Further information and requests for reagent and resources should be directed to and will be fulfilled by the Lead Contact, Rob Parton ( r.parton@imb.uq.edu.au ).

Zebrafish were raised and maintained according to institutional guidelines (Techniplast recirculating system, 14-h light/10-h dark cycle, The University of Queensland, UQ). Adults (90 dpf above) were housed in 3 or 8 L tanks with flow at 28.5°C, late-larval to juvenile stage zebrafish (6 dpf to 45 dpf) were housed in 1 L tanks with flow at 28.5°C and embryos (up to 5 dpf) were housed in 8 cm Petri dishes in standard E3 media (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl, 0.33 mM MgSO) [] at 28.5°C (incubated in the dark). All experiments were approved by the University of Queensland (UQ) Animal Ethics committee. The following zebrafish strains were used in this study: wild-type (TAB), an AB/TU line generated in UQBR Aquatics (UQ Biological Resources), cavin1b(this paper), cavin1b(this paper) and Tg(actb2:EGFP-CAAX)]. The developmental stages of zebrafish used in experiments (up to 15 dpf) are prior to specific sex determination [] and specifically stated in corresponding figure legends. All zebrafish used in experiments were healthy, not involved in previous procedures and drug or test naive.

Method Details

Animal handling and reagents Zebrafish embryos up to 7 dpf were raised and handled in standard E3 media during experimental periods (5mM NaCl, 0.17mM KCl, 0.33 mM CaCl 2 , 0.33 mM MgSO 4 ). All post-embryonic zebrafish measurements were carried out between tanks of similar population densities and conditions. All reagents were obtained from Sigma-Aldrich unless otherwise specified.

CRISPR/Cas9 generation of cavin1b mutants 48 Montague T.G.

Cruz J.M.

Gagnon J.A.

Church G.M.

Valen E. CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing. 46 Gagnon J.A.

Valen E.

Thyme S.B.

Huang P.

Akhmetova L.

Pauli A.

Montague T.G.

Zimmerman S.

Richter C.

Schier A.F. Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs. Target site with >50% G/C content and no predicted off-target site for zebrafish cavin1b (NCBI: NM_001114549.2 , corresponding Uniprot: A6NA21 ) specific sgRNA was selected using the webtool CHOPCHOP []. The method for cloning-independent synthesis of sgRNA was adopted from Gagnon et al. using the cavin1b gene-specific (containing T7 promoter site, spacer region and overlap region) and constant oligonucleotides mentioned in the Key Resources Table ]. Cavin1b-specific sgRNA was produced by incubating the gene-specific and constant oligonucleotides together in an annealing reaction via the following reaction program (in a Veriti 96-Well Thermal Cycler): 95°C for 5 min, 95°C to 85°C (−2°C /s) and 85°C to 25°C at (−0.1°C /s). Following, in a fill-in reaction, 10 mM dNTP, NEB buffer 2, 100x bovine serum albumin (BSA) and T4 DNA polymerase were added to the product of the annealing reaction and incubated for 20 min at 12°C. The resulting DNA template was purified using the QIAquick PCR Purification Kit and subsequently run on a 3% TBE agarose gel for product verification. Following, sgRNA was transcribed using the Ambion MEGAshortscript T7 Transcription Kit with TURBO DNase incubation (15 min, 37°C) and purified using Zymo Research RNA Clean & Concentrator Kit. Recombinant Cas9 protein containing a nuclear localization signal (PNA Bio Inc) was reconstituted to a solution of 1 mg/mL recombinant Cas9 protein in 20 mM HEPES, 150 mM KCl, 1% sucrose (pH 7.9) and 1 mM DTT. An injection mixture of 700-753 ng/uL Cas9 protein, 200-208 ng/uL sgRNA and 16% phenol red was prepared and incubated for 5 min at room temperature (RT) to allow for Cas9-sgRNA complex formation. Cavin1b-targeting Cas9-sgRNA was injected into the cytoplasm of the early one-cell stage WT embryos. Injection volumes were calibrated to approximately 600-800 pL of injection mixture per injection. 50 Wilson J.M.

