Nascent proteins can be positioned rapidly at precise subcellular locations by local protein synthesis (LPS) to facilitate localized growth responses. Axon arbor architecture, a major determinant of synaptic connectivity, is shaped by localized growth responses, but it is unknown whether LPS influences these responses in vivo. Using high-resolution live imaging, we examined the spatiotemporal dynamics of RNA and LPS in retinal axons during arborization in vivo. Endogenous RNA tracking reveals that RNA granules dock at sites of branch emergence and invade stabilized branches. Live translation reporter analysis reveals that de novo β-actin hotspots colocalize with docked RNA granules at the bases and tips of new branches. Inhibition of axonal β-actin mRNA translation disrupts arbor dynamics primarily by reducing new branch emergence and leads to impoverished terminal arbors. The results demonstrate a requirement for LPS in building arbor complexity and suggest a key role for pre-synaptic LPS in assembling neural circuits. •Tracking endogenous RNA shows that RNA docking predicts axon branch emergence in vivo•Axon arbor complexity in vivo depends on local protein synthesis•Axonal β-actin synthesis regulates branching by increased branch initiation•Live imaging reveals de novo synthesis of β-actin hotspots during branch formation Wong et al. track endogenous RNA granules in single axons in vivo and show that they predict positions of axonal branch formation in the tectum. They also show that perturbation of local protein synthesis in these axons autonomously regulates branch formation. CNS axons typically form highly branched terminal arbors in their synaptic target area. The branching complexity of an arbor defines the number and extent of post-synaptic partners a neuron can have and is a critical determinant of neural circuit assembly . PreviouXenopus  in axons, such as Netrin-1, BDNF, Sema3A, and Slit2 , are alsXenopus . Conversosophila  and zebrosophila  neurons,Culture studies have uncovered an association between axonal branching and LPS. For example, newly synthesized green fluorescent protein (GFP) puncta localize to the base of collateral spikes in cultured retinal axons , and theendogenous RNA granules for prolonged periods (>1 hr) in single axons in the Xenopus visual system and performed live imaging to simultaneously capture arbor dynamics and RNA trafficking in vivo. Our results reveal a close relationship between arbor dynamics and RNA trafficking and show that RNA docking predicts sites of branch emergence. Live visualization of β-actin synthesis reveals the rapid accumulation of nascent β-actin in discrete “hotspots” in branches and at branch points. Functional experiments show that LPS is required for proper axon arbor formation in vivo. Knockdown of local β-actin synthesis causes a marked reduction in the emergence of new branches and results in impoverished axon terminal arbors. Collectively, the findings provide evidence of a pivotal role of LPS in determining axon arbor architecture in vivo.In this study, we investigated the spatiotemporal dynamics of RNA movements, LPS, and axon terminal arborization in vivo. We developed a method to visualize Xenopus embryos. UTP analogs become incorporated into RNA during its synthesis and can then be monitored by live fluorescence imaging in putative ribonucleoproteins (RNPs) in retinal axons in vitro  analogs, Cy5-UTP or biotin-UTP, intracellularly by eye electroporation or blastomere injection in in vitro . The UTPin vitro A–S1K. ThNext, we examined the general characteristics of endogenous RNA motility in RGC axon terminals in the tectum in vivo. Cy5-UTP was delivered into developing RGCs along with a membrane-targeted GFP (mGFP) reporter by targeted eye electroporation at stage 28, the beginning of RGC axonogenesis. Cy5-RNA and GFP-labeled axons were imaged in the optic tectum with time-lapse microscopy (10–20 frames/min for >1 hr) during the early phase of arborization and map formation (stages 41–43). Cy5-RNA appeared as punctate granules, often highly mobile, indicative of RNPs. We refer to these as “RNA granules.” Motile RNA granules were observed in the majority of GFP-labeled retinal axon terminals with an average density of 2.6 ± 0.29 granules/10 μm  and S2. We next investigated whether distinct aspects of branching, such as branch emergence and stabilization, are associated with the spatial and temporal positioning of RNA granules. New “branches” first appear as filopodial protrusions of less than 5 μm in length that emerge from the main axon shaft. Many of these are short lived, but some elongate to >5 μm and persist. We refer to these longer structures as “branches” to distinguish them from the shorter filopodial protrusions. Motile RNA granules were often observed to pause briefly (>10 s), or “dock,” at sites of branch emergence in the axon shaft immediately preceding the appearance of a new protrusion. 