Furthermore, KLC4 is required for proper tiling of peripheral axon arbors: in klc4 mutants, peripheral axons showed abnormal fasciculation, a behavior characteristic of central axons. This result suggests that KLC4 patterns axonal compartments and helps establish molecular differences between central and peripheral axons. Finally, we find that klc4 mutant larva are hypersensitive to touch and adults show anxiety-like behavior in a novel tank test, implicating klc4 as a new gene involved in stress response circuits.Development of elaborate and polarized neuronal morphology requires precisely regulated transport of cellular cargos by motor proteins such as kinesin-1. Kinesin-1 has numerous cellular cargos which must be delivered to unique neuronal compartments. The process by which this motor selectively transports and delivers cargo to regulate neuronal morphogenesis is poorly understood, although the cargo-binding kinesin light chain (KLC) subunits contribute to specificity. Our work implicates one such subunit, KLC4, as an essential regulator of axon branching and arborization pattern of sensory neurons during development. Using live imaging approaches in  Proper development of complex neuronal morphology and assembly of neural circuits requires tight regulation of axon growth and branching. Precise control over axon branch formation is essential not only for development but also for plasticity and regeneration after injury. Branching allows one neuron to innervate numerous targets, and in many cases, neurons extend multiple axons or branches that navigate to distinct targets. Many molecular signaling pathways can influence branching , althougMolecular compartmentalization of neurons depends on precisely controlled transport of vesicle and organelle cargos to specific cell locations. Cargos are transported by motor proteins that move in a directional manner along MT tracks. Kinesin-1, the founding member of the kinesin superfamily of motor proteins, is a heterotetramer composed of homodimers of kinesin heavy chains (KHCs), which contain the MT binding and ATPase motor domains, and of kinesin light chains (KLCs), which interact with cargos. Kinesin-1 can mediate long-distance, anterograde transport of multiple cargos including mitochondria, synaptic vesicle precursors, endosomes, lysosomes, and RNA granules . Kinesinklc genes (klc1-4), with klc1, 2 and 4 expressed in the nervous system, but our knowledge of the different roles played by individual KLCs in neurons remains very limited. In Drosophila, mutants for the single klc gene are lethal in larval stages and have defects in neural development  that manifests in early childhood , a C-terminal lipid-binding amphipathic helix, and an LFP motif that mediates KLC auto-inhibition  ganglia  fish for three generations to remove any off-target mutations from the background. In situ hybridization analysis of homozygous uw314klc4 mutant embryos showed virtually no klc4 mRNA expression, suggesting that the mutant transcript undergoes nonsense-mediated decay ] (Because p-caax)]  and usedp-caax)] . Inspectp-caax)] . To quanp-caax)] . We counp-caax)] . Consistp-caax)] . The redp-caax)] .uw314klc4 mutants  and interstitial back branching (IB) (Tg(–3.1ngn1:gfp-caax) transgenic line (uw314klc4 mutant and wild-type embryos (uw314klc4 mutants.To investigate which specific dynamic processes during branching are affected by ing (IB) . To detenic line . Z-stack embryos . In bothuw314klc4 mutants, 57.7% of GCB branches and 61.9% of IB branches retracted after initiating, compared to 14.2% of GCB and 11.8% of IB branches in wild type  it retracts or changes direction to grow away from the other axon, thus minimizing contact between axons . Howeveristances . In some mutants . Fascicu mutants . Togetheuw314klc4 mutants also showed abnormal morphology suggestive of altered cell polarity. We found that some RB neurons in uw314Tg(–3.1ngn1:gfp-caax);klc4 mutant embryos and Kinesore-treated embryos projected a process from the apical cell body that crossed the midline within the dorsal spinal cord (uw314klc4 mutants had midline crossing processes compared to 1% in wild type embryos (n=608 neurons in 21 embryos)  . Similarembryos) . To furt embryos , suggestngn1:GFP-Rab5c DNA into embryos at the one-cell stage . We co-injected tivation . We prevtivation . To quanh points .uw314klc4 mutants. To visualize MT dynamics in vivo, we used the MT plus-tip binding protein EB3 fused to GFP  and double-labeled neurons with anti-HNK1 and an antibody to acetylated tubulin, a modification found on stable MTs . We anal mutants . This fiuw314Klc4 mutant larva swam for a longer total time  and performed more swim bouts  after touch stimulus than wild type larva  were given a light tail touch, evoking a swim response . The timpe larva , suggestuw314klc4 mutants seemed likely to lead to deficits in adult fish as well. Indeed, we found that uw314klc4 mutants are adult viable and fertile but are smaller in size than their wild-type siblings (uw314klc4 mutants from both AB and mixed AB/TL backgrounds and separately in-crossed their wild-type (AB) siblings. The F5 embryos were born on the same day, raised side-by-side in an identical environment, and fed the same diet. We measured the body length of these fish at 7 weeks and at 9 weeks  of individuals tested freezing for more than 3 s during first 3 min of exposure to the novel tank  was compared to their mobile swim velocity (with pauses in swimming subtracted)  . klc4uw3immobile .uw314klc4 mutants did show habituation as measured by the percentage of time spent in the upper zone of the tank  axis is an important system that mediates stress responses, and zebrafish mutants with disruptions to the HPA also exhibit anxiety behavior in the novel tank test. Mutation of zebrafish glucocorticoid receptor, a target of the stress hormone cortisol, results in increased freezing and decreased exploratory behavior. Like uw314klc4 mutants, glucocorticoid receptor mutants fail to habituate freezing behavior after repeated exposures to the novel tank  were kept in a 14/10 hr light/dark cycle. Embryos were maintained at 28.5 °C and staged as hours postfertilization (hpf) as described previously (Tg(−3.1ngn1:GFP-CAAX) line as a background, wild type is used to refer to Tg(−3.1ngn1:GFP-CAAX), +/+klc4 animals. All animals in these studies were handled in accordance with the National Institutes of Health Guide for the care and use of laboratory animals, and the University of Wisconsin Institutional Animal Care and Use Committee (IACUC). These studies were approved by the University of Wisconsin IACUC (protocols L005692 and L005704).Adult zebrafish (eviously . Wild tyeviously  (AB/TL bklc4 (5’-GAGCGTGGCACAGCTGGAGGAGG-3’) was designed by using CRISPRScan (crisprscan.org) and synthesized as previously described . Injection volume was approximately 1 nL. F0 adult fish were pair-mated to determine individuals carrying germ line mutations. A subset of individuals with germ line mutations were outcrossed to wild type AB fish. F1 fish were raised and genotyped by PCR and sequencing, using CRISP-ID (http://crispid.gbiomed.kuleuven.be/) to assist in sequence unmixing . A set of primers for exons exclusive to the long isoform of klc4 (spanning exons 7 through 12) was designed for the long isoform . PCR was then run to test for presence of the isoforms in cDNA from each embryonic age.RNA was extracted from zebrafish embryos at specified ages with Trizol (Thermo-Fisher), followed by chloroform extraction and isopropanol precipitation. cDNA was generated from embryo-derived RNA using the Superscript III reverse transcriptase kit and a 50:50 mixture of oligo dT and random hexamer primers (Invitrogen). We designed PCR primers to amplify specific klc4 mRNA, which are contained in both long and short isoform mRNAs, was synthesized by in vitro transcription using T7 RNA polymerase with a DIG RNA labeling kit (Roche). The template for transcription was made using PCR on zebrafish cDNA with the following primers: Fwd: 5’-GCGCAAATCAGTGGAGATGATAGAGC-3’, Rev: 5’-TAATACGACTCACTATAGCTCCAGATGTTTCTTCTCCTCC-3’. A second probe recognizing a region in exons 8–11, found only in the long isoform, was synthesized using PCR on zebrafish cDNA with the following primers: Fwd: 5’-CAG AGA GAA GGT GCT GGG-3’, Rev: 5’- TAATACGACTCACTATAG GGTGGTGTTCACTGTTGG-3’. Whole-mount in situ hybridization was performed as previously described  and blocked with incubation buffer  for at least 1 hr. Embryos were incubated overnight at 4 °C with monoclonal anti-HNK1 antibody in IB , rinsed in PBST, then incubated with anti-mouse AlexaFluor 488 secondary antibody  for at least 2 hr at room temperature or overnight at 4 °C. For double labeling with anti-acetylated tubulin , the IgG subtype-specific secondary antibodies anti-mouse IgG1 AlexaFluor 488 and anti-mouse IgG2b AlexaFluor 647  were used.Embryos were fixed overnight at room temperature in 4% paraformaldehyde in 0.1 M PO3.1ngn1:EB3-GFP DNA (3.1ngn1:mCherry-CAAX DNA (DNA expression constructs were made using the Multisite Gateway Cloning System (Invitrogen) into Tol2 vectors . Zebrafi-GFP DNA  to visuaCAAX DNA .Kinesore was acquired from the Hit-2-Lead library  and suspended in DMSO for a stock concentration of 100 mM. Embryos treated with Kinesore were dechorionated at 17 hpf and placed in E3 containing 150 µM Kinesore and 2% DMSO. Controls were treated with 2% DMSO in E3 for the same time period.3.1ngn1:GFP-CAAX) wild type or mutant embryos were mounted in an FEP tube  suspended in 0.02% tricaine in E3 with an agarose plug to prevent leakage.Light sheet imaging was performed on a custom multi-view light sheet microscope as described previously . The