Bunte R.M.

Carty A.J. Evaluation of rapid cooling and tricaine methanesulfonate (MS222) as methods of euthanasia in zebrafish (Danio rerio). 2 , 1 M Tris pH 8.3, 10% NP-40, 10% Tween-20, 0.1% gelatine, 20 mg/mL Proteinase K). For juvenile or adult zebrafish tissue collection, selected zebrafish was anesthetised in ethyl 3-aminobenzoate methanesulfonate (tricaine) solution, before cutting an approximately 3 mm piece of the caudal fin with a sterile razor blade and placing the fin clip in digestion buffer. The mixture was incubated at 60°C for 1 hr before reaction termination at 95°C for 15 min. Two different HRMA-compatible platforms were used (Applied Biosystems ViiA 7 Real-Time PCR System, using the MeltDoctor HRM Master Mix, and Roche LightCycler 480 System, using the LightCycler 480 High Resolution Melting Master). HRMA primers are as follows; forward: 5′- GAGAAAGAAGAGGCTGGGGA −3′ and reverse 5′- TTTTGTCAAGCAGCGTGAGG −3′. When using the LightCycler 480 System, the high resolution melt (HRM) step was initiated after a standard PCR amplification step. The HRM step consists of a denaturation step at 95°C, followed by an annealing step at 65°C. Melt data acquisition began at 65°C and ended at 97°C with 15 fluorescence readings per degree centigrade at a 0.07°C /s ramp rate. When using the ViiA 7 Real-Time PCR System, the HRM step consists of a denaturation step at 95°C, followed by an annealing step at 60°C. Melt data acquisition began at 60°C and ended at 95°C at a 0.025°C /s ramp rate. Stable cavin1b f1 mutant zebrafish lines were confirmed using Sanger sequencing with the following primers: forward: 5′- CCTCCCGGTCTCTGATGATG −3′ and reverse: 5′- TTACGCACCTTCTCCAGCAT −3′. Selected cavin1b−/−uq7rp and cavin1b−/−uq8rp lines were then bred to homozygosity. The cavin1b−/−uq7rp line was also maintained on the Tg(actb2:EGFP-CAAX)pc10 transgenic background [ 45 Williams R.J.

Hall T.E.

Glattauer V.

White J.

Pasic P.J.

Sorensen A.B.

Waddington L.

McLean K.M.

Currie P.D.

Hartley P.G. The in vivo performance of an enzyme-assisted self-assembled peptide/protein hydrogel. Founder rate and percentage of mutant allele in f1 progenies was determined via high resolution melt analysis (HRMA). In the DNA preparation step, for whole-embryo tissue collection, selected 48 hpf embryos were anesthetised by rapid cooling [] and added into the digestion buffer (1 M KCl, 1 M MgCl, 1 M Tris pH 8.3, 10% NP-40, 10% Tween-20, 0.1% gelatine, 20 mg/mL Proteinase K). For juvenile or adult zebrafish tissue collection, selected zebrafish was anesthetised in ethyl 3-aminobenzoate methanesulfonate (tricaine) solution, before cutting an approximately 3 mm piece of the caudal fin with a sterile razor blade and placing the fin clip in digestion buffer. The mixture was incubated at 60°C for 1 hr before reaction termination at 95°C for 15 min. Two different HRMA-compatible platforms were used (Applied Biosystems ViiA 7 Real-Time PCR System, using the MeltDoctor HRM Master Mix, and Roche LightCycler 480 System, using the LightCycler 480 High Resolution Melting Master). HRMA primers are as follows; forward: 5′- GAGAAAGAAGAGGCTGGGGA −3′ and reverse 5′- TTTTGTCAAGCAGCGTGAGG −3′. When using the LightCycler 480 System, the high resolution melt (HRM) step was initiated after a standard PCR amplification step. The HRM step consists of a denaturation step at 95°C, followed by an annealing step at 65°C. Melt data acquisition began at 65°C and ended at 97°C with 15 fluorescence readings per degree centigrade at a 0.07°C /s ramp rate. When using the ViiA 7 Real-Time PCR System, the HRM step consists of a denaturation step at 95°C, followed by an annealing step at 60°C. Melt data acquisition began at 60°C and ended at 95°C at a 0.025°C /s ramp rate. Stable cavin1b f1 mutant zebrafish lines were confirmed using Sanger sequencing with the following primers: forward: 5′- CCTCCCGGTCTCTGATGATG −3′ and reverse: 5′- TTACGCACCTTCTCCAGCAT −3′. Selected cavin1band cavin1blines were then bred to homozygosity. The cavin1bline was also maintained on the Tg(actb2:EGFP-CAAX)pc10 transgenic background [].