84% of the filopodial protrusions exhibited docked RNA granules at their bases in the 10 s time window preceding their emergence. The docked RNA granules often persisted at the bases of filopodia during emergence, although in some cases (5%), the dwell time was transient (<10 s), and the granules moved away before the filopodia became visible B. Indivibone fide protrusion sites. Even though some of the random positions fell indiscriminately on protrusion-forming sites, we found that RNA granules docked at just 16% of random sites, compared to 84% at protrusion sites. . The closest RNA granule to the randomized position was tracked across time to find the first instance of docking >10 s, and we then scored whether a protrusion emerges at this position within the 10 s of docking. We found that 22.0% ± 5.1% of RNA granules displaying this docking motion were followed by protrusion emergence. To test the possibility that these correlations are meaningful and not simply coincidental, we compared how frequently RNA granules docked at random positions along the axons versus an equivalent number of n sites. B, showinPrevious work has shown that only a small subset of newly formed branches persist to form stabilized mature branches, while the majority are short lived and retract within 10 min . TherefoOccasionally, branches harboring RNA granules were seen to retract abruptly. Retrospective image analysis of these events showed, strikingly, that RNA granules were rapidly trafficked retrogradely out of the branch preceding retraction  and persistent (31–60 min)—and length. The total time of the presence/absence of RNA granules at each of the three branch locations  was scored. RNA granules were present for significantly longer times in persistent branches compared with short-lived branches C. RNA grMitochondria supply the energy for organelle trafficking and mRNA translation and have previously been shown to localize to branch points in axons in vitro . To visuRNA transport and localization is intimately linked to LPS in neuronal compartments . To testIn the control condition, single axon arbors were highly dynamic, with an average of 50 filopodia and 8 branches added and removed in 10 min . By contEmbryos with eyes removed and treated with PS inhibitors for 30 min during the period of axon elongation in the optic tract (stages 35/36–37/38) did not exhibit abnormal pathfinding or stalling . Moreoveβ-actin mRNA localizes to axons  and is lTo block β-actin mRNA translation, a β-actin antisense morpholino (MO) was injected into the blastomeres fated to give rise to the CNS at the four-cell stage. This resulted in a 47% reduction in β-actin levels, in agreement with previous studies , and didTo target the β-actin knockdown to RGCs and to visualize the trajectories of single axons, we electroporated the β-actin MO together with a reporter mGFP plasmid into the eye at stage 28. At stage 45, when RGC axon arbors have reached maturity and become relatively stable, we imaged single arbors in the tectum and performed quantitative analysis. While highly complex arbors were seen in the control MO (Con MO) embryos, β-actin MO-axons exhibited much simpler arbors A. QuantiThe loss of arbor complexity could arise by a reduction in the emergence of new branches, a failure to stabilize new branches, or a combination of both. To understand the dynamic processes underlying axonal branching upon β-actin knockdown, we carried out live in vivo imaging following MO electroporation into the eye at stage 28 M. At stabefore the arrival of RGC axons (stages 35/36–37/38) and subsequently visualized the branching dynamics of axons after tectal entry at stages 41–43  effect, we delivered the MO directly into arborizing RGC axons in the tectum by electroporation at stages 41–43 E and cones 41–43 J. We foues 41–43 F–5I, indIn contrast to the severe effect on arborization, the trajectories of β-actin MO axons in the optic tract did not exhibit any major guidance defects or abnormal extension