Adult fish (3–6 months old) and embryos of either sex were used from various strains. These include transgenic lines to visualise distinct cell populations: Tg(gfap:EGFPmi2001) labels radial glia cells across the central nervous system driven by the glial fibrillary acidic protein promoter , Tg(Isl1:EGFPrw0) labels secondary motor neurons in the spinal cord (additional to cranial motor and some sensory neurons) , Tg(vsx1:GFP) labels interneurons in the spinal cord driven by the visual homeobox 1 promoter , Tg(met:GAL4; UAS:EGFP)ed6Tg  and Tg(met:mcherry 2A KalTA4)pc24Tg use the C-met promoter to drive reporter expression in primary motorneurons, which represent a distinct population from neurons expressing Islet1 . Additionally, two lines (obtained from Zebrafish International Resource Center) were used to manipulate Fgf signalling: Tg(hsp70l:dn-fgfr1-EGFPpd)1 in which heatshock induces expression of a dominant negative FgfR1 (Fgf signalling inhibition) , and spry4−/−fh117 mutants, which represent a gain of Fgf signalling function, as the key downstream negative regulator sprouty is missing. All experiments were conducted in accordance with Monash University guidelines and approved by the local ethics committee.
Spinal cord lesion
Spinal cord lesioning and injections (intraperitoneal or lesion site) were performed as described previously [1, 2] in fully anesthetized fish. Fish were fully anaesthetized in buffered 0.033% tricaine methanesulfonate (MS-222) in fish tank water, until respiratory movements of the opercula stopped (3–5 min). Halfway between the dorsal fin and the operculum, corresponding to the eighth vertebra (approximately 5 mm caudal to the operculum) of the spinal cord, a longitudinal incision was made through the muscle layer, and the vertebral column was exposed by holding the muscle tissue aside. Then the vertebral column was cut completely with micro-scissors. The wound was sealed with a drop of 3 M Vetbond. Fish were recovered from the anesthesia, by flushing the gills of the fish in a tank of fresh fish water by gently pulling the fish through the water. Fish resumed breathing within a few seconds.
Heat shock treatment for Fgf signaling inhibition
The dominant negative form of FgfR1 was induced by applying heat shock to Tg(hsp70l:dn-fgfr1-EGFP) transgenic or wildtype control animals. Animals were exposed to an increased temperature from 26 °C to 38 °C  and remained at 38 °C for 60 min, 4 h prior to spinal cord injury. Fish were exposed once daily to this heat shock regime and spinal cords collected at indicated time points.
Intraperitoneal (IP) injections of 50 μl BrdU (2.5 mg/ml in PBS; Sigma, USA) were performed in fully anaesthetized fish immediately following SCI at 0, as well as at 2 and 4 days post lesion or in age-matched control non-lesioned fish.
Recombinant human Fgf3 (0.14 μg/injection/fish)  was injected IP into fully anaesthetized Tg(Isl1:EGFP) fish every second day starting immediately after SCI for 5 or 10 days. The central region of human Fgf3 shows 72% amino acid identity with zebrafish Fgf3 .
Vivo morpholino injections
A single dose of 1 μl of 0.5 mM (5 μg/injection/fish) Fgf3 morpholino (5’CATTG TGGCATGGCGGGATGTCGGC3’) or vivo standard control morpholino (5’CCTCTTACCTCAGTTACAATTTATA3’) was injected into the lesion site immediately after spinal cord transection (Gene Tools, LLC, Oregon, USA). Fgf3 vivo morpholino injections in zebrafish larvae phenocopies the observed small otic vesicle seen in Fgf3 mutants.
At different time points (3, 6 10 and 14 days) after SCI, fish were humanely killed by deep anaesthesia with buffered 0.2% MS-222. The brains and spinal cords were exposed and fixed for 2 h in 4% paraformaldehyde (PFA) in PBS (phosphate buffered saline) at room temperature. The brains and the spinal cords were subsequently dissected out and postfixed for a further 2–3 h in 4% PFA at room temperature followed by immersion in 30% sucrose in PBS overnight at 4 °C, before embedding in OCT (TissueTek). Spinal cords were cryostat sectioned at 20 μm thickness for immunohistochemistry or 30 μm thickness for in situ hybridization.