Two-step reverse transcriptase (RT) PCR 8 Hill M.M.

Bastiani M.

Luetterforst R.

Kirkham M.

Kirkham A.

Nixon S.J.

Walser P.

Abankwa D.

Oorschot V.M.

Martin S.

et al. PTRF-Cavin, a conserved cytoplasmic protein required for caveola formation and function. RNA was isolated from zebrafish embryos (> 100 embryos randomly selected from 1 clutch) using the RNeasy Mini Kit (QIAGEN) and cDNA synthesis was performed using SuperscriptIII reverse transcriptase (ThermoFisher Scientific) as per the manufacturer’s instructions. Amplifications were performed according to Hill et al. []. Primers used for amplification detailed in Table S1 . Amplified products were separated and viewed via gel electrophoreses using 2% TAE agarose gels.

Two-step qRT-PCR −/− and WT zebrafish embryos. qRT-PCR was performed using the SYBR Green PCR Master Mix on a ViiA7 Real-time PCR system (ThermoFisher Scientific) according to the manufacturer’s instructions with three biological replicates (embryos randomly selected from 3 clutches) and three technical replicates on 96-well plates. qRT-PCR primers are listed in 51 Pearen M.A.

Eriksson N.A.

Fitzsimmons R.L.

Goode J.M.

Martel N.

Andrikopoulos S.

Muscat G.E. The nuclear receptor, Nor-1, markedly increases type II oxidative muscle fibers and resistance to fatigue. RNA isolation and cDNA synthesis was performed as described above for 5 dpf cavin1band WT zebrafish embryos. qRT-PCR was performed using the SYBR Green PCR Master Mix on a ViiA7 Real-time PCR system (ThermoFisher Scientific) according to the manufacturer’s instructions with three biological replicates (embryos randomly selected from 3 clutches) and three technical replicates on 96-well plates. qRT-PCR primers are listed in Table S1 . Gene expression was analyzed using the ΔΔCt method []. Differences in expression were calculated using a two-way ANOVA with a posthoc Tukey’s multiple comparison test.

Live imaging For quantitating and qualifying notochord lesions, real-time observation of lesion formation and measurement of general embryonic morphology, embryos were incubated in 0.2 mM phenylthiourea (PTU) solution in E3 media to maintain embryo transparency for ease of notochord lesion visualization. Zebrafish (up to 15 dpf) were anesthetized in tricaine solution in E3, mounted in 1% low melting point (LMP) agarose in tricaine solution in a lateral view unless otherwise stated (anterior left, posterior right) and imaged using a Nikon SMZ1500 fluorescence stereomicroscope. For early-stage 12 dpf larvae, zebrafish were anesthetized in tricaine and mounted in 1% LMP agarose in tricaine solution and imaged using the EVOS FL inverted fluorescence microscope (AMG).

Morphometrics of live zebrafish Captured images using the NIS Elements Version 4.20 software via live imaging of zebrafish as described above were used to measure notochord diameter (at a constant area at the tip of the embryonic yolk extension) and body length (defined as the region from the tip of the anterior end of the zebrafish to the end of the trunk before the caudal fin). Measurements were non-blind and embryos were randomly selected from 2-4 biological replicates (clutches) with no prior formal sample-size estimation. Measurements were conducted using Fiji. Statistical analysis was carried out using two-tailed unpaired t tests.