rates , indicatWe next sought to visualize newly synthesized β-actin during branching in axon terminals in the tectum in vivo. Fluorescence recovery after photobleaching (FRAP) of GFP has been used in vitro to demonstrate the local synthesis of proteins . We exprSurprisingly, when the same 10 min FRAP experiment was carried out on Venus-β-actin-expressing growth cones in the optic tract, no signal recovery was observed . This reDe novo synthesis of β-actin could occur uniformly along the entire axon and/or branch providing a continuous supply of new actin monomers for branching axons. Alternatively, it could take place in focal hotspots, potentially providing new nucleation sites for actin polymerization. To investigate the spatial distribution of newly synthesized β-actin, we used FRAP with high-resolution time-lapse microscopy to map the subcellular location of newly synthesized β-actin in arborizing axons in vivo A.Figure Venus-β-actin showed a markedly different subcellular localization to the control Venus reporter. The control Venus reporter exhibited low intensity and fairly ubiquitous levels of signal recovery as expected for free diffusion, whereas the Venus-β-actin exhibited more concentrated spots of high intensity that we refer to as LPS hotspots . The posTo perform an unbiased quantitative analysis of the hotspots, we measured the variation in the fluorescence signal, which is predicted to increase with the presence of hotspots. The standard deviation (SD) of fluorescence values was used to generate a fluorescence variation index (FVI). The SD value of the fluorescence for the branches were internally normalized to the SD of the fluorescence for the axon shaft, where a ubiquitous recovery pattern would yield a FVI ≈ 1 and the presence of hotspots would result in a FVI > 1 D. In agrTo discover whether β-actin is synthesized at docked RNA granules, we carried out high-resolution FRAP experiments and recorded the recovery of Venus-β-actin signal simultaneously with Cy5-RNA localization in branching axons in vivo. Hotspots of β-actin recovery were found highly associated with docked RNA granules, beginning 10–20 s post-FRAP . To quanWe used live imaging to show that new axon branches commonly emerge at sites where RNA granules and mitochondria dock. We found that branch lifetime correlates positively with sustained RNA and mitochondrial invasion. Our functional analysis shows that PS is required for the elaboration of complex arbors and that local β-actin synthesis contributes to the addition and stabilization of new branches. Translation reporter experiments show that β-actin is focally synthesized and retained in concentrated microdomains at the bases and interiors of axonal branches. Our findings are consistent with a model in which RNA localization and nascent β-actin help to direct branch emergence and expand arbor complexity in response to target post-synaptic cell contact.Intracellular delivery of fluorescent UTP labels endogenous RNAs and has enabled us to perform tracking of the intra-axonal movements of RNA granules. These granules are highly fluorescent and photostable and can be followed for prolonged periods (>420 min). Because the fluorescent UTP is incorporated into RNA during transcription in vivo, the RNAs may associate with normal regulatory proteins inside the nucleus to form specific RNP complexes before export to the cytoplasm. The dynamic behavior of these granules therefore should provide an accurate reflection of their endogenous activities. The MS2 genetic system is a valuable alternative for labeling endogenous RNAs for live imaging  and has Our fast image-capture analysis enabled us to simultaneously track RNA, translation, and branching events and revealed the highly dynamic nature of developing axon arbors with multiple branches emerging and retracting over minutes. This is in broad agreement with previous studies using slower capture rates . We founPrevious work has shown that LPS is required for growth cone turning in vitro in response to various factors, including Netrin-1 and BDNF . InteresDefects in axon pathfinding were not detected in the current study after β-actin translation knockdown. This is consistent with the finding that Vg1RBP knockdown does not cause pathfinding defects but disrupts axon arborization . It is pHow might a small quantity of newly synthesized β-actin play a physiologically significant role in branch dynamics, promoting branch formation and stabilization, especially when there is a large pool of pre-existing actin? Locally synthesized β-actin has been estimated to constitute <1% of the actin in sympathetic neuron axons  and 7% olocal translation. Dysregulated translation, both too much or too little, can have profound effects on both axonal and dendritic branching and synapse formation .Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Christine E. Holt 2, 41 μM CaCl2) at 14-22°C, and staged according to the table of Nieuwkoop and Faber . To construct the Venus control plasmid, actb 5'UTR, the coding sequence and the majority of the 3'UTR were deleted from the Venus-β-actin construct except the last 46 nucleotides, leaving the polyadenylation signal sequence and a short stretch of poly(A) in place. For the morpholino insensitive β-actin/mGFP dual promoter construct, full-length actb was cloned into the multiple cloning site of pCS2+ plasmid. The β-actin morpholino targeted sequence at the first 16bp of the coding sequence was mutated with QuikChange II Site-directed Mutagenesis Kit (Agilent Technologies) with the primer (5'-CAACCCTCAGATCACAATGGAGGATGACATAGCCGCACTGGTCGTTG-3'). The β-actin amino acid sequence encoded remained the same and only the codons were changed. mGFP driven by the eIF1α constitutive promoter was cloned immediately upstream of the CMV promoter in reverse orientation.Total mRNA extracted from Stage 32 embryos using RNeasy Mini Kit (QIAGEN) was reverse transcribed into cDNA library with SuperScript III First-Strand Synthesis System (ThermoFisher) using Oligo(dT) as primer. To obtain the Venus-β-actin fusion construct, full-length The β-actin antisense morpholino (5'-CAATATCGTCTTCCATTGTGATCTG-3') as previously described  and the Targeted eye electroporation was performed as previously described . Stage 21)RNA dynamics  and S1: 2)Mitochondria dynamics : 1 μg/μl3)Cy5-RNA and mito-GFP colocalization : 5mM Cy53)Axon navigation  and S6: 4)Mature axon arbor visualization A–4G: 0.55)Axon branching dynamics , and S4:6)FRAP experiments , and S7:Electroporation mixtures and number of electric pulses delivered:For the pathway  x 0.5 mm internal diameter (ID), Harvard Apparatus) and a pressurized microinjector . For the culture experiment shown in Stages 40/41 embryos were anaesthetized in 0.4mg/ml MS222 in 1X MBS, and decapitated to harvest total RNA using the QIAshredder and RNeasy Mini Kit (QIAGEN) from whole heads. RNA was extracted following manufacturer’s instructions with on-column DNase digestion. The RNA Integrity Numbers (RINs) for control and biotin-UTP samples were 9.4 ± 0.4 and 9.6 ± 0.2, respectively. These values indicate that the extracted RNA samples were of high quality.150 μL of Streptavidin Mag Sepharose  slurry was transferred into two separate Eppendorf tubes. The beads were washed three times with 1ml of buffer L1 . The beads were then incubated in 1125 μL of buffer L1 and 125 μL of 5X Denhardt’s Solution (Sigma-Aldrich) for 1 hr, and subsequently washed three times in buffer L2 . An equal amount  of the total RNA harvested from 100 whole heads of uninjected control/biotin-11-UTP injected embryos was added to each tube containing the beads and the volume was brought up to 200 μL with buffer L2. The RNA was incubated with the beads at 4°C for 2 hr before being washed three times with buffer L2, six times with buffer L3  and six times with buffer L4 . For competitive elution of the bound molecules, 200 μl of 2.5mM biotin solution was added to the beads and was heated at 95°C for 4 min. The eluents were then purified with the RNeasy-mini kit. To increase the concentration of the RNA samples for bioanalyzer RNA analysis, 15 μL of RNase-free water was used for eluting the RNA from the RNeasy-mini columns, with the eluents from the first round of elution rerun into the column for the second round.150 μL of Streptavidin Mag Sepharose slurry was transferred into two separate tubes. The beads were washed three times with 1ml of buffer H1 . The beads were then incubated in 1ml of buffer H1 for 1 hr. Buffer H1 was removed and an equal amount  of the total RNA harvested from 100 whole heads of uninjected control/biotin-11-UTP injected embryos were added into each tube containing the beads. The volume was brought up to 200 μL with buffer H1. The RNA were incubated with the beads at 4°C for 2 hr before being washed three times with buffer H1, six times with buffer H2 , six times with buffer H3  and six times with buffer H4 . For elution