det3.1ngn1:GFP-CAAX) embryos with either an Olympus FV1000 laser-scanning confocal microscope using a UplanSApo 20 x Oil objective (NA 0.85) or an Opterra Swept-Field confocal (Bruker Fluorescence Microscopy) with a Nikon S Fluor 40 x Oil immersion objective (NA 1.30). Imaging was started between 18 and 19 hpf, when peripheral RB axons are extending out of the spinal cord. Optical sections (1–1.25 µm) covering a 30–36 µm Z depth were captured every 60–90 s for 2–4 hr.For live confocal imaging, embryos were anesthetized in 0.02% tricaine and mounted in 1% low melting agarose in 10 mM HEPES E3 medium as described previously . Axon ouHigh speed imaging of EB3-GFP comets was performed with the Opterra Swept-Field confocal microscope equipped with a Nikon Plan Apo VC 100 x oil immersion objective (NA 1.40). Embryos were imaged at stages between 18 and 26 hpf, during peripheral axon outgrowth. EB3-GFP comets in nascent branches were imaged at stages between 22 and 25 hpf with a Nikon Plan Apo 60 x oil immersion objective (NA 1.40). Z-stacks of 5–30 1 μm optical sections were captured at 2–5 s time intervals, for total durations between 7 and 10 min. High speed imaging of Rab5 vesicle dynamics was done with the Opterra Swept-Field confocal and the Nikon Plan Apo 100 x oil objective (NA 1.40). Embryos were imaged between 22 and 26 hpf. Z-stacks of 5–30 μm optical sections were captured at 2–5 s time intervals for a total duration of 7–10 min. Photoactivation and imaging of PA-GFP-Rab5 were performed on the Olympus FV1000 confocal using a 60 X UplanSApo oil immersion objective (NA 1.35). A region of interest encompassing the cell body was activated with the 405 nm laser during the scan of one Z-plane in the cell body. Z-stacks of 1 µm optical sections were captured every 30–170 s for durations ranging from 26 to 34 minutes .https://ibidi.com/chemotaxis-analysis/171-chemotaxis-and-migration-tool.html) to generate velocity measurements and rose plot histograms. Rose plots were re-plotted in MATLAB (MathWorks) using secplot .To measure the speed of axon outgrowth, the area immediately behind the growth cone was measured using the Manual Tracking plugin in FIJI (Fabrice Cordelières). The generated data was loaded into the Chemotaxis and Migration Tool . Comet or vesicle parameters were measured from the resulting kymographs. To measure Rab5 accumulation at branch points, movies were divided into 4 time periods (t1-t4), each containing 7–8 min. Maximum intensity projections were made for t1-t4 and the fluorescent signal at branch points was measured for each maximum intensity projection. Background signal from the branch point prior to activation was subtracted from measured t1-t4 values, and then all values were normalized to the signal at the t1 period. Comparisons between experimental conditions were made using the Mann-Whitney test for non-parametric data.Hyperstacks of EB3-GFP or GFP-Rab5c images were assembled using FIJI  and segmuw314klc4 homozygous siblings. Larva were placed in 3 L tanks side-by-side and subjected to the same light cycle and feeding schedule during rearing. At 7 weeks and 9 weeks, fish were briefly anesthetized with 0.02% tricaine, placed on white waterproof surface, measured with a ruler, and photographed.Zebrafish were raised for this experiment by in-crossing wildtype or klc4 mutant larva did not respond to an initial touch , and these samples were excluded from analysis.To assess responsiveness to touch, 3 dpf zebrafish larva were individually placed in a 6 cm dish and allowed to acclimate for 2 min. A tail touch was administered to the end of the tail using a gel loading tip attached to a long applicator stick. The tail touch was given from a side angle to avoid looming stimulus. A high-speed camera was used to acquire 50 frames per second for a total of 1050 frames (21 s). The response time of each larva was measured from the first movement after a touch to the cessation of movement for more than 15 frames (inter-bout pauses only lasted ~7–8 frames). Some wild type and Feeding experiments were conducted as in The evening before an experiment, fish were moved to the behavior experiment room and allowed to acclimate overnight. Exposure to novel tank was carried out in the morning, and the order of which genotype received exposure first was randomized. The novel tank had 3 opaque sides to block potential views of other fish and one clear side for imaging. The water in the novel tank was changed between groups. Fish were netted from their 3 L home tank, released into the novel tank, and allowed to explore for 3 min before being removed and placed into a recovery tank. All tested animals were of AB background.wild type: group 1  group 2, , group 3 , group 4 , uw314klc4: group 1 , group 2, , group 3 , group 4 .Two weeks prior to an experiment, adults were sorted into separate 3 L tanks with 5–6 individuals of the same genotype in each. Experiment was conducted in a similar manner as described above, but fish were allowed to explore for 10 min before removal. Novel tank exposure was repeated on the same groups twice a week for 3 weeks. Exposures were conducted a minimum of two days apart. The experiment was repeated for another 3 weeks with an additional two groups of 6 fish per genotype. All tested animals were of AB background. When possible, groups were divided into similar ratios of male and female fish for a total of 11 females and 12 males within each genotype. The group compositions were as follows: For the habituation experiment, automated tracking of zebrafish through ToxTrac  was usedStatistical analyses and graphs were generated with Graphpad Prism. See figure legends for details on sample sizes, specific statistical tests used, error bars and p values. Data were checked for normality using Anderson-Darling test, D’Agostino & Pearson test, and Shapiro-Wilk test before deciding on a statistical test. Non-normal data were tested for significance using a Mann-Whitney test. In some cases, the N values were too low to perform a statistical test . In Figu Using zebrafish as an in vivo model, this study reveals for the first time that mutations in the kinesin light chain gene klc4, which are known to cause a form of early onset hereditary spastic paraplegia in human, affect specific aspects of neuronal development and nervous system functions. High-resolution movies of developing sensory neurons in vivo and behavioral assays support the key findings that the motor subunit Klc4 plays essential functions in the control of neuronal morphogenesis and compartmentalization as well as associated behaviors. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, including Fabienne E. Poulain as Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Didier Stainier as the Senior Editor.Thank you for submitting your article "KLC4 shapes axon arbors during development and mediates adult behavior" for consideration by eLife. Essential revisions can be summarized as follows:After discussing their reviews with one another, and as you will see from the individual reviews below, all three reviewers agree that your study describes novel and interesting functions of Klc4 in sensory axon development and has therefore a potential high impact. However, reviewers share four major concerns that need to be fully addressed before your manuscript can be considered further for publication in 1) The study lacks a detailed analysis of axonal phenotypes in klc4 mutants and would be strengthened by analyzing single arbor dynamics and/or using additional morphological criteria to define branches and analyze axonal density.2) The study lacks mechanistic insight into the roles of KLC4 and the phenotypes observed. Reviewers agree that at least some mechanistic experiments are necessary to explain how klc4 mutations cause axonal phenotypes. These include analyzing the transport of specific cargoes during axonal development and microtubule behavior during axonal branching. The localization and cell-autonomous function of klc4 could also be analyzed.3) There is a disconnect between the description of sensory axonal defects in embryos and the characterization of behavioral defects in adults. Behavioral assays that rely on functional sensory neurons instead (touch-response in larvae for example) would be more appropriate for this study.4) As described in detail by reviewer 2 (see below), multiple concerns about statistical analyses were raised and need to be addressed.Reviewer #1 (Recommendations for the authors):Overall, the manuscript is very well written and the conclusions are well supported by the data. The light sheet and swept confocal microscopy movies provide a unique view of sensory axon development in vivo and are beautiful! Below are a few questions / comments that I had while reading the manuscript:1) The data in figures 3 and 5 show nicely that axon branches are not stabilized and that axonal growth is shifted posteriorly in klc4 mutants. Is the collapse of branches direction-dependent in the mutant? Is there a difference in branch stabilization/pruning depending on whether the branch elongates anteriorly or posteriorly?2) The presence of apical protrusions crossing the midline in klc4 mutants suggests a defect in RB neuron polarity. Is the centrosome mislocalized in klc4 mutants?3) The existence of both Klc4 isoforms is interesting, especially since corresponding transcripts seem to be differently regulated during development. Since the short isoform is not predicted to bind any cargo, is anything known about its functions? Are both isoforms expressed in RB neurons?4) It would be interesting to analyze the localization of Klc4 in sensory neurons, although this might be difficult to do. Would it