Lesion qualification and quantification In qualifying notochord lesions, three qualities of severity are identified via brightfield imaging using the Nikon SMZ1500 fluorescence stereomicroscope at x90 magnification, with embryos orientated at a constant position (anterior to the left). Mild lesions appear slightly delaminated with less apposed neighboring notochordal cells. Moderate lesions appear as lesions with larger delamination and are occasionally flanked by a small amount of fragmented vacuoles. Severe lesions appear as dense cellular regions with a larger delamination compared to moderate lesions, and are flanked by, and/or consist of small fragmented vacuoles. These lesions are represented in Figure 1 H. In quantitating notochord lesions, the notochord of a live zebrafish embryo was viewed at x90 magnification via live imaging of zebrafish as described above. The number of lesions in one particular notochord is recorded in Microsoft Excel and the qualities of severity of each lesion were recorded in the same Microsoft Excel spreadsheet using a severity index describing mild, moderate and severe lesions with the arbitrary values of 2, 3 and 4 respectively. The severity index score was calculated by adding the arbitrary values of mild (2), moderate (3) and severe (4) and taking the sum of values for each individual notochord. The arbitrary values are approximations of relative lesion site size and degree of fragmentation in vacuoles. Embryos used for measurements of lesion number and severity index score in relevant experiments were randomly selected from 2-4 biological replicates (clutches) with no prior formal sample-size estimation. Measurements were non-blind. Statistical analyses were carried out using two-tailed unpaired t tests, ordinary one-way ANOVA with posthoc Tukey’s multiple-comparison tests or two-way ANOVA with posthoc Tukey’s multiple-comparison tests. The proportion of lesion severity was calculated by taking the proportions of the total number of mild, moderate and severe lesions counted and expressing the values as proportion percentages in a total number of embryos. Embryos used for measurements of proportion of lesion severity in relevant experiments were randomly selected from 3-4 biological replicates (clutches) with no prior formal sample-size estimation. Measurements were non-blind. Statistical analyses were carried out using chi square tests.

High magnification live imaging For high magnification and resolution imaging and live timelapse of notochord lesions, zebrafish embryos were incubated at 28°C in either or both BODIPY FL C5-Ceramide and BODIPY TR methyl ester (ThermoFisher Scientific) for 24 and/or 2 hr respectively, anesthetized in tricaine, mounted in 1% LMP agarose in tricaine solution and imaged using a Zeiss LSM 710 upright confocal microscope equipped with a x40/1.0 W N-Achroplan M27 water immersion lens.

Notochord vacuole size and number measurement Captured images using the Zeiss LSM 710 upright confocal microscope on the Carl Zeiss ZEN 2012 (black edition) software via the high magnification live imaging of zebrafish technique as described above were used to measure individual vacuole size in Fiji. The embryos were labeled using BODIPY TR methyl ester and imaged in a dorsal view (frontal plane) at the anterior end of the yolk extension (avoiding the imaging of notochord areas containing lesions). Images containing notochord lesions were excluded from measurements. Embryos used for measurements were randomly selected from 4 biological replicates (clutches) with no prior formal sample-size estimation. Measurements were non-blind. Statistical analyses were carried out using two-tailed unpaired t tests

Latrunculin treatment of embryos Live cavin1b−/− or WT zebrafish were treated with 1.25 μM Latrunculin A in E3 media (Sigma-Aldrich) for 35 min and subsequently washed 4 times in E3 media.

Whole mount phalloidin staining 9 Nixon S.J.

Wegner J.

Ferguson C.

Méry P.F.

Hancock J.F.

Currie P.D.

Key B.

Westerfield M.

