SHORT REPORT

Cofilin regulates axon growth and branching of Drosophilaγ-neuronsSriram Sudarsanam*,‡, Shiri Yaniv‡, Hagar Meltzer‡ and Oren Schuldiner§

ABSTRACTThemechanisms that control intrinsic axon growth potential, and thusaxon regeneration following injury, are not well understood.Developmental axon regrowth of Drosophila mushroom body γ-neurons during neuronal remodeling offers a unique opportunity tostudy themolecularmechanisms controlling intrinsic growth potential.Motivated by the recently uncovered developmental expression atlasof γ-neurons, we here focus on the role of the actin-severing proteincofilin during axon regrowth. We show that Twinstar (Tsr), the flycofilin, is a crucial regulator of both axon growth and branching duringdevelopmental remodeling of γ-neurons. tsr mutant axonsdemonstrate growth defects both in vivo and in vitro, and alsoexhibit actin-rich filopodial-like structures at failed branch pointsin vivo. Our data is inconsistent with Tsr being important for increasingG-actin availability. Furthermore, analysis of microtubule localizationsuggests that Tsr is required for microtubule infiltration into the axontips and branch points. Taken together, we show that Tsr promotesaxon growth and branching, likely by clearing F-actin to facilitateprotrusion of microtubules.

KEY WORDS: Actin dynamics, Axon branching, Cofilin, Twinstar,γ-neurons, Mushroom body, Neuronal remodeling

INTRODUCTIONThe limited regeneration of injured adult neurons within centralnervous systems is due to a combination of inhibitory environments(Tedeschi and Bradke, 2017) and reduced intrinsic growth ability(Mahar and Cavalli, 2018). Intrinsic growth abilities decrease withage, concomitant with lower regeneration capacity (Wang et al.,2007). Therefore, uncovering the factors that determine intrinsicgrowth potential in young developing neurons, holds the promise offurthering our understanding regarding factors that normally restrictregeneration.Neuronal remodeling is a conserved mechanism, which includes

the elimination of axons and synaptic connections, often followedby formation of new connections to sculpt mature neural networks(Luo andO’Leary, 2005; Yaron and Schuldiner, 2016). Remodelingof the Drosophila mushroom body (MB), a center for olfactory

associative learning, is an excellent model to study developmentalaxon regrowth (from now on – regrowth) as neurons need to rapidlychange their growth state from pruning (degeneration) to regrowth(regeneration; Rabinovich et al., 2016). The MB is comprised ofthree types of sequentially born neurons – γ, α′/β′ and α/β – ofwhich only γ-neurons undergo developmental remodeling (Fig. 1A;Lee et al., 1999). While we have previously shown that axonregrowth is a genetically controlled program, dependent upon thenuclear receptor transcription factors Unfulfilled (UNF, also knownas Hr51; Yaniv et al., 2012) and Ecdysone-induced protein 75B(Eip75B; Rabinovich et al., 2016), the molecular machinery thatgoverns growth in this context is largely unknown. Importantly, wehave demonstrated that regrowth is not only molecularly distinctfrom initial axon outgrowth, but also shares molecular mechanismswith regeneration following injury (Yaniv et al., 2012). To continueto dissect the genetic program that controls axon regrowth, we haverecently uncovered the detailed transcriptional landscape ofdeveloping γ-neurons (Alyagor et al., 2018). We found thatseveral actin regulators exhibit significant expression dynamicsduring neuronal remodeling, positioning them as candidates forstructural components of axon regrowth.