and purification of the bound molecules, the same procedures as for the low stringency pull-down assay were carried out.Analyses of purified RNA were carried out with the Agilent 2100 Bioanalyzer and RNA 6000 Pico Kit (Agilent Technologies) following the manufacturer’s instructions.g for 2 min at 4°C before commencing the cycling protocol. The PCR cycling conditions used followed the instructions provided by the manufacturer . Real-time PCR runs were performed on a LightCycler 480 . The following primers were used; for rps13, 5'-CTTCAAACTGGCCAAGAAGG-3' and 5'-GGCCAGAGCCTTAGACTTGA-3'; for actb, 5'-TACTCTTTTGTTGGCGCTTG-3' and 5'-GGGCAACACTGAGAGGGTAG-3'; for sdha, 5'-AGACTCAACATGCAGAAGACCA-3' and 5'-TCCATTGCAGAATTGATGACAC-3'.RNA samples from the streptavidin pull down were reverse transcribed with the SuperScript III First-Strand Synthesis System. Triplicate 10 μL reactions were prepared according to manufacturer’s instructions . Plates were then centrifuged at 1,500 x Stages 40/41 embryos were anaesthetized in 0.4mg/ml MS222 in 1X MBS, and decapitated to harvest total DNA using the QIAamp DNA Mini Kit (QIAGEN) from whole heads. DNA was extracted following manufacturer’s instructions with RNase A digestion. DNA was fragmented using the NEBNext dsDNA Fragmentase (New England BioLabs) to facilitate biotin-streptavidin binding and electrophoresis.The same buffers and procedures were used as the biotin-RNA pull down experiments for both low and high stringencies. An equal amount of fragmented DNA from the control and biotin-UTP samples was used for the DNA pull down. The eluted solution from the pull down was purified with the QIAquick PCR Purification Kit (QIAGEN). Analyses of purified DNA were carried out with the Agilent 2100 Bioanalyzer and High Sensitivity DNA Kit (Agilent Technologies) following manufacture instruction.2O) overnight at 20°C and then washed 3 times with ddH2O and dried for 1 hr, followed by coating with laminin  in L-15 medium (GIBCO) for 1 hr at room temperature. Injected stage 33-34 embryos were washed 3 times in 0.1X MBS with 1% Antibiotic-Antimycotic (ThermoFisher). Embryos were then anesthetized with 0.4mg/ml MS222  and secured on their lateral side with custom made pins on a Sylgard-coated dish. Both eyes were dissected out using dissection pins, washed in 60% L-15, and plated on pre-coated dishes containing 60% L-15 culture medium (60% Phenol Red-free L-15 medium supplemented with 1% Antibiotic-Antimycotic). Cultures were incubated at 20°C for 18 hr before imaging. Cultured axons were imaged for 5 min with 60X UPLSAPO objectives (NA 1.3) with a PerkinElmer Spinning Disk UltraVIEW ERS, Olympus IX81 inverted spinning disk confocal microscope. The example in 50 mm glass-bottom dishes (Matek) were coated with poly-L-lysine  diluted in double distilled water , DMSO, cycloheximide (100 μM) or anisomycin (160 μM) solution for 30min before imaging.AB_2566826; 1:50) was incubated with the embryos at 4°C for overnight. The immunostained embryos were washed three times with PBT for 10 min, once with PBT for 30 min, twice with 1X PBS for 5 min before being mounted in 1X PBS for imaging.Adapted from a previously reported in vitro approach with the use of puromycylation of nascent proteins and immunolabeling of puromycin . Live emAB_2305186; 1:8000) or rabbit anti-β-catenin  antibody at 4°C for overnight. The blots were then incubated with HRP-conjugated secondary antibodies  in room temperature for one hour, followed by ECL-based detection (Invitrogen).Brains and eyes were harvested from Stages 40/41 embryos that were anesthetized with 0.4mg/ml MS222 in 1X MBS. The tissues were mechanically homogenized with radioimmunoprecipitation assay  buffer supplemented with protease inhibitor cocktail (Roche) by repeated pipetting, and solubilized by constant rotation at 4°C for 30 min. Remaining non-solubilized material was then pelleted by centrifugation at 13000rpm for 10 min and only the supernatant was then retained. Protein concentration was determined by Bradford assay (Bio-Rad) and spectrophotometry. Bovine serum albumin  was used to create a standard curve for protein concentration and for normalizing the concentration between samples. The lysates were resolved by 12% TGX precast gels (BioRad) at constant 20mA, transferred to nitrocellulose membrane (BioRad) at constant 110V and subjected to western blot analysis by incubating with a rabbit anti-β-actin  in 1 ml of ethanol (Sigma-Aldrich) was injected