Parton R.G. Zebrafish as a model for caveolin-associated muscle disease; caveolin-3 is required for myofibril organization and muscle cell patterning. Our staining protocol was modified from previous publication []. Zebrafish were anesthetized using tricaine and fixed in 4% paraformaldehyde overnight. The embryos were washed into methanol and incubated overnight at −20°C. The embryos were then washed into PBS/0.1% Tween-20 (PBST) and blocked in a blocking solution (1% BSA, 1% DMSO, 0.2% saponin and 1% horse serum) for 2 hr at RT. The embryos were then incubated in Alexa Fluor 594 Phalloidin (ThermoFisher Scientific) in 1:20 dilution overnight at 4°C and washed in the blocking solution over the course of 2 hr. Following, the embryos were washed into PBST, then PBS, and stored at 4°C. For imaging, stained embryos were mounted in 1% LMP agarose in PBS and imaged using a Zeiss LSM 710 upright confocal microscope equipped with a x40/1.0 W N-Achroplan M27 water immersion lens. 52 McCloy R.A.

Rogers S.

Caldon C.E.

Lorca T.

Castro A.

Burgess A. Partial inhibition of Cdk1 in G 2 phase overrides the SAC and decouples mitotic events. Stained embryos were imaged and captured from a dorsal view (frontal plane) at the anterior end of the yolk extension as described above and images were analyzed using Fiji. To measure corrected total fluorescence, the integrated density of the imaged notochords were measured and subtracted with the mean of background mean gray value of 3 randomly selected vacuoles x the area of the imaged notochord as modified from previous publications []. Images containing notochord lesions were excluded from measurements. Embryos used for measurements were randomly selected from 2-3 biological replicates (clutches) with no prior formal sample-size estimation. Measurements were non-blind. Statistical analyses were carried out using a two-tailed unpaired t test and an ordinary one-way ANOVA with posthoc Tukey’s multiple-comparison test.

Live zebrafish notochord cell laser ablation −/− or WT zebrafish expressing EGFP-CAAX were anesthetized and mounted in 1% LMP agarose on MatTek glass bottom dishes with a dorsal view (frontal plane) and imaged using the LSM 710 Meta inverted confocal microscope equipped with a multiphoton Mai Tai eHP 760-1040nm laser. Laser ablation was carried out as previously described under the 40x, 1.3 NA Plan Apochromat oil immersion objective at 28°C [ 21 Liang X.

Michael M.

Gomez G.A. Measurement of mechanical tension at cell-cell junctions using two-photon laser ablation. 21 Liang X.

Michael M.

Gomez G.A. Measurement of mechanical tension at cell-cell junctions using two-photon laser ablation. 2+(Ytop (t)-Ybottom (t))2). The amount of strain [ε(t)] after ablation is then measured using the following formula: ε(t) = L(t)-L(0). Fitting of the data was acquired using the following equation: ε(t) = L(t)−L(0) = F0/E⋅(1−e−[(E/μ)∗t]) where F0 is the tensile force present at the junction before ablation, E is junction elasticity and μ is viscosity coefficient related to the viscous drag of the cell cytoplasm. We utilized the following equations; initial recoil = dε(0)dt/dt = F0/μ and K value = E/μ as fitting parameters. Fitting of the recoil data was carried out using GraphPad PRISM. Embryos used for measurements were randomly selected from 2 biological replicates (clutches) with no prior formal sample-size estimation. Measurements were non-blind. Statistical analyses were carried out using two-tailed unpaired t tests. Live 2 dpf cavin1bor WT zebrafish expressing EGFP-CAAX were anesthetized and mounted in 1% LMP agarose on MatTek glass bottom dishes with a dorsal view (frontal plane) and imaged using the LSM 710 Meta inverted confocal microscope equipped with a multiphoton Mai Tai eHP 760-1040nm laser. Laser ablation was carried out as previously described under the 40x, 1.3 NA Plan Apochromat oil immersion objective at 28°C [] with modifications. 3-5 ablation sites were randomly selected along the length of the yolk extension and a time series of approximately 8 s (time interval 0.95 s) was performed post ablation. Notochords containing lesions were excluded from the experiment. Data analysis of recoil measurements after laser ablation, instantaneous recoil velocity at time = 0 and K value was performed as previously published []. Briefly, the MTrackJ plugin from Fiji was used to analyze the time-lapse images after application of a median filter (1 pixel). The strain or deformation ε(t) of the notochord cell-cell junction was measured as a function of time after ablation by tracking the XY coordinates of each vertex resulting from the ablated junction over time and data was collected in Microsoft Excel. The length of the contact L(t) for each time point was calculated using the following formula: L(t) = sqrt ((Xtop (t)- Xbottom (t))+(Ytop (t)-Ybottom (t))). The amount of strain [ε(t)] after ablation is then measured using the following formula: ε(t) = L(t)-L(0). Fitting of the data was acquired using the following equation: ε(t) = L(t)−L(0) = F0/E⋅(1−e) where F0 is the tensile force present at the junction before ablation, E is junction elasticity and μ is viscosity coefficient related to the viscous drag of the cell cytoplasm. We utilized the following equations; initial recoil = dε(0)dt/dt = F0/μ and K value = E/μ as fitting parameters. Fitting of the recoil data was carried out using GraphPad PRISM. Embryos used for measurements were randomly selected from 2 biological replicates (clutches) with no prior formal sample-size estimation. Measurements were non-blind. Statistical analyses were carried out using two-tailed unpaired t tests.