Axon growth is thought to be driven by growth cones that consistof an actin-rich peripheral zone with filopodia and lamellipodia, anda microtubule (MT)-rich central zone. The ability of growth cones tonavigate depends on a dynamic interplay between F-actin and MTs(Blanquie and Bradke, 2018). F-actin dynamics is the combinedresult of nucleation, elongation and severing of filaments (Omotadeet al., 2017; Shekhar et al., 2016). The actin depolymerization factor(ADF)/cofilin family consists of small globular proteins (three inmammals) that depolymerize and/or sever F-actin filaments, and arealso implicated in G-actin sequestration (Bernstein and Bamburg,2010; Flynn et al., 2012; Wioland et al., 2017). Counterintuitively,ADF/cofilin proteins are known to enhance axon growth and havebeen implicated in neurite formation during development (Flynnet al., 2012), as well as growth cone motility (Endo et al., 2003),branching (Bernstein and Bamburg, 2010; Kanellos and Frame,2016) and, most recently, axon regeneration (Tedeschi et al., 2019).Interestingly, actin stabilization by pharmacological manipulationor by mutating cofilin slows neurite growth and axon regeneration(Flynn et al., 2012; Tedeschi et al., 2019). The two main hypothesesas to how cofilin promotes axon extension are: (1) by increasingF-actin assembly, either by ‘actin treadmilling’ (Shekhar andCarlier, 2017), expected to increase the supply of severed G-actin tothe growing barbed ends, or by removal of capping, thus providingnew barbed ends available for elongation, and (2) by clearing actinto allow microtubules protrusion (Bradke, 1999).

Twinstar (Tsr, the sole Drosophila cofilin), which was shown tobind actin in vitro and to promote its depolymerization and severing(Shukla et al., 2018), is highly and dynamically expressed in MBγ-neurons throughout remodeling (Alyagor et al., 2018). Here, weexplore its role during axon regrowth and branching.Received 31 March 2019; Accepted 18 February 2020

Department of Molecular Cell Biology, Weizmann Institute of Sciences, Rehovot7610001, Israel.*Present address: The Solomon H. Snyder Department of Neuroscience; JohnsHopkins University.‡These authors contributed equally to this work

§Author for correspondence (oren.schuldiner@weizmann.ac.il)

O.S., 0000-0002-7350-9380

This is an Open Access article distributed under the terms of the Creative Commons AttributionLicense (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use,distribution and reproduction in any medium provided that the original work is properly attributed.

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RESULTS AND DISCUSSIONTsr is required for axon growth of MB γ-neuronsWe have recently uncovered the expression profiles of developingMB γ-neurons at a fine temporal resolution (Alyagor et al., 2018).Out of 126 actin-related genes in Drosophila, 77 are expressed

above a threshold (for more details, see Alyagor et al., 2018), 45 in adynamic pattern in developing γ-neurons (Fig. S1A; data taken fromAlyagor et al., 2018), out of which 21 are expressed in a patternsuggestive of a potential role in axon regrowth. In this study, wedecided to focus on the actin filament-severing protein Tsr, the flycofilin, whose expression is upregulated during metamorphosis(Fig. S1A), with maximal values reached at the onset of regrowth [at24 h after puparium formation (APF)].

Tsr has been previously shown to be required for axon growth ofboth γ- and α/β-neurons (Ng and Luo, 2004). Additionally, tsraxons displayed abnormal protrusions and swellings. However, thenature of these swellings, and whether Tsr is required for initial axonoutgrowth, regrowth or both, remained unknown. We thereforegenerated tsr homozygous mutant clones using the mosaic analysiswith a repressible cell marker (MARCM) technique. In line withresults from previous studies (Ng and Luo, 2004), MARCMγ-neuron neuroblast clones (NBCs) homozygous for tsrN121 ortsrN96A, both considered null alleles derived from imprecise excisionof the same P-element (but not molecularly defined), displayedreduced cell numbers and a severe growth phenotype in whichaxons stopped at the branchpoint (Fig. 1E,F,M; Fig. S1B). In orderto differentiate between a defect in initial outgrowth or regrowth, weexamined clones in third-instar larva (L3) and found that most, butnot all, larval tsr γ-axons stalled near the peduncular branch point(Fig. 1B,C). The growth defects could be rescued by expressing afull-length Tsr transgene within the mutant cells (Fig. 1D,G,M). Toour surprise, NBCs of the later-born α/β neurons appeared normal intsr mutants (Fig. S1C–E), even though Tsr has previously beenimplicated in their correct growth (Ng and Luo, 2004). Thisinconsistency could be due to the use of different Gal4 drivers(R44E04-Gal4 in this study versus OK107 in the original paper),and thus labeling different α/β sub-populations, or be due to thetiming of the heat-shock induced recombination (L3 here versuspupae in Ng and Luo).

