• Maintenance of Zebrafish
A breeding colony of zebrafish {Danio rerio) were maintained on a 14 hour light/10 hour dark cycle (Westerfield, 1993). Embryos were collected by natural spawning and staged according to Kimmel et al. (1995). Normal embryos were generated from the wild-type lines kcwt (King's College wild-type), uwt (University College wild-type), *AB (AB wild- type), Tübingen and Tup Longfin. Non pigmented embryos were generated by raising in 0.2 mM phenylthioarbam ide at 28.5 °C. After dechorination, and throughout manipulations, embryos were maintained in embryo medium containing 10 mM Hepes (from IM stock of Hepes in dHjO pH7.2) (hence forward referred to as buffered EM).
• Observation of live embryos
Embryos were observed in fish water under a Leica dissecting microscope and manually dechorinated with #5 watchmaker's forceps. Where required, embryos were anaesthetised with 0.02% Tricaine (3-amino benzoic acidethylester) and mounted for viewing in 3% Methyl Cellulose in EM.
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• Wounding and culturing of epiboly stage embryos
Embryos were dechorinated in petri dishes lined with 1 % Agarose in EM. For wounding, transplantation pipettes were pulled from 1 mm borosilicate glass capillaries (World Precision Instruments, IB 100-4) and broken off using watchmaker's forceps to an inner diameter of approximately 100 pm. The pipettes were loaded onto an oil-filled pipette holder (World Precision Instruments, 5430-10) and held by a 3-axis micromanipulator (Narishige, MN-153) connected by a continuous stream of mineral oil to a 50 pi Hamilton syringe driven by a micrometer-controlled syringe pump (Stoelting, 51218). Embryos at approximately 50% epiboly were transferred to wounding wells, preformed using an acrylic mould (made by Anatomy, UCL workshops) in 1% agarose in EM. The mould left wells approximately 1.0 mm deep by 1.0 mm wide, with the bottom surface of the well sloping slightly down from the back of the well. Embryos were placed into these wells and orientated with the animal pole uppermost. To make the wound, tissue from the animal pole was gently drawn in and out of the pipette about 3 or 4 times until the tissue became removed from the surrounding tissue and yolk. This leaves a circular wound approximately 100 pm in diameter (Fig 3.2 A, C). After wounding, embryos were left in their wells and allowed to heal in buffered EM for defined timepoints at 28.5°C.
• Wounding and culturing of 24 hour and later embryos
Embryos were dechorinated in EM and anaesthetised with 0.02% Tricaine. Embryos were mounted in 1% low melting point agarose in buffered EM, and oriented dorsal upward for wounding between the otic vesicles or on their flank for wounding along the tail. Embryos were wounded using an electrolytically sharpened tungsten needle. After wounding, individual embryos were carefully transferred from the agarose mounting using
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watchmaker's forceps into buffered EM where they were allowed to heal in petri-dishes for defined timepoints at 28.5°C.
• Processing embryos for scanning electron microscopy (SEM)
Embryos were fixed overnight in freshly prepared, ice-cold 1/2 strength Kamovsky's fixative (Kamovsky, 1965) at 4°C. After fixation the embryos were washed in O.IM sodium cacodylate buffer for 20 minutes, post-fixed in a solution of 1% osmium tetroxide / 0.1 M sodium cacodylate at 4°C for 20 minutes, and rinsed twice in 0.1 M sodium cacodylate for 10 minutes each. Specimens were then dehydrated through a graded series of alcohols - 30%, 50%, 70%, 90%, 95% and 100% - 5-10 minutes in each with two final rinses in 100% ethanol. Embryos were then washed in 50:50 100% ethanol:acetone for 10 minutes, and then washed twice in 100% acetone for 10 minutes before being critical-point dried in CO2. Once dry, embryos were mounted on carbon adhesive discs on SEM stubs
(Agar Scientific) and coated with approximately 30nm of gold and finally viewed using a JEOL 5410LV.
• Processing embryos for light histology and transmission electron microscopy
(TEM)
Processing of embryos for TEM was essentially identical to that described for SEM up to and including dehydration through a graded series of alcohols. After dehydration embryos were washed 3 times in 100% ethanol for 5 minutes each and then washed 3 times in propylene oxide for 5 minutes each. Embryos were then transferred into a mix of 50:50 propylene oxide:Araldite resin and incubated for 45 minutes at room temperature with agitation. Embryos were then transferred to fresh Araldite in soda vials and allowed to sink
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in individual plastic moulds and Araldite was added to fill the capsule. The moulds were cured overnight at 60°C.