be possible to mosaically express low levels of tagged klc4 in mutant sensor neurons and analyze its distribution?Reviewer #2 (Recommendations for the authors):The authors use Crisper technology to generate a zebrafish line that lacks expression of KLC4. This animal is then used throughout, along with pharmacological inhibition of KLC binding to adaptor proteins, to address the roles of KLC4 in the development of the morphology of Rohon-Beard (RB) sensory neurons in embryos and assess behavioral alterations in adult fish. Modern imaging methods are applied to study neuronal morphology in embryos and a battery of behavioral tests in adults. The study provides first line of inquiry assessment of the role of KLC4 in the development of RB morphology, focusing on the failure to establish proper axon arbors in the periphery. While each data set has intrinsic value and is novel, none is followed up to provide mechanistic insights into KLC4 functions in neurons. The value of the submission rests in providing a characterization of the effects of KLC4 knockout, but it does not further the understanding of the mechanistic contributions of KLC4 in the processes impacted by the genetic deletion of KLC4. While the significance of the question posed is well presented and clear, multiple issues regarding interpretation, presentation and analysis decrease enthusiasm for the submission in its current form.For the authors' consideration:1. The current in situ data uses a riboprobe that does not differentiate between the short and long forms of the KLC4 transcript. Given the large difference between short and long transcripts of KLC4, and expected differences in function of the resultant proteins, it would assist in the interpretation of the results to assess short and long transcript expression in RB neurons. Ideally, this would be followed up by specific knock down of the long form to dissect its contributions from the short form.2. At first evaluation, it is not clear how the axon branching metric  is specific for axon branching. In the images provided there seem to be consistently fewer axons present as a function of distance from the central fasicle in the KLC mutant and kinesore treated embryos . How can the metric dissociate the number of main axons that arrived at the ROI from branches made from those axons?Based on the high quality of the imaging, it seems that it would be possible to use morphological criteria to define axon branches and measure the density along individual axons. This form of assessment would be much more stringent than the ROI crossing method used. Indeed, this form of assessment is used in the live imaging studies addressing rates or branch formation and stability.3. Considering the above comment (2) regarding the consistent observable decrease in the apparent number of axons and their lengths into the periphery noted above, it is surprising that an increase in the rate of axon extension was detected in the live imaging analysis . It is suggested that the authors analyze the density of axons and their total length as a function of distance from the central axon fascicle, noting that the ROI based approach in supplemental Figure 2 should serve this purpose by simply changing the distance from the fascicle into the periphery at constant intervals (currently only 65 microns was addressed).Based on the images presented I would expect that a decrease in axon density and length will be determined in the KLC4 mutant. If so, then these data would stand in conflict with the observed increase rate of axon extension, but may indicate that earlier aspects of axon formation were also impacted in the KLC4 mutant. The formation of axons from the cell body may share mechanistic similarity to the emergence of branches, and thus may be delayed in the mutant due to failure to stabilize the new axon at its early stages of formation which could then result in the difference suggested by the images provided without being confounded by the subsequent increase in axon extension rate. It would also be insightful to perform this analysis at multiple points in ontogenesis.4. An interpretational caveat is that with both the genetic mutant and the drug treatment all cells are expected to be affected. From the in situ is it not clear to this reviewer if there is no expression of KLC4 in the environment the axons/branches are migrating through. Might this issue be resolved using zebrafish genetics with lines allowing for specific knockdown in RB neurons? or some other approach to address whether the effects are cell intrinsic.5. Statistical analysis. There are multiple concerns regarding the statistical presentation and analysis.The section in the methods requires much elaboration, starting with which test was used to determine the normalcy of data sets, and addressed by subsequent comments.Figures4 and 10 require statistical analysis of the data. "Plots using 95% confidence intervals (CI) were considered to be significant when the 95% CI ranges did not overlap." Is not considered a stringent analytical method, although a valid "rule of thumb". The authors are urged to seek assistance from a statistician.6E, no 95% CI shown as claimed in the legend. The mean is not likely to be significant as the WT only have 3 points and one is an outlier relative to the other 2. I also do not see how the mean in the +/+ could be as shown, as claimed in the legend, it seems to be the median. I would estimate the mean to be somewhere around 20 um. For the mutant data, the distribution does not strike me as normal. Were these data analyzed for normality? Also, there is no statistical analysis shown, appreciating that with an n=3 for the WT this is problematic and power would have to be increased through greater sampling.The legend to Figure 8 states that t-test with Mann-Whitney correction are used. I am not familiar with this correction for a t-test and was not able to find references to such a correction for t-test. Was Welch correction intended? The statistical description in the method is very sparse as presented and details should be presented. How the basic assumptions of the rather limited t-test were met by data prior to selection of specific t-test should be clearly denoted.Figure 9. In panel C, a t-test is used but (1) the data are bounded values, and (2) the distribution in WT is clearly non normal with many data points at the boundary . A t-test is not applicable as basic assumptions are broken, and do not appear to have been determined. E and H have no p values or test shown in legend and it is not clear why individual data points are not shown here as in other panels. F and G have no test denoted. The meaning of * and multiples thereof is only defined for one panel. Panel G has *** not defined at all.6. Strictly speaking, the authors are inferring contact repulsion but do not use the imaging data sets to directly address it . The observed changes in angles between sister branches could be due to a variety of other mechanisms altering the ability of sister branches to generate larger angles.The self-contact examples in Figure 6 are not particularly compelling. Perhaps, presentation of all relevant frames in the timelapse with greater annotation and description would assist a reader.In Figure 6B, in contrast to what the text claims, this reviewer does not see a clear growth cone bifurcation followed by fasiculation due to lack of self-repulsion. It appears a small back branch from the second panel from the left aligns with the main axon.Also, the "appearing to collapse onto one another" (line 232) could be interpreted as a consequence of the prior analysis of branch stability.7. Figure 8D,E. "Comet Frequency" in comets/um/min. It is not clear if this reflects new comets observed during the imaging or all observed comets/um/min.The imaging sets would benefit from additional analysis to extract the duration of EB3 comets  and total distance from initiation to loss of the comet.It is not clear why central axons are analyzed for only these data sets and not others. As the focus of the paper is on peripheral axons the suggested additional data sets for peripheral axons would suffice.As a whole, I would suggest that the data in Figure 8 from EB3 imaging indicate increased polymerization of plus tips along axons in the mutant. This is not immediately reconcilable with decreases branch stability as one would presume that microtubules would be more prone to catastrophe and depolymerization in conjunction with decreased branch stability. The data are broadly consistent with the detected increase in axon extension rate. However, analysis of EB3 comets needs to be provided in branches  in order to be linked to the main observed phenotype of decreased branch stability.8. While of general interest, the behavioral assays do not address RB neuron function or circuitry, which is embryonic. The fit of these data to the rest of the study is not clear. Could tail startle be assessed  Curr Biol 27:697-704) for addressing RB/RB-related circuit function? Any RB function assays would provide a much better fit to the rest of the data presented.Importantly, these behavioral deficits cannot be attributed to changes in circuit formation, branching, axon extension etc as the mutant is not neuron specific, the relevant circuitry has not been analyzed, and no aspect of CNS neurons or glia or anything else has been analyzed. The concern is compounded by the differences in overall growth of the fish.9. One fundamental aspect of the study that is missing is consideration of KLC4 localization in the RB neurons. Whether it localizes to the RB axons remains unknown, or whether it ever targets to branches. One would presume so, but this needs to