We next examined single-cell clones (SCCs), in which theanatomical resolution is improved. In contrast to what was seen withneuroblast clones, tsrN121 SCCs extended their axons normally at L3(Fig. 1H,I). The fact that SCCs undergo initial growth normally, incontrast to NBCs, is likely due to protein and/or RNA perdurance ofTsr, a phenomenon that has been previously shown (Yu et al.,2013). Briefly, in NBCs, tsr RNA and protein are diluted bynumerous cell divisions, while in SCCs gene deletion occurs duringthe last division and thus protein and RNA might still be present.

The fact that tsr SCCs grow normally in larvae (and also at 6 hAPF, n=17, not shown) but exhibit a varying degree of growthdefects in adults (Fig. 1J,K,N; Fig. S1F–J), suggests that Tsr isrequired for regrowth independently of its requirement during initialaxon outgrowth. Furthermore, overexpressing a wild-type (WT) Tsrtransgene (UAS-tsr.N) within tsrN121 MARCM clones completelyrescued the regrowth defect (Fig. 1L,N). However, expression of thephosphomimetic Tsr.S3E, presumed to be inactive (Ng and Luo,2004), did not (Fig. 1N; Fig. S2B). Interestingly, expression of thenon-phosphorylatable Tsr.S3A, presumed to be constitutivelyactive, resulted in partial growth rescue (Fig. 1N; Fig. S2A),suggesting that phosphoregulation of Tsr is critical for its functionduring axon regrowth.

Tsr promotes branching of adult γ-neuronsWhile examining tsrN121 SCCs, we noticed that even axons thatextended almost normally exhibited elevated occurrences ofswellings that resemble lamellipodia-like protrusions. To testwhether these might represent failed branching sites, we

Fig. 1. Tsr is required for axon growth of MB γ-neurons. (A) Schematicrepresentation of γ-neuron developmental remodeling. Den, dendrites; p,peduncle; d, dorsal lobe; m, medial lobe; APF, after puparium formation.(B–L) Confocal Z-projections of WT (B,E,H,J), tsrN121 (C,F,I,K) or tsrN121

additionally expressingUAS-tsr.N (D,G,L) in γ-neuronMARCMNBCs (B–G) orSCCs (H–I) for L3 or adult flies. Asterisks mark γ-lobe edge. Gray is OK107-Gal4 (B–D)- or R71G10-Gal4 (H–I)-driven mCD8::GFP. Green is R71G10-Gal4 (E–G,J–L)-driven mCD8::GFP. Magenta represents FasII staining.(M) Box-plot quantification of γ-axon regrowth, depicted as a regrowth index.See Yaniv et al. (2012) for quantification method. (N) Box-plot quantification ofphenotypic severity shown as ratio of the SCC length to the entire lobe length.Scale bars: 20 µm. ***P<0.005.

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quantified branches (>5 µm) and found that tsr mutant axons hadsignificantly fewer branches than WT axons (Fig. 2A,B,D).Consistent with their effect on axon growth, expression of bothUAS-tsr.N and UAS-tsr.S3A within tsr mutant axons rescued thebranching defect, interestingly to a similar extent, while UAS-tsr.S3E did not (Fig. 2C,D; Fig. S3).Taken together, we propose that Tsr promotes axon elongation as

well as branching. Whether these two functions are linked and utilizethe same mechanism of action remains unclear. Interestingly, a recentstudy suggests that axon branching and growth of γ-neurons could belinked. The authors propose that branching allows axons to bypassobstacles more efficiently (Razetti et al., 2018). In addition, a recentpaper demonstrated that the MT regulator Efa6 is also required forboth normal branching and growth, further implying that these twoprocesses may be mechanistically related, even in cultured neurons,where presumably the extracellular environment plays a lesser role(Qu et al., 2019). Even inNBCs, tsrmutant axons predominantly stopat the branch point, suggesting that branching is key to successfulgrowth. This might also be consistent with the lack of growth defectsin α/β neurons as they have much simpler morphology.