into the eye cavity until completely filled . The embFor lateral-view imaging, the brain was dissected and mounted in 1X PBS. The contralateral brain hemisphere was imaged with confocal microscopy. For ventral-view preparation F, the veCapped mRNA was in vitro transcribed using mMessage mMachine SP6 Transcription Kit (ThermoFisher), and polyadenylated using Poly(A) Tailing Kit (ThermoFisher).Embryos electroporated with mRFP and Venus-Poly(A)/Venus-β-actin cDNAs were raised until Stages 35/36-37/38 , Stages For 2) in a sequentially manner. The axon was then photobleached for 20 s with an irradiance of 1.5kW/cm2 using the 488 nm wavelength laser and the recovery of Venus fluorescence recorded at 1Hz for 300 s at low irradiance levels (< 2W/cm2) to avoid additional photodamage of the fluorescent proteins and ensure survival of the axons. A second axon outline image of the mRFP was taken directly after FRAP imaging was finished. All imaging steps were performed under episcopic illumination. Imaging was done with the full field of view of the EM-CCD camera (512 × 512 pixel2), which corresponds to a region of 60 × 60 μm2, and an additional EM gain of 200 to ensure high sensitivity.For For ∗p < 0.01, ∗∗p < 0.01, ∗∗∗p < 0.001. ##p < 0.01, ###p < 0.001. Details of statistic results such as p values, degree of freedom, and U/t/F values are presented in the figure legends.A filopodium was defined as a protrusion with a length < 5 μm while a branch was defined as a protrusion with a length > 5 μm . Data wep values) were analyzed with the Roche’s LightCycler 480 software. Standard curves to calculate the amplification efficiency were run independently from the actual experiments. The readout of the control samples were normalized to the biotin-UTP samples to obtain relative concentrations, which were then compared with two-way ANOVA for each RNA species/category (n = 3 independent experiments). For DNA analysis, the Agilent 2100 Bioanalyzer and High Sensitivity DNA Kit did not yield any measurable signals in both control and biotin-UTP samples (n = 3 independent experiments).For RNA analysis, the concentrations of total RNA, 18S rRNA and 28S rRNA in arbitrary units were obtained from the Agilent 2100 Bioanalyzer and RNA 6000 Pico Kit. For RT-qPCR, quantification reads . Docking was defined as stationary or oscillatory movements within a distance of the diameter of the RNA granule or the mitochondrion. To assess the association between RNA/mitochondria docking behavior at the axon shaft and protrusion formation, the proportion of protrusions with a docking time longer than 10 s (RNA) or 1 min (mitochondria) prior to filopodial emergence was retrospectively quantified (“n” represents the number of protrusions). These temporal criteria were defined by at least 3 frames of time-lapse imaging (3-6 s/frame for RNA and 30 s/frame for mitochondria). Mitochondria dynamics were captured at a slower rate of 30 s per 3D-stack per frame to prevent phototoxicity as time-lapse imaging was often carried out over periods of > 1 hr. The occurrences of these docking behaviors at random sites were quantified by computing random axon positions at random time points with random number generators. The results from the protrusion-forming positions were compared to random position with paired t test. For RNA trafficking speed and direction, the terminal 70 μm of GFP-labeled RGC axons was quantified for 1 min (“n” represents the number of RNA granules). RNA granules that moved less than 1 μm were defined as “stationary” and granules that changed the direction of trafficking at least once during the 60 s period of quantification were defined as “bi-directional.”High resolution image stacks of Cy5-RNA, mito-GFP and mRFP were imported into Volocity Quantitation package (PerkinElmer). The axons were identified in a three dimensional manner with the ‘Find objects’ function with mRFP signal. A three dimensional crop was performed to prevent the noise outside of axons in z-layers from being included in the colocalization analysis. The cropped image stack was imported into FIJI  and a 3DThe imaging parameters were kept the same for each round of experiment to allow background-corrected quantitative immunofluorescence comparisons across conditions. One-way ANOVA with Tukey multiple comparisons test was carried out. As an estimation of non-specific incorporation of Alexa488 conjugated anti-puromycin antibody into embryo whole brain, the 99.9% confidence intervals