be assessed. It is hard to interpret results with any degree of mechanistic insight without knowledge of the localization of the molecule addressed.10. Final considerations: Overall, this submission contains interesting and valuable data sets that are however preliminary in nature as none is followed up to achieve a deeper mechanistic understanding. This manuscript presents a characterization of phenotypes in KLC4 mutant zebrafish RB neurons but does not provide insights into how KLC4 is mechanistically contributing to the observed phenotypes, or whether it is acting cell intrinsically. Having said this, the discussion carefully considers the potential implications of the individual findings and notes relevant future directions.Reviewer #3 (Recommendations for the authors):Further characterization of axonal defects:– While the population-based methods for characterizing axonal defects are elegant and informative, these defects could potentially be further appreciated with single cell resolution, What do the arbors of individual neurons look like in these mutants? Does the mutant affect all sensory neurons equally, or is a sub-population of neurons more severely affected? It should be possible to analyze single neurons with transient transgenesis in the zebrafish model. This could be particularly helpful since aberrant fasciculation may make it difficult to distinguish multiple fasciculated axon branches when the whole population of neurons is labeled. Identifying defects at a single neuron level could also make it possible to carry out rescue experiments to address the cell autonomy of axon arborization defects.Explanation of axonal defects mechanistically:– The impact of this paper would be most enhanced by characterizing the axonal trafficking of cellular cargos in klc4 mutants, particularly if defects in specific trafficked cargoes could plausibly explain the axonal defects. In the discussion of the paper, the authors note that they have previously found that endosomal trafficking regulates branching. Characterizing endosomal trafficking, as well as trafficking of mitochondria, lysosomes, golgi, etc, thus has the potential to make this study substantially more mechanistically satisfying.– The changes in microtubule dynamics of klc4 mutants are interesting, but how trafficking defects affect these dynamics, and how defects in MT dynamics affect branching, is unclear. To address how trafficking affects MTs, the authors could attempt to identify MTOCs in axons. For example, since golgi outposts are thought to be MTOCs, is their trafficking into axons affected by klc4 mutation? To address how MT defects affect branching, the authors could characterize MT dynamics during branching-for example, do mutants display any defects in the frequency of MT invasion into nascent branches?Behavior:– As mentioned above, the analyses of adult behavioral defects, while interesting and rigorously carried out, do not complement the morphological analysis in larval sensory neurons. It would be exciting if axonal arborization defects in a specific neuron type could be linked to behavioral defects. Could the authors attempt to characterize touch-response behaviors in larval fish? This would be a particularly exciting if it were possible to do cell-autonomous rescue experiments, thus directly testing if defects in sensory neuron morphology affect behavior. Essential revisions1) The study lacks a detailed analysis of axonal phenotypes in klc4 mutants and would be strengthened by analyzing single arbor dynamics and/or using additional morphological criteria to define branches and analyze axonal density.To get more detailed information about axon density, we increased our image sampling by using a grid ROI instead of a single line ROI. This analysis approach provides information about axon density as a function of dorsal-ventral location as suggested by Reviewer 2, and also gives information about both growth cone bifurcation and interstitial branching. Details are described in response to Reviewer 2, points 2 and 3 below, and data added to Figure 2F, G, and Supplemental Figure 2. Although all neurons were labeled in our live imaging of axon growth and branching experiments, we were able to distinguish axons from individual neurons, and the analysis of branch dynamics was done on individual neurons. Further description of our reasoning on this point is in our response to Reviewer 3, point 1.2) The study lacks mechanistic insight into the roles of KLC4 and the phenotypes observed. Reviewers agree that at least some mechanistic experiments are necessary to explain how klc4 mutations cause axonal phenotypes. These include analyzing the transport of specific cargoes during axonal development and microtubule behavior during axonal branching. The localization and cell-autonomous function of klc4 could also be analyzed.klc4 mutants did not have a decrease in endosome delivery to branch points, high-speed imaging of vesicle dynamics revealed some alterations in transport of these vesicles in klc4 mutants. These alterations in endosome transport are not likely to be the only effects of klc4 mutation on cargo dynamics or to fully explain the mechanism by which KLC promotes branch stabilization. However, an exhaustive analysis of all potential cargos would require many months to years of work and is outside the scope of this manuscript. Further description is in response to Reviewer 3, point 2.We added new experiments to image endosomal dynamics , trafficking of Rab5-labeled endosomes from the cell body to branch points , microtubule behavior during axon branching , and acetylation of microtubules in nascent branches . We chose Rab5-labeled endosomes because of our previous work showing a correlation between their transport and RB axon branching. Although the klc4 mutants, there is no difference in frequency of comets entering nascent branches, suggesting a relative decrease. We used anti-acetylated tubulin labeling to show that klc4 mutants have fewer nascent branches with stable, acetylated tubulin. Further description is in our response to Reviewer 2, point 7 and Reviewer 3, point 3.We also added new experiments analyzing microtubules in nascent branches . We used live imaging with EB3-GFP to measure frequency of comets entering nascent branches. Although comet frequency in established axons is increased in klc4 mutation are likely cell autonomous because klc4 is not expressed in the somites or the skin, which are the tissues in contact with RB peripheral axons, and RB neurons are the predominant cells in the dorsal spinal cord that express klc4 at the stages when we see the axon phenotypes.As described in our response to Reviewer 2, point 4, we believe the effects of Traffic 20:851-866). Because the vast majority of kinesin light chains and heavy chains exist in an auto-inhibited conformation that is freely diffusible, the diffuse signal resulting from expression of tagged proteins obscures visualization of motors bound to cargos. Thus, we are unfortunately not able to image KLC4 protein localization with current available approaches.As described in response to Reviewer 1, point 4 and Reviewer 2, point 9, there are major obstacles that prevent visualization of KLC4 protein localization within neurons. There are no available antibodies to zebrafish KLC4. We tried using expression of KLC4-GFP fusion constructs (as suggested by Reviewer 1), but expression leads to diffuse labeling throughout the cytoplasm. This problem has been previously reported by others attempting to visualize kinesin-1 heavy chains  There is a disconnect between the description of sensory axonal defects in embryos and the characterization of behavioral defects in adults. Behavioral assays that rely on functional sensory neurons instead (touch-response in larvae for example) would be more appropriate for this study.klc4 mutant larvae showed increased number of swim bouts and longer duration swim runs after a touch stimulus, suggesting the mutant larvae are hypersensitive to touch. We added these data to Figure 11. We moved the data on adult body size and feeding behavior to Figure 11 —figure supplement 1A-C. However, we kept the data on adult anxiety-like behavior in the main Figures 11 and 12. We believe these results will be of interest to the community and potentially useful as a new model to study stress response behaviors and circuits. Further explanation of our reasoning for keeping these data in the paper is in the response to Reviewer 2, point 8 and Reviewer 3, point 4.We added new experiments testing the larval touch response. We found that 4) As described in detail by reviewer 2 (see below), multiple concerns about statistical analyses were raised and need to be addressed.We address each concern in the response to Reviewer 2, point 5.Reviewer #1 (Recommendations for the authors):Overall, the manuscript is very well written and the conclusions are well supported by the data. The light sheet and swept confocal microscopy movies provide a unique view of sensory axon development in vivo and are beautiful! Below are a few questions / comments that I had while reading the manuscript:1) The data in figures 3 and 5 show nicely that axon branches are not stabilized and that axonal growth is shifted posteriorly in klc4 mutants. Is the collapse of branches direction-dependent in the mutant? Is there a difference in branch stabilization/pruning depending on whether the branch elongates anteriorly or posteriorly?We scored the directionality of the axon branches that failed to stabilize and did not find a significant difference in the percent that were anteriorly or posteriorly directed. We have added this result to the text of the Results.2) The presence