Tsr mutant γ-neurons show reduced neurite dynamicsTo further probe the effects of Tsr on growth dynamics, we wantedto subject neurons undergoing growth to time-lapse imaging.Unfortunately, live imaging of SCCs during remodeling ispractically impossible because the process occurs in a deep pupalstructure. Likewise, SCCs are extremely difficult to visualize in theex vivo brain culture system that we have devised (Rabinovich et al.,2015). In order to circumvent this, we turned to primary cultures ofsparsely labeled but densely plated WT or mutant γ-neurons(Marmor-Kollet and Schuldiner, 2016). Brains containing WT ortsrN121 MARCM clones were dissociated and plated for 2 daysin vitro (DIV) before being subjected to time-lapse imaging(Fig. 2E,F). WT neurons exhibited dynamic extension–retraction(Fig. 2E,G; Movie 1), while, in contrast, tsrN121 neurons exhibitedsignificantly reduced neurite dynamics (Fig. 2F,G; Movie 2).Additionally, tsrN121 neurons grew shorter neurites (Fig. 2H) andbranched less (Fig. 2I), which is similar to the in vivo effects shownabove. Additionally, subjection of primary cultures of WTγ-neurons to latrunculin (known to promote actin disassembly,Spector et al., 1983) or jasplakinolide (known to interfere with

Fig. 2. Tsr is required for γ-neuron branchdynamics. (A–C) Confocal Z-projections of WT(A), tsrN121 (B) or tsrN121 additionally expressingUAS-tsr.N (C) γ-neuron MARCM SCCs.A′–C′ are tracings of the primary axon in red,with secondary branches in cyan. Arrowheadspoint to axonal structures that appear to befailed branch points. Gray is R71G10-Gal4-driven mCD8::GFP. (D) Box-plot quantificationof secondary branches (>5 µm) per 100 µm ofprimary axon. (E,F) Time-lapse series (15 minincrements) of confocal Z-projections of WT (E)or tsrN121 (F) primary γ-neurons derived from L3brains. White and yellow arrows point toextending or retracting branches, respectively.Gray is R71G10-Gal4-driven mCD8::GFP.(G) Box-plot quantification of cell dynamicsexpressed as change in neurite length dividedinto extensions and retractions per 1 h imagingaveraged over 3 h. (H) Box-plot quantificationsof total neurite length per cell. (I) Box-plotquantification of number of branches per cell.Scale bars: 20 µm. *P<0.05; ***P<0.005.

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disassembly, Cramer, 1999) both resulted in decreased sproutingand growth in vitro (Fig. S3C–F). Taken together, these data suggestthat precise actin dynamics and the balance between assembly anddisassembly are crucial for normal neurite extension and branching.

Tsr does not promote regrowth by increasing G-actin orbarbed ends availabilityTo inspect the hypothesis that Tsr promotes regrowth by increasingG-actin levels, as suggested by the ‘treadmilling’ model (Shekharand Carlier, 2017), we performed epistasis experiments in which weaimed to increase the availability of G-actin. Increasing G-actinavailability by overexpressing either Act5C (the fly ortholog ofβ-actin) or Chic, the fly profilin (which delivers G-actin to actinelongation factors), both significantly exacerbated the regrowthdefect of tsrN121 SCCs, in contrast to our original prediction basedon the aforementioned hypothesis (Fig. 3A–D,H; Fig. S4A,B).Based on these results, wewanted to determine whether profilin andcofilin antagonize each other in the context of axon regrowth. In aparallel project, we demonstrated that perturbing Chic (by RNAi ormutant analysis) also results in a severe γ-axon regrowth defect(Yaniv et al., 2020; Fig. S4C), which can be rescued byoverexpressing UAS-Chic. Accordingly, and again in contrast toour original prediction, knocking down Chic levels by RNAi withintsr mutant clones ameliorated growth defects (Fig. 3E,H), indeedsuggesting they counteract the function of each other.Next, we aimed to examine the hypothesis that Tsr-mediated