of the “No Puromycylation Control” was 0.016%–0.025% of the control condition (n = 18). “n” represents the number of embryos.Developed films from western blot detection were scanned and imported into FIJI. The color was inverted and the background-corrected signals for β-actin and β-catenin were measured. The level of β-actin was normalized to β-catenin to yield a ratiometric readout. Paired t test was used to assess knockdown efficiency of β-actin protein level (n = 5 independent experiments).For lateral view imaging preparation, the optic tract length was defined by a straight line from the optic chiasm to the distal-most axon terminals in the optic tract/tectum . Ten equThe number of protrusions added and removed was counted on the terminal 50 μm of mGFP/mRFP-labeled RGC axons for 10 min . The addition and removal of protrusions were then compared statistically. Paired t test was used for intragroup comparison, unpaired t test (2 groups) or one-way ANOVA with Tukey multiple comparisons test (3 groups) was used for intergroup comparisons.Quantification of axon growth was performed up to one hour of live imaging. Stalling was defined by axon moving less than 5 μm over 1 hr; misprojection was defined by axon navigating aberrantly away from the normal trajectory and death was defined by axon retracting or showing beaded morphology. Fisher’s exact test (2 groups) or Chi-square test (3 groups) was used for comparing percentages of axons exhibiting different behaviors. Unpaired t test (2 groups) or one-way ANOVA with Tukey multiple comparisons test (3 groups) were used for axon velocity comparisons.3D projections of axon arbors acquired at 40X were semi-automatically traced through the z axis using the Simple Neurite Tracer plugin  in FIJI.0) to pre-photobleaching fluorescence (Fp). For For For H.H.-W.W. and C.E.H. conceived the project and wrote the manuscript. H.H.-W.W. designed the experiments. H.H.-W.W. performed and analyzed experiments, except for those presented in


CircRNAs are a group of endogenous noncoding RNAs. The quickly developing high throughput RNA sequencing technologies along with novel bioinformatics approaches have enabled researchers to systematically identify circRNAs and their biological functions in cells. Deep sequencing of rRNA-depleted RNAs treated with RNase R, which digests linear RNAs and leaves circRNAs enriched, is an efficient way to identify circRNAs. However, very few of RNase R treated data are at hand but a large amount of total RNA-Seq data with no sequencing costs is available, for circRNA predictions. In this study, we systematically investigated the prediction bias from total RNA-Seq data as well as the influence of sequencing depth, sequencing quality and single-end or paired-end sequencing strategy on the predictions. We also identified circRNA properties that may contribute to the improved prediction performance. Our analysis shows that circRNA predictions from total RNA-Seq data gain ∼50% true positive. Sequencing error dramatically worsens the predictions, rather than single-end sequencing strategy or low sequencing depth. However, false positive can be carefully controlled by using data with good quality and narrowing down circRNAs guided by their properties. Circular RNAs (circRNAs) have been discovered as a novel group of endogenous noncoding RNA. Differing from linear RNAs, circRNAs lack 5′ cap and 3′ poly(A) tail and form covalently closed loop structures with circularization of exon, intron or intergenic regions, which makes them more conserved and stable , 2. CircCircRNAs were first found in a viroid as early as 1970s , unfortuPresently, there are few studies investigate the prediction bias, as well as the factors that contribute to the improved predictions, from total RNA-Seq data. In this study, we used a de novo circRNA prediction algorithms CIRI  and KNIFet al. employed Hs68 fibroblast to compare the performance of five circRNA prediction algorithms [http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) showed the good quality of four single-end RNA-Seq data, including SRR444655_1, SRR444655_2, SRR444975_1 and SRR444975_2, except for a declined quality at the 3′ termini of the reads , 2,207 (CIRI2.0), and 2,854 (KNIFE) circRNAs with at least two reads spanning the splicing sites  OnlIn the first simulation based on the data SRR444655 Figure , CIRI1.2In the second simulation Figure , the numIn the third simulation Figure , the numTo validate our