Intravenous microinjection of embryos 53 Bassett D.I.

Bryson-Richardson R.J.

Daggett D.F.

Gautier P.

Keenan D.G.

Currie P.D. Dystrophin is required for the formation of stable muscle attachments in the zebrafish embryo. For Evans Blue dye injection (EBD), pericardial injection of approximately 5 nL of 0.1 mg/mL EBD via the common cardinal vein was performed on zebrafish embryos as previously described []. Embryos were incubated at 28°C for 3 hr to ensure sufficient EBD circulation and uptake. Anesthetized embryos were mounted and imaged as described in live imaging of zebrafish above. Embryos were randomly selected from 1 biological replicate (clutch). For Alexa Fluor 488 5-UTP microinjections, zebrafish embryos were labeled with BODIPY TR methyl ester as described above, followed by a pericardial injection of approximately 5 nL of Alexa Fluor 488 5-UTP solution via the common cardinal vein. Embryos were incubated at 28°C for 2 hr to ensure sufficient fluorophore circulation and uptake. Anesthetized embryos were mounted and imaged as described in high magnification live imaging of zebrafish above. Embryos were randomly selected from 1 biological replicate (clutch).

Mechanical stimulation of embryos For bleaching experiments, 1 dpf zebrafish embryos were incubated in a mild bleaching solution (40 μL 10% sodium hypochlorite solution in 50 mL E3 media) for 10 min at RT. Embryos were then washed three times in E3 media and placed in 0.2 mM PTU solution until 112 hpf at 28°C. At 112 hpf, embryos were manually dechorionated and notochord lesion quantitation and qualification were performed as described above. For electrical stimulation experiments, 3 dpf zebrafish embryos were first placed in 3.5 cm culture dishes, anesthetized in tricaine and notochord lesion quantitation and qualification were performed as above. Embryos were then washed three times in E3 media and subjected to electrical stimulation using a constant voltage electrical stimulator (Square Pulse Stimulator S44, Grass Instruments) with the following settings: stimulation rate: 4 pps x 0.1, delay: 9 ms x 0.1, duration: 9 ms x 0.1, voltage: 12 V x 10 for 10 min. Electrodes were manually circulated evenly around the culture dish to ensure even stimulation in all embryos. For unstimulated controls, electrodes connected to the stimulator with the power turned off were manually circulated evenly around the culture dish for 10 min. Zebrafish were then incubated at 28°C before notochord lesion quantitation and qualification repeated 24 hr post stimulation. For prolonged electrical stimulation, 3 dpf zebrafish embryos were anesthetized in tricaine, mounted in 1% LMP agarose and subjected to electrical stimulation using a constant voltage electrical stimulator (Square Pulse Stimulator S44, Grass Instruments) with the following settings: stimulation rate: 4 pps x 0.1, delay: 9 ms x 0.1, duration: 8 ms x 0.1, voltage: 11.6 V x 1 for 40 min under a Nikon SMZ1500 fluorescence stereomicroscope at RT, or for approximately 76 min (see below). Video was taken using NIS Elements Version 4.20 AVI acquisition tool at 7 fps. Constant notochord area was determined by approximating a length of 6-somites from the anus of the zebrafish using Fiji. Embryos were randomly selected from 3 biological replicates (clutches). For high resolution live timelapse of lesion formation, 65 hpf zebrafish embryos were anesthetized in tricaine, embedded in 1% LMP agarose and subjected to approximately 76 min of prolonged electrical stimulation with the same parameters above on a constant voltage electrical stimulator using