of apical protrusions crossing the midline in klc4 mutants suggests a defect in RB neuron polarity. Is the centrosome mislocalized in klc4 mutants?Development 139:3590-3599). We expect that we would also see apical mislocalization of the centrosome in klc4 mutants. However, because showing a third condition under which these two events are correlated would be a relatively incremental conceptual advance, and because centrosome imaging is one of several time-intensive experiments requested by reviewers, we focused our efforts on the experiments deemed to be essential revisions .This is an interesting question. We previously showed a correlation between mislocalized centrosomes and apical protrusions with two other manipulations that lead to apical protrusions in RB neurons: inhibition of LIM homeodomain transcription factors and lamininA1 mutation  The existence of both Klc4 isoforms is interesting, especially since corresponding transcripts seem to be differently regulated during development. Since the short isoform is not predicted to bind any cargo, is anything known about its functions? Are both isoforms expressed in RB neurons?klc4-expressing neurons, including RB neurons. We added those data in Figure 1. The short isoform does not have unique sequence that isn’t present in the long isoform, so we are not able to make a short isoform specific riboprobe. To our knowledge, nothing is known about the function of the short isoform.We made a riboprobe that recognizes sequence found only in the long isoform. We found that the expression pattern of the long isoform is very similar to that of the riboprobe that binds both isoforms, indicating that the long isoform is likely present in all 4) It would be interesting to analyze the localization of Klc4 in sensory neurons, although this might be difficult to do. Would it be possible to mosaically express low levels of tagged klc4 in mutant sensor neurons and analyze its distribution?Traffic 20:851-866). Because the vast majority of kinesin light chains and heavy chains exist in an auto-inhibited conformation that is freely diffusible, the diffuse signal resulting from expression of tagged proteins obscures visualization of motors bound to cargos. Thus, we are unfortunately not able to image KLC4 protein localization with current available approaches.We tried using expression of KLC4-GFP fusion constructs, but expression leads to diffuse labeling throughout the cytoplasm. This problem has been previously reported by others attempting to visualize kinesin-1 heavy chains :The authors use Crisper technology to generate a zebrafish line that lacks expression of KLC4. This animal is then used throughout, along with pharmacological inhibition of KLC binding to adaptor proteins, to address the roles of KLC4 in the development of the morphology of Rohon-Beard (RB) sensory neurons in embryos and assess behavioral alterations in adult fish. Modern imaging methods are applied to study neuronal morphology in embryos and a battery of behavioral tests in adults. The study provides first line of inquiry assessment of the role of KLC4 in the development of RB morphology, focusing on the failure to establish proper axon arbors in the periphery. While each data set has intrinsic value and is novel, none is followed up to provide mechanistic insights into KLC4 functions in neurons. The value of the submission rests in providing a characterization of the effects of KLC4 knockout, but it does not further the understanding of the mechanistic contributions of KLC4 in the processes impacted by the genetic deletion of KLC4. While the significance of the question posed is well presented and clear, multiple issues regarding interpretation, presentation and analysis decrease enthusiasm for the submission in its current form.For the authors' consideration:1. The current in situ data uses a riboprobe that does not differentiate between the short and long forms of the KLC4 transcript. Given the large difference between short and long transcripts of KLC4, and expected differences in function of the resultant proteins, it would assist in the interpretation of the results to assess short and long transcript expression in RB neurons. Ideally, this would be followed up by specific knock down of the long form to dissect its contributions from the short form.klc4-expressing neurons, including RB neurons. We added those data in Figure 1. The short isoform does not have unique sequence that isn’t present in the long isoform, so we are not able to make a short isoform specific riboprobe. We also do not have a means to specifically knock down the short isoform. Splice blocking morpholinos could potentially be used to knock down the long isoform, but given the issues with morpholinos and interpretation of morpholino phenotypes, this would not be a definitive experiment.We made a riboprobe that recognizes sequence found only in the long isoform. We found that the expression pattern of the long isoform is very similar to that of the riboprobe that binds both isoforms, indicating that the long isoform is likely present in all 2. At first evaluation, it is not clear how the axon branching metric  is specific for axon branching. In the images provided there seem to be consistently fewer axons present as a function of distance from the central fasicle in the KLC mutant and kinesore treated embryos . How can the metric dissociate the number of main axons that arrived at the ROI from branches made from those axons?The metric does not distinguish between primary peripheral axons and those arising from secondary, tertiary or higher order branches. Most RB peripheral axons form as a branch from the central axon close to the cell body, and often this primary branch bifurcates shortly after spinal cord exit. Thus, all the axon segments in the periphery should be considered branches. We modified our language in the manuscript to avoid use of the term “main axon” because there is no clear definition of this term.Based on the high quality of the imaging, it seems that it would be possible to use morphological criteria to define axon branches and measure the density along individual axons. This form of assessment would be much more stringent than the ROI crossing method used. Indeed, this form of assessment is used in the live imaging studies addressing rates or branch formation and stability.To get more detailed information about axon density, we increased our image sampling by using a grid ROI instead of a single line ROI. This analysis approach provides information about axon density as a function of dorsal-ventral location (as suggested by the reviewer in point 3 below). This approach also provides information about both growth cone bifurcation and interstitial branching. We added the new data to Figure 2F, G, and Figure 2 —figure supplement 1.3. Considering the above comment (2) regarding the consistent observable decrease in the apparent number of axons and their lengths into the periphery noted above, it is surprising that an increase in the rate of axon extension was detected in the live imaging analysis . It is suggested that the authors analyze the density of axons and their total length as a function of distance from the central axon fascicle, noting that the ROI based approach in supplemental Figure 2 should serve this purpose by simply changing the distance from the fascicle into the periphery at constant intervals (currently only 65 microns was addressed).This is an excellent suggestion, and we now added this additional, multiple ROI analysis.Based on the images presented I would expect that a decrease in axon density and length will be determined in the KLC4 mutant. If so, then these data would stand in conflict with the observed increase rate of axon extension, but may indicate that earlier aspects of axon formation were also impacted in the KLC4 mutant. The formation of axons from the cell body may share mechanistic similarity to the emergence of branches, and thus may be delayed in the mutant due to failure to stabilize the new axon at its early stages of formation which could then result in the difference suggested by the images provided without being confounded by the subsequent increase in axon extension rate. It would also be insightful to perform this analysis at multiple points in ontogenesis.Neural Dev 6:27), and we do believe that process shares mechanistic similarity to the formation of subsequent branches. Interestingly, the grid ROI analysis showed fewer branches in the mutants at all dorsal-ventral levels, indicating that the initial formation of peripheral axons may also be affected in the mutants, as the reviewer suggests. We added discussion of our interpretation of these results to the manuscript.There are fewer axons present in the mutants because there is less branching. In both wild type and mutants, axons that are shorter at one snapshot in time likely formed more recently than the longer axons. Live imaging is required to make conclusions about extension rate, and our live imaging showed that in the mutants, the axons that do form grow faster than wild type axons. We previously showed that the peripheral axons most often arise as a branch off the central axon  were considered to be significant when the 95% CI ranges did not overlap." Is not considered a stringent analytical method, although a valid "rule of thumb". The authors are urged to seek assistance from a statistician.We consulted with a statistician, who advised us to calculate the area under the curve. For Figure 4, in the portion of the graph where the 95% confidence intervals do not overlap , we calculated the area under the curve, which gives us a mean and standard error of the mean and allows a