findings we also employed CIRI2.0 and KNIFE algorithms to predict circRNAs from total RNA-Seq data SRR444975 and used three simulations to evaluate the prediction bias. The results on SRR444975 were consistent with our findings on SRR444655 .Our analysis above suggested that single-end reads, low sequencing depth and poor sequencing quality correlated with the decreased prediction performance. However, still a proportion of circRNAs was efficiently identified as true ones in the simulated imperfect RNA-Seq data. To identify the factors that may contribute to such efficient predictions, we evaluated four types of circRNAs properties - including distance between back-splicing sites, enrichment score, junction reads ratio, and reads count – between two groups of circRNAs (See methods). The first group (S1) covered the true circRNAs that were efficiently identified in the imperfect RNA-Seq data, and the second group (S2) covered true circRNAs that were not identified in the imperfect RNA-Seq data Figure . KolmogoThis evaluation was performed on three types of simulated imperfect data . The results showed that the shape of CDF for distance of splicing sites in S1 was similar to that in S2 Figure .In this study, we performed comparison analyses to systematically evaluate the bias of circRNA predictions from total RNA-Seq data. Our analysis showed that it was feasible to fully take advantage of the large amount of total RNA-Seq data in the public databases for circRNAs predictions. Though such prediction suffers from false positive, it can be carefully controlled by using total RNA-Seq data with good quality and preferentially selecting the circRNAs with high junction reads ratio and reads count.Our analysis demonstrates that only total RNA-Seq data with good base quality is qualified for circRNA predictions. The number of true circRNAs decreased obviously along with the increasing sequencing errors Figure , moreoveIn our study, we used CIRI1.2, CIRI2.0 and KNIFE to predict circRNAs. Different scircRNA prediction algorithms have different results. A recently published study  comparedOur study has provided a comprehensive view on the predictions using the imperfect total RNA-Seq data. Our analysis demonstrates how public total RNA-Seq data with no sequencing costs, can be effectively utilized to promote the researches on circRNAs.http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Using BWA-MEM alignment algorithm [m is the reads count of predicted circRNA from the Rnase R treated sample, and n is the reads count of predicted circRNA from total RNA-Seq sample. A predicted circRNA with enrichment score greater than 0 is considered as true positive.RNA-Seq data of human fibroblast cell line Hs68, including total RNA sample  and RNase R treated sample  , was dowlgorithm , we mapplgorithm . CIRI1.2The imperfect RNA-Seq data were simulated based on total RNA-Seq data SRR444655 or SRR444975 in three ways: (i) Two files, the first single-end reads and the second single-end reads from SRR444655 or SRR444975, were simulated as two independent total RNA-Seq data with single-end sequencing. (ii) 80%, 50% and 20% reads were sampled from paired-end reads of SRR444655 or SRR444975, which simulated the gradually decreased sequencing depth. (iii) The bases in each read from SRR444655 or SRR444975 were replaced by other bases differing from them. For example, A is replaced by T, C, G, or N. 2, 5, 10 or 20 bases were substituted in each read, respectively, which simulated the gradually decreased sequencing quality. Simulated RNA-Seq data were carried out the same circRNA predictions above.n is the reads count of predicted circRNAs, and N is the reads count of predicted RNAs (including circRNA and linear RNA) around the circular region, a region between two junction sites.Four types of circRNAs properties, including distance between back-splicing sites, enrichment score, junction reads ratio, and reads count, were investigated. Junction reads ratio can be calculated by,Here true circRNAs were separated into two groups based on whether they were efficiently predicted in the imperfect RNA-Seq data. The first group covered the true circRNAs that were identified in the imperfect RNA-Seq data, and the second group covered ones that were not identified in the imperfect RNA-Seq data. Kolmogorov-Smirnov test (K-S test) was employed to respectively compare cumulative distribution function (CDF) of these properties between two groups of circRNAs.