The plastic moulds containing embedded embryos were cut away using a razor blade leaving an Araldite stub containing the embryos in random orientation. Thick, 10 p,m, sections were cut on a microtome (Reichert, OmU3), dried onto Tespa-coated slides, and stained with Toluidine Blue for 2 minutes at 65°C. For TEM, ultra-thin sections (60-70 nm) were cut on an ultra-microtome (Reichert ultra-cut E) and floated off into distilled water, prior to mounting on copper grids. Sections were stained with lead citrate (BDH) and viewed on a Jeol Transmission Electron Microscope (TEM)-1010.
• Preparing embryos for phalloidin staining
Embryos were fixed overnight in 4% paraformaldehyde at 4°C, then rinsed 3 times for 10 minutes in PBS, permeabilised for 30 minutes at room temperature on a shaker in PBS containing 0.5% Triton X-100 (PBT), washed in PBS, and then stained in PBT containing 250ng/ml FITC-phalloidin or Alexa 566 phalloidin (Sigma) for several hours at 4°C with gentle agitation. Embryos were then rinsed three times in PBT for 10 minutes each and mounted in Citifluor (UKC, Kent, U.K.) under a coverslip and filamentous actin was imaged using a Leica confocal laser scanning microscope (Leica SP).
• Live staining of embryos with fluorescent vital dyes
Epiboly stage embryos were dechorinated in buffered EM, and placed in a 1.5 ml eppendorf cap coated with 1% agarose in EM. Embryos were incubated for 5 minutes in the dark in a solution of Alexa 488-conjugated lectin from Helix pomatia (edible snail) (Molecular Probes L-11271) to a final concentration of 100 pg/ml (stock concentration 2 mg/ml in
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distilled H2O). The lecEmbryos were then rinsed twice in buffered EM for 5 minutes and
mounted in 1 % low melting point agarose in buffered EM, in a purpose-built depression slide made by sticking a glass ring onto a microscope slide using silicone grease (Dow Coming). Buffered EM was added to the well to cover the embryo and the embryos were viewed using 20x and 40x water-immersion lenses on a Leica confocal laser scanning microscope (Leica SP).
• Preparing embryos for immunostaining of microtubules
Embryos were transferred to glass vials containing Microtubule fixative (see Appendix No 1) for 4-5 hours at room temperature. The specimens were then transferred to 100% methanol and stored in fresh methanol overnight at 4°C. The following day, embryos were rehydrated to PBS through 3:1, 1:1,1:3 methanohPBS and followed by several PBS washes throughout the day. Embryos were incubated overnight in 1:500 dilution of mouse monoclonal anti-a-tubulin (Sigma T-5168) in PBS. Embryos were washed in PBS for the following day and then incubated in a 1:200 dilution of Fluorescein-conjugated goat anti mouse IgG (H+L) (Pierce No. 31544). The following day embryos were washed several times in PBS and mounted in Citifluor under a glass coverslip and viewed using 20x, 40x and 63x oil-immersion lenses on a Leica confocal laser scanning microscope.
• Treatment of embryos with specific inhibitors
Embryos were wounded and allowed to heal in the presence of several inhibitors to test the function of various signals during wound healing. To ensure that blockers were effective at the time of wounding, embryos were pre-incubated in inhibitors for up to 2 hours prior to
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Japan), embryos were dechorinated two hours prior to wounding, at approximately sphere - dome stage (4 hours post fertilisation), and incubated at 28.5°C in 750 |xM Y27632 in buffered EM (stock solution of lOmM in DMSO). Embryos had one of two fates. Embryos were either wounded and allowed to heal for 30 minutes, whilst still bathing in Y 27632, before being removed from the medium and processed for scanning electron microscopy. In other cases, immediately following wounding, embryos were stained for 5 minutes in lectin and subsequently time-lapse imaged during wound closure using the CLSM (Leica SP) as described above.
For treatment with the microtubule depolymerising blocker nocodazole, embryos were dechorinated approximately one hour prior to wounding (40% epiboly), and incubated in 5 p,g/ml nocodazole in EM (stock solution 2mg/ml in DMSO) (Sigma M-1404). Some embryos from each experimental batch were fixed after one hour and processed for anti-a- tubulin immunocytochemistry to confirm disruption of microtubules after one hour. The remaining embryos were wounded in the presence of nocodazole, and either allowed to heal for 10 minutes and processed for anti-a-tubulin to study microtubule orientation, or allowed to heal for 30 minutes, and processed for SEM to analyse wound closure.