t-test to be run on the data. The t-test showed that the difference in area under the curve was significant (p=0.0047). In Figure 12 , we calculated the area under the curve in panels B-F and found all to be significant, with p values p=0.0005 or p<0.0001. We added these analyses to the Methods section and figure legends.6E, no 95% CI shown as claimed in the legend. The mean is not likely to be significant as the WT only have 3 points and one is an outlier relative to the other 2. I also do not see how the mean in the +/+ could be as shown, as claimed in the legend, it seems to be the median. I would estimate the mean to be somewhere around 20 um. For the mutant data, the distribution does not strike me as normal. Were these data analyzed for normality? Also, there is no statistical analysis shown, appreciating that with an n=3 for the WT this is problematic and power would have to be increased through greater sampling.We regret the inaccuracy of the legend which likely occurred during changes to that figure. The figure has been updated to show the mean with standard error of the mean. As the reviewer noted, these data were not analyzed for significance because of the paucity of fasciculation events in wildtype . We did not report the normality test because we were not running a statistical test on the data. However, a normality test showed that mutant distribution was normal by Anderson-Darling, D’Agostino and Pearson, and Shapiro-Wilk. Normality could not be assessed for wild type because of the small number of fasciculation events. Our statistical consultant confirmed that it would not be appropriate to run a statistical test on the data because the events are so rare in wild type. They advised us to note in the legend that length measurements are conditional on event occurring, which we have done. We believe that reporting these measurements is still informative to readers even if they can’t be tested for statistical significance.The legend to Figure 8 states that t-test with Mann-Whitney correction are used. I am not familiar with this correction for a t-test and was not able to find references to such a correction for t-test. Was Welch correction intended? The statistical description in the method is very sparse as presented and details should be presented. How the basic assumptions of the rather limited t-test were met by data prior to selection of specific t-test should be clearly denoted.We apologize for the inaccurate description. The Mann Whitney test is not a corrected t-test, but rather an alternative to the t-test for non-normal data. This test is the same as the Wilcoxon rank sum test. This does not change the results, as the Mann-Whitney test is still an appropriate test for these data. We added more description to the statistical section of the Methods. Before selection of a test, the data were tested for normality by the previously described methods. In most cases, data were not normally distributed in all samples, leading to use of the Mann-Whitney test. In cases where data were determined to be normal, a standard t-test was used. Data tested by Mann-Whitney or t-tests were independent of each other and comprised ordinal or continuous numbers. Data tested by t-test were confirmed to have no difference in variance by performing an F test to compare variances.Figure 9. In panel C, a t-test is used but (1) the data are bounded values, and (2) the distribution in WT is clearly non normal with many data points at the boundary . A t-test is not applicable as basic assumptions are broken, and do not appear to have been determined. E and H have no p values or test shown in legend and it is not clear why individual data points are not shown here as in other panels. F and G have no test denoted. The meaning of * and multiples thereof is only defined for one panel. Panel G has *** not defined at all.We regret that the legend for Figure 9, Panel C  was incorrect as a Mann-Whitney test was performed and not a t-test. The Mann-Whitney test accepts ordinal values, which should include bounded variables. The Mann-Whitney test is also equivalent to the Wilcoxon rank sum test. To be thorough, we also performed a Kruskal-Wallis test, which also works on independent data that are non-normal and ordinal or continuous. We found that the Kruskal-Wallis test agreed with the Mann-Whitney test and found that there was a significant difference in the number of larvae eaten between wildtype and mutant fish, with p<0.0001.For E and H , these graphs represent 3 individual experiments. Multiple animals were tested in each trial, but the N is the number of individual behavioral trial days. N=3 is too low to test for normality, therefore it was not appropriate to perform a statistical analysis on the data. The Mann-Whitney test  fails with an N this low—if the N is less than 7, the Mann-Whitney will always give a p value greater than 0.05 no matter how much the groups differ. We were able to test significance of the total time spent in the upper zone  and the time spent freezing . We believe it is informative to also report the percent fish that entered the upper zone in each experiment  and the percent that froze , even though these can’t be tested for significance. We have updated the data display to show individual points for each experiment, along with mean and standard error of the mean. We have updated the legends to include all statistical test descriptions and p values.6. Strictly speaking, the authors are inferring contact repulsion but do not use the imaging data sets to directly address it . The observed changes in angles between sister branches could be due to a variety of other mechanisms altering the ability of sister branches to generate larger angles.The reviewer’s point is well taken. We have modified our language in the text accordingly.The self-contact examples in Figure 6 are not particularly compelling. Perhaps, presentation of all relevant frames in the timelapse with greater annotation and description would assist a reader.We updated the images in Figure 6 to show a clearer example of self-contact. Space constraints prohibit including all relevant frames in the timelapse, but they can be viewed in the accompanying video.In Figure 6B, in contrast to what the text claims, this reviewer does not see a clear growth cone bifurcation followed by fasiculation due to lack of self-repulsion. It appears a small back branch from the second panel from the left aligns with the main axon.We have updated the images in Figure 6B to more clearly show branches collapsing onto each other.Also, the "appearing to collapse onto one another" (line 232) could be interpreted as a consequence of the prior analysis of branch stability.When following the dynamic axon behaviors in the movies, we can distinguish between branches that have retracted versus those that fasciculate onto other axons.7. Figure 8D,E. "Comet Frequency" in comets/um/min. It is not clear if this reflects new comets observed during the imaging or all observed comets/um/min.These data include all observed comets. We clarified this point in the figure legend .The imaging sets would benefit from additional analysis to extract the duration of EB3 comets  and total distance from initiation to loss of the comet.We now include data on both distance (in microns) and duration (in seconds) of comet runs in Figure 9 —figure supplement 1C-F, and describe the results and interpretation in the Results section.It is not clear why central axons are analyzed for only these data sets and not others. As the focus of the paper is on peripheral axons the suggested additional data sets for peripheral axons would suffice.Because the focus is on peripheral axons, we moved the data on EB3-GFP comets in central axons to Figure 9 —figure supplement 1.As a whole, I would suggest that the data in Figure 8 from EB3 imaging indicate increased polymerization of plus tips along axons in the mutant. This is not immediately reconcilable with decreases branch stability as one would presume that microtubules would be more prone to catastrophe and depolymerization in conjunction with decreased branch stability. The data are broadly consistent with the detected increase in axon extension rate. However, analysis of EB3 comets needs to be provided in branches  in order to be linked to the main observed phenotype of decreased branch stability.klc4 mutants, there is no difference in frequency of comets entering nascent branches, suggesting a relative decrease. Second, we used anti-acetylated tubulin labeling to show that nascent branches in klc4 mutants are less likely to have stable, acetylated tubulin. We added further interpretation of these data to the Discussion section.We used two approaches to analyze microtubules in nascent branches . First, we used EB3-GFP to image microtubule polymerization into nascent branches. Although comet frequency in established axons is increased in 8. While of general interest, the behavioral assays do not address RB neuron function or circuitry, which is embryonic. The fit of these data to the rest of the study is not clear. Could tail startle be assessed  Curr Biol 27:697-704) for addressing RB/RB-related circuit function? Any RB function assays would provide a much better fit to the rest of the data presented.Importantly, these behavioral deficits cannot be attributed to changes in circuit formation, branching, axon extension etc as the mutant is not neuron specific, the relevant circuitry has not been analyzed, and no aspect of CNS neurons or glia or anything else has been analyzed. The concern is compounded by the differences in overall growth of the fish.klc4 mutant larvae showed increased number of swim bouts and longer duration