Sections were labelled using standard immunohistochemical procedures to determine expression and localization of different proteins at the lesion site. Sections were post-fixed for 10 min in 4% PFA, followed by blocking solution (PBS-triton X containing 5% normal goat serum (Invitrogen, CA, USA)) for 1 h at room temperature. Antigen retrieval was performed by incubating the sections for 15 min in 2 M HCl prior to blocking for BrdU immunohistochemistry. Primary antibodies were diluted in blocking solution and sections were incubated overnight at 4 °C. After rinsing in PBS, sections were incubated for 2 h at room temperature with secondary antibodies diluted in blocking solution. Sections were mounted in Fluoromount (Dako, USA). Primary antibodies used were: mouse anti-NeuN (1:1000; Millipore); rabbit anti-pMAPK (mitogen-activated protein kinase 1:1000; Cell signalling); mouse anti-bromodeoxyuridine (1:400, Roche); rabbit anti-GFP (1:500; Invitrogen); mouse anti-β-tubulin (1:1000, Promega); rabbit anti-Ki67 (1:400, Thermo). Secondary antibodies used were: goat anti-rabbit or goat anti-mouse Alexa Fluor-488 or Alexa Fluor-594 (1:1000; Molecular Probes). Nuclei were visualised by staining with DAPI (4′,6-diamidino-2-phenylindole) (Sigma).
Probe generation and in situ hybridization
In situ hybridization and probe generation was performed as previously described [14, 15]. Briefly, plasmids were linearized, transcribed and labelled, using T7 or SP6 polymerase (Roche) and a DIG RNA labelling mix (Roche). In situ hybridization was performed using standard procedures on 30 μm cryostat sections. Following staining, tissues were imaged using a Z1 AxioImager compound microscope. Prior to performing in situ hybridization, sections with cells expressing GFAP:EGFP or Isl1:EGFP were imaged allowing us to examine gene expression of the same glia or neuronal cells before and after in situ staining.
Fgf exposure in larva
For Fgf exposure, Tg(met:GAL4; UAS:EGFP)ed6Tg or Tg(Isl1:GFPrw0 / met:mcherry 2A KalTA4pc24Tg) double transgenic embryos were used at 24 h postfertilisation (hpf). Embryos were swum in 1.5 μg/ml Fgf3/8 or 2 diluted in embryo medium, or embryo medium alone (control) for 48 h. The embryo medium was replaced after the first 24 h.
Following Fgf swimming exposure whole zebrafish embryos were mounted in 1% low melt agarose, covered by embryo medium containing 0.033% MS-222, and imaged using a 20X objective at the Zeiss LSM710 confocal microscope at 1 μm optical intervals. After imaging, embryos were fixed, sectioned and processed for GFAP immunoreactivity as described above. Sections were examined by brightfield or fluorescence microscopy using a Z1 AxioImager (Zeiss, Berlin, Germany) epifluorescence microscope. Photomicrographs (1300 × 1030 dpi) were obtained with various Plan-Neofluar objectives (Zeiss), and acquired as digital images using an AxioCam (Zeiss) digital camera with AxioVision software (v. 4.4; Zeiss). In order to confirm co-localization between different proteins, single optical plane sections of samples were acquired using the Apotome module and a 40X objective, using AxioVision software. All images were taken focused through the medial section containing the central canal identified in the DAPI channel without looking at the stained channels.
PC12 rat pheochromocytoma cell culture
The PC12 cell line derived from rat pheochromocytoma (adrenal medulla) was kindly provided by A/Prof Julian Heng (Harry Perkins Institute of Medical Research). The PC12 cells were grown in Dulbecco’s modified Eagle’s medium supplemented with antibiotics, 10% heat inactivated fetal bovine serum and 10% horse serum (HS). Cells were incubated at 37 °C in 5% CO2 in air, and the medium was changed every 3–4 days. Cells were passaged when 90% confluent using PBS-EDTA (ethylenediaminetetraacetic acid). Cells were induced to differentiate by growing on polylysine-coated plates at a density of 5000 cells/well in a 24 well plate either in the presence or absence of 50 ng/ml hFgf2, hFgf3 or hFgf8 (R&D) without serum for 3 days. After 72 h, cells were fixed and immunostained using primary mouse anti-βIII-tubulin antibody (1:2000; Promega) and secondary anti-mouse Alexa – Fluor 564 antibody (1:1000; Molecular Probes) for quantification and length measurement of neurite outgrowth.