severing might promote growth by increasing the fraction of barbedends that are accessible to polymerization. We thus expressed RNAitransgenes targeting capping protein-α (Cpa) and capping protein-β(Cpb) (Ogienko et al., 2013). Inconsistent with the abovehypothesis, we found that knocking down Cpa (but not Cpb)

enhanced the tsrN121 regrowth defect (Fig. 3F–H, Fig. S4D). Giventhat Cpa and Cpb are known to function as an obligatory heterodimer(Schafer and Cooper, 1995), our results probably stem from differentRNAi efficiency, something we unfortunately cannot test due tounavailability of antibodies targeting capping proteins.

Taken together, these results suggest that increasing G-actinconcentration (by overexpressing Act5C or Chic) or increasingthe availability of barbed ends (through knockdown of Cpa)exacerbates growth defects, while reducing F-actin (by knockingdown Chic) ameliorates it. Unfortunately, the currently availablereporters to examine the G-actin to F-actin ratio did not result inmeaningful results due to high background, likely due to the denseneuropil structure. Nonetheless, together these data suggest that Tsrdoes not promote regrowth by increasing actin polymerization.Instead, since increasing G-actin further impairs regrowth in Tsrmutants, we suggest that Tsr may be important for clearing actinpolymers.

Microtubule localization is disrupted in tsr mutantsWe next turned to examine whether Tsr is required for theelimination of actin structures, thereby allowing MT elongation(Flynn et al., 2012; Tedeschi et al., 2019). First, we determined thatF-actin levels were indeed increased within tsr SCCs compared toWT (Fig. 4A–C). Next, we co-expressed membrane-bound tandemtomato (mtdT) and a tubulin reporter (α-tubulin84B.tdEOS), which,in WT SCCs, demonstrated that MTs and membrane markers seemto overlap (Fig. 4D). In contrast, MTs did not seem to extend intothe enlarged filopodia-like structures (Fig. 4E) in tsr mutants.Interestingly, quantification of the colocalization (assessed by thePearson’s coefficient) revealed that colocalization was decreased inboth axon branch points and tips but remained unaffected in the

Fig. 3. Tsr does not promote regrowth byincreasing G-actin levels or by uncapping.(A–G) Confocal Z-projections of WT (A), tsrN121

(B) or tsrN121 additionally expressing UAS-act5c (C),UAS-Chic (D), chic RNAi (E), cpa RNAi (F) or cpbRNAi (G) in γ-neuron MARCM SCCs. The dashed linerepresents the average ratio of tsrN121 SCC to thelength of the γ lobe, which is 0.4. Green is R71G10-Gal4-driven mCD8::GFP. Magenta represents FasIIstaining. (H) Box-plot quantification of phenotypicseverity shown as ratio of SCC length to entire lobelength. Scale bar: 20 µm. *P<0.05; ***P<0.005;N.S., not significant.

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main axon shaft (Fig. 4F). These results suggest that Tsr seversF-actin to clear actin aggregates, which, in turn, is crucial for properMT protrusion. To demonstrate this in a more direct manner, weattempted to express the MT reporter α-tubulin84B.tdEOS incombination with the F-actin reporter F-tractin but unfortunatelydue to rapid bleaching were unable to extract meaningful high-resolution images. Therefore, while our findings support thehypothesis that Tsr reduces F-actin accumulations to allow MTelongation, further exploration is necessary. These findings areconsistent with in vitro studies demonstrating increased F-actin levelsin ADF/cofilin mutants. These increased F-actin levels resulted inaccumulations that were suggested to obstruct and misdirect MTscausing a decrease in neurite formation (Flynn et al., 2012).To conclude, we have delved deeper into the role of Tsr during

axon formation, including in regrowth and branching. We havefollowed up on several in vitro studies, and showed that Tsrregulates growth and branching in vivo, presumably by severingF-actin thereby allowing the protrusion of MTs into the potentialgrowth cone.