swim runs after a touch stimulus, suggesting the mutant larvae are hypersensitive to touch. We added these data to Figure 10. We moved the data on adult body size and feeding behavior to Supplemental Figure 10. However, we kept the data on adult anxiety-like behavior in the main Figures 10 and 11. It is true that these behaviors cannot be linked directly to cell biological defects in RB neurons. We are not claiming a direct link. Instead we are making use of two separate approaches to address questions about KLC4 function: (1) We take advantage of the superficial position of the RB neurons, which makes them ideal for imaging, and their distinctly polarized morphology to uncover cell biological processes influenced by KLC4. (2) We use adult behavioral analysis to more broadly assess roles of KLC4 in other neural circuits. We believe that publication of the adult behavior phenotypes will benefit the scientific community and be useful as a new model to study stress response behaviors and circuits.We added new experiments testing the larval touch response at 3 dpf, a behavior that is mediated by sensory neurons. We found that 9. One fundamental aspect of the study that is missing is consideration of KLC4 localization in the RB neurons. Whether it localizes to the RB axons remains unknown, or whether it ever targets to branches. One would presume so, but this needs to be assessed. It is hard to interpret results with any degree of mechanistic insight without knowledge of the localization of the molecule addressed.Traffic 20:851-866). Because the vast majority of kinesin light chains and heavy chains exist in an auto-inhibited conformation that is freely diffusible, the diffuse signal resulting from expression of tagged proteins obscures visualization of motors bound to cargos. Thus, we are unfortunately not able to image KLC4 protein localization with current available approaches.As mentioned in response to reviewer 1, we tried using expression of KLC4-GFP fusion constructs, but expression leads to diffuse labeling throughout the cytoplasm. This problem has been previously reported by others attempting to visualize kinesin-1 heavy chains :Further characterization of axonal defects:– While the population-based methods for characterizing axonal defects are elegant and informative, these defects could potentially be further appreciated with single cell resolution, What do the arbors of individual neurons look like in these mutants? Does the mutant affect all sensory neurons equally, or is a sub-population of neurons more severely affected? It should be possible to analyze single neurons with transient transgenesis in the zebrafish model. This could be particularly helpful since aberrant fasciculation may make it difficult to distinguish multiple fasciculated axon branches when the whole population of neurons is labeled. Identifying defects at a single neuron level could also make it possible to carry out rescue experiments to address the cell autonomy of axon arborization defects.We did analyze individual neurons labeled with transient transgenesis for the Rab5 and microtubule imaging experiments, and the single-cell labeling in Figure 7 to follow the trajectory of apical processes. As the reviewer points out, this is important because fasciculation of multiple axons would confound results. We used stable transgenics for the live imaging of axon growth, fasciculation and branch dynamics. This was absolutely necessary for the fasciculation analysis because all axons must be labeled to detect non-self-fasciculations. We are able to do the growth rate and branch dynamics analyses on individual neurons using the stable transgenics because we can easily resolve individual neurons by following videos of axons growing over time.Explanation of axonal defects mechanistically:– The impact of this paper would be most enhanced by characterizing the axonal trafficking of cellular cargos in klc4 mutants, particularly if defects in specific trafficked cargoes could plausibly explain the axonal defects. In the discussion of the paper, the authors note that they have previously found that endosomal trafficking regulates branching. Characterizing endosomal trafficking, as well as trafficking of mitochondria, lysosomes, golgi, etc, thus has the potential to make this study substantially more mechanistically satisfying.klc4 mutants did not have a decrease in endosome delivery to branch points, high-speed imaging of vesicle dynamics revealed some alterations in transport of these vesicles in klc4 mutants. These alterations in endosome transport are not likely to be the only effects of klc4 mutation on cargo dynamics or to fully explain the mechanism by which KLC promotes branch stabilization. However, an exhaustive analysis of all potential cargos would require many months to years of work and is outside the scope of this manuscript.We added new experiments to image endosomal dynamics , trafficking of Rab5-labeled endosomes from the cell body to branch points , microtubule behavior during axon branching , and acetylation of microtubules in nascent branches . We chose Rab5-labeled endosomes because of our previous work showing a correlation between their transport and RB axon branching. Although the – The changes in microtubule dynamics of klc4 mutants are interesting, but how trafficking defects affect these dynamics, and how defects in MT dynamics affect branching, is unclear. To address how trafficking affects MTs, the authors could attempt to identify MTOCs in axons. For example, since golgi outposts are thought to be MTOCs, is their trafficking into axons affected by klc4 mutation? To address how MT defects affect branching, the authors could characterize MT dynamics during branching-for example, do mutants display any defects in the frequency of MT invasion into nascent branches?klc4 mutants, there is no difference in frequency of comets entering nascent branches, suggesting a relative decrease. We used anti-acetylated tubulin labeling to show that klc4 mutants have fewer nascent branches with stable, acetylated tubulin.We have tried to detect MTOCs using an antibody to γ tubulin, but that labeling in whole embryos has a very low signal to noise. It was not possible for us to detect signal above noise. We added new experiments analyzing microtubules in nascent branches . We used live imaging with EB3-GFP to measure frequency of comets entering nascent branches. Although comet frequency in established axons is increased in Behavior:– As mentioned above, the analyses of adult behavioral defects, while interesting and rigorously carried out, do not complement the morphological analysis in larval sensory neurons. It would be exciting if axonal arborization defects in a specific neuron type could be linked to behavioral defects. Could the authors attempt to characterize touch-response behaviors in larval fish? This would be a particularly exciting if it were possible to do cell-autonomous rescue experiments, thus directly testing if defects in sensory neuron morphology affect behavior.klc4 mutant larvae showed increased number of swim bouts and longer duration swim runs after a touch stimulus, suggesting the mutant larvae are hypersensitive to touch. We added these data to Figure 11. We moved the data on adult body size and feeding behavior to Figure 11 —figure supplement 1. However, we kept the data on adult anxiety-like behavior in the main Figures 11 and 12. We believe these results will be of interest to the community and potentially useful as a new model to study stress response behaviors and circuits.This is an excellent suggestion and we have now added larval touch response assays . We found that


The Mini-SIPS, a condensed version of the Structured Interview for Psychosis-Risk Syndromes (SIPS), is intended to efficiently identify for clinicians the minimum information needed to support a DSM-5 Attenuated Psychosis Syndrome (APS) diagnosis.The instrument and the DSM-5 criteria are accessible through the online training program.Most individuals (67.5%) in the first 212 to complete the training program indicated an intended use of the Mini-SIPS exclusively for clinical purposes. Performance on the post-training quiz was excellent for those with and without prior training in structured diagnostic interviewing.The Mini-SIPS, and accompanying training program, are offered as public-domain clinical resources to the mental health community.The online version contains supplementary material available at 10.1186/s12888-022-04406-z. Based on retrospective descriptions dating from the time of Bleuler , the lasDeveloped beginning in 1996, the SIPS has been employed in North America extensively and in Europe, Asia, Africa, and South America . ReliabiApproximately ten years ago, the American Psychiatric Association DSM-5 Task Force and its Psychotic Disorders Workgroup engaged in an extensive evaluation of the reliability, validity, and utility of the CHR construct and elected to include the most common of the three CHR syndromes , 13 in tShortly after the SAMHSA CHR clinics began operation, our group began receiving requests for a shorter version of the SIPS specifically designed for clinical rather than research use. Clinics were finding that 1–2 h was a lengthy period for diagnostic assessment, especially when third-party payors would often not reimburse for the full time required. In addition, some of the ratings recorded, while useful for characterizing CHR subjects in research studies, were not essential for making the clinical diagnosis. Moreover, staff turnover was unfortunately not uncommon at some of the clinics, and the extensive training needed for new hires posed resource challenges. We acknowledged that