electrodes mounted on a x40/1.0 W N-Achroplan M27 water immersion lens at RT. Timelapse was taken using a Zeiss LSM 710 upright confocal microscope on the Carl Zeiss ZEN 2012 (black edition) software. For high intensity electrical stimulation, individual 3 dpf WT or cavin1b−/− embryo was placed in a glass dish and electrically stimulated using an electrical stimulator (Square Pulse Stimulator S44, Grass Instruments) with the following settings: stimulation rate: 6 pps x 1, delay: 9 ms x 0.01, duration: 10 ms x 10, voltage: 5.5 V x 10 for 80 s. Five seconds before the end of stimulation, 2.5% glutaraldehyde in 2X PBS was added to the dish containing stimulated embryo in equal volume with the E3 media and placed for 3 min in a Pelco Biowave under vacuum and irradiated at 80 W. Electron microscopy was carried out as described below. Tricaine anesthetized embryos were used as a control.

Tricaine treatment of embryos At 1 dpf, zebrafish embryos were dechorionated by incubation in 1 mg/mL pronase solution at RT for 5 min, then washed three times in E3 media. Embryos were anesthetized in a standard tricaine solution (0.16 mg/mL in E3 media) for up to 24 hr at 28°C in the locomotion inhibition experiments. For tricaine washout experiments, during the washout step, embryo plates containing tricaine solution were rinsed and washed three times with fresh E3 media. Quantitation and qualification of notochord lesions were carried out as described above.

Behavioral analysis of zebrafish 54 Laird A.S.

Mackovski N.

Rinkwitz S.

Becker T.S.

Giacomotto J. Tissue-specific models of spinal muscular atrophy confirm a critical role of SMN in motor neurons from embryonic to adult stages. −/− larva was placed in each well of 24-well plates filled with 1 mL of E3 media. The plates were incubated at 28°C in the dark prior to the experiments. Larva spontaneous swimming behaviors were recorded for 1 hr at RT under normal lighting condition and without specific stimulation. Time points of 5, 6 and 7 dpf were used (same zebrafish for each time point). At the end of the experiment, each larva was checked in order to exclude potential dead animals from the data. Data were exported and processed using Microsoft Excel to analyze and compare total distance traveled during the 1 hr recording period. Zebrafish used for measurements were randomly selected from 2 biological replicates (clutches) with no prior formal sample-size estimation. Measurements were non-blind. Statistical analyses were carried out using two-tailed unpaired t tests. Swimming behavioral analysis was carried out using the ZebraBox Revolution following manufacturer’s instructions ( http://www.viewpoint.fr/en/p/equipment/zebrabox ) and as previously described []. One WT or cavin1blarva was placed in each well of 24-well plates filled with 1 mL of E3 media. The plates were incubated at 28°C in the dark prior to the experiments. Larva spontaneous swimming behaviors were recorded for 1 hr at RT under normal lighting condition and without specific stimulation. Time points of 5, 6 and 7 dpf were used (same zebrafish for each time point). At the end of the experiment, each larva was checked in order to exclude potential dead animals from the data. Data were exported and processed using Microsoft Excel to analyze and compare total distance traveled during the 1 hr recording period. Zebrafish used for measurements were randomly selected from 2 biological replicates (clutches) with no prior formal sample-size estimation. Measurements were non-blind. Statistical analyses were carried out using two-tailed unpaired t tests.