Lysates preparation and immunoblot
For the p-MAPK signalling analysis, cells were plated at a density of 1 × 106 cells/ 10 cm plate the day before the experiment. On the experimental day the medium was replaced with medium without serum and hFgf2/3/8 was added at different time points as indicated, and then lysed in lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM EDTA pH 8, 1 mM EGTA pH 8, 1.5 mM MgCl2, 200 μM Na3VO4, 150 nM aprotinin, 1 μM leupeptin and 500 μM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride). Protein concentration was determined using the Bradford assay (BioRad). An equal amount of protein was taken for each immunoblot. Equal amounts of protein from each sample were loaded and resolved by SDS-polyacrylamide gel electrophoresis through 10% gels. The gels were electrophoretically transferred to a nitrocellulose membrane. Membranes were blocked, blotted with the corresponding primary antibody (rabbit anti-pMAPK p44/p42 variant or MAPK 1:1000, Cell Signalling;) followed by secondary antibody linked to horseradish peroxidase. Immunoreactive bands were detected by chemiluminescence reaction.
BrdU-positive cell quantification following adult SCI
The number of Isl1:EGFP only labelled cells or BrdU/Isl1:EGFP double labelled cells in the spinal cord sections were counted within a 200 μm2 grid located ~ 100–300 μm proximal to the lesion site from both sides of the lesion. This was done in images taken of every second serial longitudinal 20 μm thick section using the Z1 AxioImager (Zeiss, Berlin, Germany) with the ApoTome. Results were expressed as the mean ± SEM (n = 5 fish per group). Statistical significance determined using one-way ANOVA followed by multiple comparisons using the Tukey’s test.
Islet1-positive cell and Islet1 / C-met neurite quantification in larva
The number of Isl1:EGFP labelled cells and the total neurite area of the Isl1:EGFP/ C-met:mCherry labelled cells were quantified from one side of the spinal cords in 3 dpf old larvae. For Islet1 positive cells counts, transverse 20 μm sections were taken from the area between the back fin and the anal fin, and single optical plane images were taken on a Z1 AxioImager with the ApoTome. Results were expressed as the mean ± SEM (n = 10 fish per group) and statistical significance determined using one-way ANOVA followed by multiple comparisons using the Tukey’s test.
For neuronal filament sprouting analysis, confocal stacks were loaded into Imaris (Bitplane) and neurite area was quantified using the FilamentTracer module. In the filament creation wizard, the seed points are first detected and thresholds were set to determine neurite starting points. The module then connected the seed points to create the spine and subsequent neurites. Neurite area was determined with manual thresholding (identical across image files) based on the actual fluorescence of the transgenic line. Results were expressed as the mean ± SEM (n = 8 fish per group) and statistical significance determined using one-way ANOVA followed by multiple comparisons using the Tukey’s test.
C-met neurite quantification in adult
The number of C-met labelled neurites in Tg(met:GAL4; UAS:EGFP) fish was quantified at 350 μm distance distal and proximal to the lesion site 10 days following SCI. Results were expressed as the mean ± SEM (n = 7 fish per group; at least 27 sections of spinal cord from each group) and statistical significance was determined using the Student’s t-test.
Proliferation and neurite outgrowth in PC12 cell line
The percentage of Ki67 labelled proliferating cells and the number of total cells extending β-tubulin labelled neurites was quantified and expressed as mean ± SEM of at least 10 fields / well in triplicate wells in at least n = 3 independent experiments. One-way ANOVA followed by multiple comparisons using the Tukey’s test was used to test for differences between experimental groups.
NeuN positive cell counts
The number of NeuN positive cells were quantified and expressed as mean ± SEM. NeuN neuronal staining counted within a 200 μm2 grid located located from the centre of the lesion (n ≥ 7 per group). One-way ANOVA followed by multiple comparisons using the Tukey’s test was used to identify significant differences between groups in the experiments.