research-level precision or breadth of assessment was often not required in the clinical context and agreed to work on a briefer version for clinical use and a brief on-line training program, described here. We also report characteristics of the first two hundred individuals who completed the training program. The Mini-SIPS instrument is available online .The first step was to judge which elements of the SIPS were minimally required for the clinical diagnostic task: to distinguish patients at CHR from those who were already psychotic and from those who were neither at CHR nor psychotic. Even though the negative, disorganization, and general symptom ratings are useful patient descriptors, only the attenuated positive symptom ratings are required for a diagnosis of CHR or for a SIPS diagnosis of psychotic disorder. It was also apparent that the 0–6 rating scale in the original SIPS version of these items, while helpful for grading symptom severity in longitudinal research within CHR individuals, could be simplified if only clinical diagnosis were of interest. Moreover, three of the five SIPS positive symptom ratings  mapped onto different subtypes of attenuated delusions, allowing further simplification into a single delusion-like symptoms item. Lastly, since many of the SAMHSA clinics were using the DSM-5 Other Specified Schizophrenia Spectrum and Other Psychotic Disorder (298.8) and associated ICD-10 (F28) codes for billing purposes, ratings for the two less common CHR syndromes not contributing to a DSM-5 APS diagnosis were not essential.Features included in the SIPS and Mini-SIPS are shown in Fig. One feature of the SIPS, the verbatim query questions relating to attenuated positive symptoms, were deemed essential to detecting symptoms that might otherwise go undiscussed without the structured queries.The Mini-SIPS training program was designed by the authors of the study and deployed online using the Yale University Qualtrics Program, a University-supported website and licensed version of the Qualtrics Research Suite. An institutional review board of the Yale Human Research Protection Program (FWA00002571) determined that the research and its inclusion of an on-line participant consent as part of the training program were exempt under 45 CFR 46.104 (2)(ii) and ethics approval was therefore waived. All methods were carried out in accordance with relevant guidelines and regulations in the Declaration of Helsinki—Ethics approval and consent to participate section. The research group advised mental health professionals about the availability of the training program by sending out emails via listservs and mentioning it during talks at scientific conferences and SAMHSA clinic coordination meetings. Participation in training was voluntary, unpaid, and otherwise unsolicited.Based on the considerations described above, we reduced the 48-page SIPS 5.6.1 research version to a four-page clinical version (the Mini-SIPS). Page 1 offers rating instructions at a level of detail consistent with the more limited aims of the clinical instrument. Page 2 retains all of the attenuated positive symptom queries from the SIPS. Page 3 permits rating the three DSM-5 positive symptom types on the restricted severity range and includes checkboxes for recording based on the interview as to whether sufficiently severe symptoms meet the additional requirements of the DSM-5 criteria. Page 4 guides frank psychosis and DSM-5 APS diagnoses based on the symptom and functioning ratings.All participants provided written informed consent. The training program indicates that individuals should have read the DSM-5 criteria for APS and have prior experience in psychiatric diagnostic interviewing. Participation in the online program is anonymous; however, participants are asked questions about their educational background, current profession, work setting, and demographics. The training program discusses the importance of focusing on positive symptoms and incorporating other factors when considering an APS diagnosis, such as being at least 12 years of age, a minimum IQ of 70, and considering presence of severe neurological disorder or traumatic brain injury that may complicate diagnosis.The online training program reflects the procedure detailed on page 1 of the Mini-SIPS, while also providing some additional tips for conducting the interview. The online program proceeds to describe the three qualifying symptoms and the range of possible symptom ratings. Guidance is also provided for other considerations, such as ruling out those with frank psychosis and documenting whether an APS diagnosis is suggested or not.Once participants consent and view the training materials, they proceed to the quiz, the final step in the program. The quiz is comprised of 14 questions and includes vignettes of all three symptoms at each of the three severity ratings as well as questions regarding diagnostic criteria and interviewing approaches. Feedback is immediately provided to all participants after each question is answered. The feedback includes their answer, the correct answer, an explanation, and the original question in case participants want to review it again. Once the quiz is complete, participants are asked if they want to be sent a record of completion of this training course. If so, they provide an email address at which to receive an electronic certificate.M = 39.59, SD = 11.54). The participants varied somewhat in their intended use of the training, largely participating in the training for clinical practice (67.5%) compared to those who intended to use the training for research purposes or both clinical and research purposes (10.8% and 27.7% respectively). Most of the participants (72.2%) work in a community-based setting . The remaining participants came from academic settings. As shown in Table We aggregated data for the first 212 participants who completed the training program. All participants reported English as their primary language, with 82.5% identifying as female and the others identifying as male (16%) or non-binary (1.4%). The majority of the participants identified as white and not Hispanic or Latino . Only 30.2% of participants completed the training assessment in 30 min or less, the target duration for the program. However, a majority (52.8%) of participants completed in an hour or less, and 75% of participants completed in 2 h or less. Given that the training program is on-line and self-paced, it is possible that some participants paused the training program at one or more points; such “breaks” are included in the total completion time.Participants differed greatly in how much time they took to complete the training program . A Welch’s two sample t test showed that those who had training in structured interviewing for psychiatric diagnoses  received a higher post-assessment score (M = 89.0) than those that did not have such training  = -2.92, p = 0.004). Those who had training in structured interviewing for psychiatric diagnoses were more likely to work in an academic setting (37.3%) as compared to those that did not have such training , but were similarly likely to have received master’s or doctoral degrees (86.4%) as compared to those that did not have such training . Furthermore, those who had received training in structured interviewing for clinical high risk for psychosis syndromes  trended toward a significantly higher score (M = 88.0) than those who did not have such training  = -1.68, p = 0.09). Those who had received training in the SIPS or CAARMS were similarly likely to work in an academic setting (30.8%) as compared to those that did not have such training  and were also similarly likely to have received master’s degrees or higher (90.8%) as compared to those that did not have such training .Participants performed well on the post-training assessment in terms of percent accuracy across the 14 quiz questions , to harmonize the two research instruments where possible and to generate the two instruments’ differing sets of CHR criteria from the same research interview. This effort is expected to have relatively little impact on DSM-5 APS diagnosis guided by the Mini-SIPS, but when it has been fully completed and validated we will consider whether to issue a revised version of the Mini-SIPS incorporating minor modifications.Finally, our current method employing anonymous on-line training is both a strength and a limitation. The strength is that the ease of use for trainees promotes dissemination which in turn we hope will promote public health. The limitation is that we cannot enforce our recommendation that trainees have prior experience in psychiatric diagnostic interviewing. This limitation imposes a burden on clinics who may employ staff without such prior experience to provide supervision and oversight. We stress that the Mini-SIPS by itself does not establish a psychiatric diagnosis; it is a guide or tool that assists in the diagnostic process. The responsibility for the ultimate diagnostic determination in clinical practice lies not with the tool but with the appropriately-credentialed practitioner.The Mini-SIPS and accompanying training program are offered as public-domain resources to the mental health community. The brief duration of the Mini-SIPS guide to diagnostic assessment may improve the bottom line for clinics serving patients with DSM-5 APS and permit resources to be more focused on treatment. The availability of the training program as an on-line resource may facilitate awareness of the DSM-5 APS diagnosis and enable community mental health sites to maintain a trained staff despite staff turnover.Additional file 1: Supplementary Materials 1. Mini-SIPS Training Program data _April 27 2021.