• Imaging software for fixed tissue and time-lapse movies
Imaging of fluorescently-tagged live embryos and fixed tissue were performed using a Leica TCS SP confocal system. For live embryo studies, images were compiled from up to 10 confocal optical sections depending on the depth of the wound region; each section was
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averaged twice and samples were collected every 30 or 60 seconds. The time-lapse series were assembled and analysed using NIH image software 1.6 (http://rsb.info.nih.gov/nih- image/). Optical sections corresponding to the same time point were projected into a single image and the background noise was reduced using a median filter. For fixed tissues, images were compiled from up to 15 optical sections accumulated up to 4 times and saved as a single projection using NIH image software. The final images were optimised and assembled into a plate using Adobe Photoshop 5.5.
• Testing the role of Calcium during wound closure
Shield-stage embryos were incubated in either 0.3 x or 1 x Danieau solution (Ix Danieau: 58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO^, 0.6 mM Csl(NO^)2, 5 mM Hepes, pH 7.6),
(Shih and Fraser, 1995), and wounded as described above. Embryos were then allowed to heal in 0.3 or 1 x Danieau's and fixed after either 0, 10 or 20 minutes and assessed for wound closure by SEM.
• Immunostaining for anti-phospho-histone-3
Wounded embryos and embryos at various stages of epiboly were fixed overnight in 4% paraformaldehyde at 4°C. The following day specimens were washed several times in PBS, blocked for 1 hour in 10 % goat serum in PBS and incubated overnight at 4°C with anti-phospho-histone-3 (a gift from Louise Mahadevan) at 1:500 dilution in PBST. The following day embryos were washed in PBS, approximately 8 times for 30 mins, then
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reacted against Fluorescein conjugated goat anti-rabbit (Pierce No. 31572) at 1:200 in PBS at room temperature for 4 hours. Specimens were washed in PBS and incubated with 250ng/ml Alexa 566 phalloidin (Sigma) for 4 hours at room temeperature before washing overnight in PBS and mounting in Citiflor for viewing on the CLSM the following day.
• Whole-mount immunocytochemistry for anti-active ERK on Zebrafish embryos
Embryos were fixed in 10% formalin in PBS for 2 hours whilst shaking gently. Embryos were then washed for 10 mins in PBS, and then treated with 10 )xg/ml proteinase K in PBS for 5 mins, washed for 10 minutes in PBS and postfixed in 10% formalin in PBS for 20 minutes, and then washed again in PBS for 10 minutes. Embryos were then washed for 2 x 1 hour in BBT and blocked for one hour in 5% horse serum in BBT. Embryos were then incubated overnight at 4°C in monoclonal anti-activated-MAP kinase (Sigma M-8159) at 1:1000 dilution in BBT with 5% horse serum. The following days the embryos were washed all day in BBT with a final block with 5% horse serum. Embryos were incubated overnight in peroxidase-conjugated goat anti-mouse 2° antibody (Stratatech Sci/Jackson Labs 115-035-044) at 1:100 in BBT with 5% goat serum. The following day embryos were washed in BBT for one hour and several 30 minute washes in PBT before DAB reaction.
• Whole-mount immunocytochemistry for anti-active p38 and anti-active JNK on Zebrafish embryos
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Embryos were fixed overnight in 4% paraformaldehyde in PBS at 4°C. The following day embryos were washed several times in PBS, before being transferred to PBT to continue washing. Embryos were blocked for 1 hour in 5% goat serum in PBT before being incubated with primary antibody overnight at 4°C. For detection of active p38, NEB Phospho-p38 MAP Kinase (Thrl80/Tyrl82) polyclonal Rabbit Antibody was used at 1:10 dilution in PBT (NEB 921 IS). For detection of active-JNK, Promega Anti-ACTIVE JNK pAb Rabbit polyclonal was used (Promega V7931) at 1:100 dilution in PBT.
The following day embryos were washed all day in PBST and then reacted against Fluorescein conjugated goat anti-rabbit (Pierce No. 31572) at 1:200 in PBS at 4°C overnight. The following day embryos were washed several times in PBS before mounting in Citiflor and viewed on a CLSM.
• Treatment with MAP kinase inhibitors
1 mg of MEK Inhibitor U0126 (Promega V I 121) was resuspended in 234 pi of DMSO to produce a stock solution of 10 mM. Embryos were incubated in a working dilution of 100 pm in buffered EM for one hour prior to wounding and throughout the procedure.
1 mg of SB 203580 (Calbiochem 559389) was resuspended in 265 pi DMSO to give a stock solution of 10 mM. Working dilutions of 10 pm and 100 pm were used to inhibit p38 activity (Fujii et al., 2000).
C h a p te r s Introduction