250, DFOS-101AP), mouse anti-Sox14 (Kirilly et al., 2009) (a gift fromDr. Fenwei Yu, Temasek Life Sciences Laboratory, National University ofSingapore, 1:200) and rat anti-Drl (Lundgren et al., 1995) (a gift fromDr. J. B. Thomas, Molecular Neurobiology Laboratory, Salk Institute forBiological Studies, La Jolla, CA, USA, 1:200). Secondary antibodiesconjugated to Cy2, Cy3, Cy5 or DyLight 647 (Jackson ImmunoResearch;715-165-151, 711-165-152, 711-225-152, 712-225-153 and 712-175-153)were used at 1:100, 1:500, or 1:500, respectively. C4da neurons wereimaged either in live animals or in stained larval or pupal fillets using a LeicaSp2 or Zeiss LSM780 confocal microscope and processed with AdobePhotoshop. Staining intensities of β-gal and Fos in the nuclei of c4daneurons were measured using Adobe Photoshop. Two-tailed Student’s t-testwas used for statistical analyses.

AcknowledgementsWe thank Drs T. Adachi-Yamada, A. Singh, A. DiAntonio, J. B. Thomas,D. Bhomann, F. Yu, H. Ying, C. Hama, the Bloomington Drosophila Stock Centerand Fly Stocks of National Institute of Genetics for fly stocks and antibodies;Sriharsha Gowtham and Xiaobing Deng for technical support; members of the Jablab and the Zhu lab for thoughtful discussion and comments; and Dr M. Connellfor proofreading.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: S.Z., Y.-N.J.; Formal analysis: S.Z., R.C.; Investigation: S.Z.,R.C., P.S.; Data curation: S.Z., R.C., P.S.; Writing - original draft: S.Z.; Writing -review & editing: S.Z., R.C., P.S., Y.-N.J.; Supervision: S.Z., Y.-N.J.; Projectadministration: S.Z., Y.-N.J.; Funding acquisition: S.Z., Y.-N.J.

FundingThis work was supported by National Institutes of Health grants 1R35NS97227(to Y.-N.J.) and R01NS085232 (to S.Z.), by the Howard Hughes Medical Institute(to Y.-N.J.), and by start-up funds from the SUNY Upstate Medical University (toS.Z.). Deposited in PMC for release after 6 months.

Supplementary informationSupplementary information available online athttp://dev.biologists.org/lookup/doi/10.1242/dev.163592.supplemental

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RESEARCH ARTICLE Development (2019) 146, dev163592. doi:10.1242/dev.163592

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