MATERIALS AND METHODSGeneration of MARCM clonesMARCM clones of MB neurons and single cell clones were generated by a1 h heat-shock of newly hatched larvae (24 h after egg laying) and examinedat the indicated developmental stages, as described previously (Lee et al.,1999).

Immunostaining and imagingDrosophila brains were dissected in cold Ringer’s solution, fixed using 4%paraformaldehyde for 20 min at room temperature (RT), and washed inphosphate buffer with 0.3% Triton-X (PBT) (three immediate washes andthree 20-min washes). Non-specific staining was blocked by 5% inactivatedgoat serum in PBT. Brains were subjected to primary antibody overnight at4°C. Primary antibodies included: chicken anti-GFP (GFP-1020; AVES),1:500; and mouse monoclonal anti-FasII (1D4), 1:25 (DevelopmentalStudies Hybridoma Bank; DSHB). Brains were washed with PBT (threeimmediate washes and three 20-min washes), and then stained withsecondary antibodies for 2 h at room temperature. Secondary antibodiesincluded: goat Alexa Fluor 647-conjugated anti-mouse-IgG (A-21236;Invitrogen), 1:300; and donkey FITC-conjugated anti-chicken-IgG (703-095-155; Jackson Immunoresearch), 1:300. Brains were mounted onSlowfade (S-36936; Invitrogen) and imaged on Zeiss LSM 800 confocalmicroscope. Images were processed with ImageJ (NIH).

Drosophila melanogaster rearing and strainsAll fly strains were reared under standard laboratory conditions at 25°C onmolasses-containing food. Males and females were chosen at random.Unless specifically stated otherwise, the relevant developmental stage isadult, which refers to 3–5 days post eclosion.

tsrN121, tsrN96A, UAS-tsr.N, UAS-tsr.S3A, UAS-tsr.S3E, UAS-act5c, UAS-LifeAct-Ruby, UAS-F-tractin.tdTomato, UAS-α-tubulin84B-tdEOS and RNAilines (cpa, cpb, chic) were all obtained from the BloomingtonDrosophila stockcenter (Indiana University, USA). UAS-Chic was kindly provided by FlorenceBesse (Institut de Biologie Valrose, Nice, France) (Medioni et al., 2014).

Drosophila genotypes used in this study are listed in Table S1.

Fig. 4. Microtubule localizationis perturbed in tsr mutants.(A,B) Confocal Z-projections of WT(A) or tsrN121 (B) adult γ-neuronMARCM SCCs. Yellow dashedsquares demark representativeregions used for quantification ofLifeAct intensity in the axon shaft andtip. Green and cyan are R71G10-Gal4-driven mCD8::GFP or LifeAct-Ruby, respectively. Gray is R71G10-Gal4-driven mCD8::GFP (A1,B1) orLifeAct-Ruby (A2,B2). Magenta isFasII staining. Scale bar: 20 µm. (C)Box-plot quantification of LifeAct toCD8::GFP intensity ratio in markedregions in A,B. (D,E) Confocal Z-projections of WT (D) or tsrN121 (E)adult γ-neuron MARCM SCCs. Insetscorrespond to dashed boxes asexamples for regions used forcolocalization analyses – in this case,an axon tip. Magenta and green areR71G10-Gal4-driven mtdT orα-tubulin84B-tdEOS, respectively.Gray is R71G10-Gal4-driven mtdT(D1,E1) or α-tubulin84B-tdEOS (D2,E2). Scale bar: 10 µm. (F) Box-plotquantification of mtdT (membranes)and α-tubulin84B-tdEOS (MT)colocalization. In each neuron at leastone axon tip, branch point and axonshaft were quantified. Colocalizationwas tested using Pearson’scoefficient. While in tsrN121 there isless colocalization in both the axon tipand branch point, there is nodifference in the main axon shaft.**P<0.01; ***P<0.005; N.S., notsignificant.