Touch-evoked response assay 4 and 5 dpf zebrafish embryos were placed in a petri dish filled with E3 media. From which, single embryos were placed in individual petri dish on a determined spot. Tactile stimuli were applied to the tail of the embryo with a pair of no. 5 forceps, up to 3 times. Only embryos that responded with straight swimming upon touch stimuli were recorded. Embryos that did not respond after 3 touch stimuli or exhibited turning behaviors after touch stimuli were not used for the experiment. Captured images were used to determine distance traveled in measures of body length. Embryos used for measurements were randomly selected from 3 biological replicates (clutches) with no prior formal sample-size estimation. Measurements were non-blind. Statistical analyses were carried out using two-tailed unpaired t tests.

Calcein staining of larvae 55 Du S.J.

Frenkel V.

Kindschi G.

Zohar Y. Visualizing normal and defective bone development in zebrafish embryos using the fluorescent chromophore calcein. This protocol is adapted from Du et al. []. Zebrafish early-stage larvae were incubated in 0.2% calcein solution (pH 7.0) for 10 min and washed in aquarium system water three times. Anesthetized embryos were mounted in 1% LMP agarose and imaged as described above. Imaging was carried out using a Nikon SMZ1500 fluorescence stereomicroscope.

Electron microscopy of zebrafish notochord 56 Deerinck, T.J., Bushong, E.A., Thor, A., and Ellisman, M.H. (2010). NCMIR methods for 3D EM: a new protocol for preparation of biological specimens for serial block face scanning electron microscopy. https://www.ncmir.ucsd.edu/sbem-protocol/. This protocol is a modified version originally by Deerinck et al. [], and designed to enhance membrane contrast using reduced osmium tetroxide, thiocarbohydrazide-osmium, uranyl acetate and en bloc lead nitrate staining. A solution containing 2.5% glutaraldehyde in 2X PBS was added to the dish in equal volume with anesthetized zebrafish embryos and placed for 5 min in a Pelco Biowave under vacuum and irradiated at 80 W. Embryos were reduced in size by removing the head and tail, and again irradiated in fresh fixative (2.5% glutaraldehyde), under vacuum, for a further 6 min. Embryos were washed 4 × 2 min in 0.1 M cacodylate buffer. A solution containing both potassium ferricyanide (1.5%) and osmium tetroxide (2%) in 0.1 M cacodylate buffer was prepared and samples immersed for 30 min at RT. Following 6 × 3 min washes in distilled water, samples were then incubated in a filtered solution containing thiocarbohydrazide (1%) for 30 min at RT. After subsequent washing in distilled water (6 × 2 min), samples were incubated in an aqueous solution of osmium tetroxide (2%) for 30 min. Samples were washed again in distilled water (6 × 2 min) and incubated in 1% uranyl acetate (aqueous) for 30 min at 4°C. Further distilled water washes (2 × 2 min) were completed before adding a freshly prepared filtered 0.06% lead nitrate in aspartic acid (pH 5.5) solution warmed to 60°C. The lead nitrate solution containing tissue blocks was further incubated for 20 min at 60°C before rinsing in distilled water (6 × 3 min) at RT. Samples were dehydrated twice in each ethanol solution of 30%, 50%, 70%, 90% and 100% absolute ethanol for 40 s at 250 W in the Pelco Biowave. Epon LX112 resin was used for embedding the tissue with infiltration steps at 25%, 50%, 75% resin to ethanol in the Pelco Biowave under vacuum at 250 W for 3 min and finishing with 100% resin (twice), before the final resin embedding and placed in a 60°C oven for 12 hr. Blocks were sectioned on a Leica UC64 ultramicrotome at 60 nm and mounted on formvar coated 3 slot Cu grids. Thin sections (60 nm) were viewed on a Jeol JEM-1011 at 80kV.