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Sprouting assayDissociation and plating of cells has been previously described (Marmor-Kollet and Schuldiner, 2016). Briefly, L3 brains were dissociated in 10 mg/ml collagenase (Sigma), washed three times with PBS and resuspended inSchneider’s medium (Sigma) supplemented with 10% heat-inactivated fetalbovine serum and antibiotics. Dissociated cells were plated in glass-bottom96-well plates coated with poly-L-lysine (MatTek) at ∼10 brains/well in avolume of ∼30 µl in culturing medium. At 1 h after plating, medium wasadded to give a total volume of 200 µl. Fluorescent cells were imaged 2 daysafter plating (DIV) and neurite length was calculated using the SimpleNeurite Tracer plugin in Fiji. When indicated, 10 µM latrunculin B or 10 µMjasplakinolide (Life Technologies) were added 1 DIV and incubated anadditional 24 h before imaging.

Sprouting live imaging was performed on cultured neurons labeled withCD8:GFP and Ftractin-tdT starting at 48 h in vitro (2DIV). They wereimaged over a period of 10 h, at 15 min intervals. For analysis of dynamics,total neurite length was measured at every 15 min interval (using the SimpleNeurite Tracer plugin) for a period of 3 h. Changes between time pointswere summed for each 1 h period and averaged over 3 h.

Statistical analysisWe strove for n>10 brain hemispheres; n values are shown in thequantifications within the figures. In the sprouting experiments, n representscells. No samples were excluded from the analyses.

For the quantification of developmental regrowth in MARCM clones(Fig. 1M), we used a method previously described (Yaniv et al., 2012). Inshort, we determined the γ lobe occupancy by comparing the clonal (GFP)versus non clonal (FasII staining) in the Z-plane cross section. To calculatethe regrowth index, we then divided the lobe occupancy of the clonal axonsat a distal section by a proximal section.

For analysis of SCCs (Figs 1N and 3H), the furthest point of innervation intoadult γ lobe was measured on the confocal Z-stack and divided by the totallength of the γ lobe (determined by FasII staining). See Fig. S1F for details.

For in vivo branching (Fig. 2D), the number of secondary branches over>5 µm were counted and normalized to 100 µm of axon length.

For LifeAct–Ruby intensity studies (Fig. 4C), in each image the intensityof both LifeAct–Ruby and GFP were measured at two different points alongthe axon as well as at the axon tip and normalized to the background.

For MT and membrane-bound RFP colocalization studies (Fig. 4F), ineach neuron, at least two branch points, two axon terminals (if available) andtwo points along the main axon were measured for both GFP and RFP.Colocalization, represented by Pearson’s coefficient, was measured usingthe Coloc2 Fiji plugin.

Statistical analysis was performed by a one-way ANOVA including allgroups followed by a Tukey post-hoc test (Figs 1M,N and 3H) or Student’st-test (Figs 2G–I and 4C,F).

For all box plots, boxes encompass the values in between the 1st–3rdquartiles; whiskers are ±1.5 interquartile range (IQR), the median isrepresented by the line, the mean by the blue triangle, and outliers by reddiamonds.

AcknowledgementsWe thank F. Besse and the Bloomington Stock Center for reagents; monoclonalantibodies were obtained from the Developmental Studies Hybridoma Bankdeveloped under the auspices of theNICHDandmaintained by the University of Iowa.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: S.S., S.Y., H.M., O.S.; Methodology: S.S., S.Y., H.M., O.S.;Validation: O.S.; Formal analysis: S.S., S.Y., H.M., O.S.; Investigation: S.S., S.Y.,H.M.; Resources: O.S.; Data curation: S.Y., H.M.; Writing - original draft: S.Y., O.S.;Writing - review& editing: H.M., O.S.; Supervision: S.Y., O.S.; Project administration:O.S.; Funding acquisition: O.S.

FundingThis work was supported by the European Research Council (ERC), consolidatorgrant ‘AxonGrowth’ (#615906). Fly food for this project was funded by the Women’s

Health Research Center. O.S. is the Incumbent of the Prof Erwin Netter ProfessorialChair of Cell Biology. Open access funding provided by Weizmann Institute ofScience. Deposited in PMC for immediate release.

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/doi/10.1242/jcs.232595.supplemental

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