Zebrafish and microscopy
Experiments were carried out in accordance with Institutional Animal Use and Care Committee regulations and approvals. Adult stocks of strain AB* zebrafish were maintained at 28.5°C and euthanized by deep tricaine anaesthesia followed by exposure to ice-cold water. Embryos were raised in E3 buffer (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4). Microscopy was carried out after tricaine anaesthesia to prevent movement. Images of free-floating whole larvae were obtained using an inverted microscope, and confocal images were obtained after embedding anaesthetized larvae in 3% low melting point agarose in E3 buffer.
RNA was separated on a 1.2% agarose formaldehyde/MOPS gel and transferred to Nytran-N membrane (Schleicher & Schuell BioScience, Keene, NH). eno2 antisense cRNA probe was generated by in vitro transcription of a plasmid template containing 307 bp of the eno2 open reading frame and 5′UTR using digoxigenin-conjugated UTP to label the antisense cRNA probe (Roche, Indianapolis, IN, USA). Pre-hybridization was carried out in UltraHyb (Ambion, Austin, TX< USA) supplemented with Torula RNA (1 mg/ml final concentration; Sigma, St Louis, MO, USA), followed by addition of cRNA probe (20 ng/ml final concentration) to the hybridization buffer. Blots were washed in 2× SSC, 0.1% SDS and 0.1× SSC, 0.1%, SDS. Probe was detected with alkaline-phosphatase (AP) conjugated anti-DIG FAb (Roche, Indianapolis, IN, USA), in maleic acid buffer, 1% blocking substrate, and revealed by exposure to photographic film after incubation with a light-emitting AP substrate (CDP-star, Roche, Indianapolis, IN, USA).
Whole mount mRNA in situ hybridization
Embryos were raised in E3 buffer, 0.003% 1-phenyl-2-thiourea to prevent pigment formation, fixed overnight at 4°C in 4% paraformaldehyde (PFA) in phosphate-buffered saline pH 7.5 (PBS), washed in PBS, dehydrated in methanol and stored at –20°C. Prior to hybridization, embryos were treated with acetone at –20°C, re-hydrated in 50 and 30% methanol and then diethylpyrocarbonate (DEPC)-treated PBS, followed by protease-K (10 µg/ml) at room temperature for 5–30 min, followed by post-fixing in 4% PFA and washing in PBTw (PBS, 0.1% Tween-20, 0.2% bovine serum albumin). Pre-hybridization was carried in 50% formamide, 2× SSC, 0.3% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate (CHAPS), 1 mg/ml torula RNA, 50 µg/ml heparin, followed by the addition of 150 ng/ml cRNA probe. Embryos were washed in 50% formamide, 2× SSC, 0.3% CHAPS, then 2× SSC, 0.3% CHAPS, followed by 0.2× SSC, 0.3% CHAPS. Probe was detected using AP conjugated anti-DIG antibody diluted in maleic acid buffer, and revealed by incubation in BM Purple (Roche, Indianapolis, IN).
mRNA in situ hybridization in brain sections
Zebrafish brains were fixed in 4% PFA at 4°C for 6 h, washed with PBS and cryoprotected in PBS-sucrose. Ten micrometer thick cryosections on SuperFrost slides (Fisher, Pittsburgh, PA), were post-fixed in 4% PFA, carbethoxylated with 0.1% DEPC in PBS (33
), washed in 5× SSC, and post-fixed in 4% PFA. Following incubation in hybridization buffer (UltraHyb, Ambion, Austin, TX) supplemented with Torula RNA (4 mg/ml final concentration; Sigma, St Louis, MO) for 1 h at 68°C, DIG-labelled RNA probe (final concentration 0.2 ng/µl) was added to the buffer and sections were incubated in a humid chamber overnight at 68°C, following which they were washed in 2× SSC, 1× SSC then 0.1× SSC. Probe was detected using an AP-conjugated anti-DIG FAb diluted 1:1000 in maleic acid buffer, 1% blocking substrate (Roche), and revealed by incubation in a colorigenic AP substrate (BM-Purple
, Roche, Indianapolis, IN, USA).
RT–PCR, 5′RACE and 3′RACE
For RT–PCR, total RNA from whole adult zebrafish brains was subjected to reverse transcription using SuperScript III
(Invitrogen, Carlsbad, CA, USA). PCR amplification was carried out using primers complementary to eno2
exon1 F: 5′-CGC TCT GTT TTC CTC TCT CTT CCG-3′ and Tau R: 5′-AAT CCT GGT GGC GTT GGC CT-3′; beta actin F: 5′- ATG GAT GAG GAA ATC GCT GC-3′ and R: 5′- AGC CTC AGT GAG CAC GAC AG-3′; eno2
F: 5′-ATG TCT GTT GTA AGC ATC AT -3′ and R: 5′-GAT GTG TCT GTC TGC ATT TG -3′. 5′ and 3′ RACE were carried out as described (17
), using zebrafish eno2
primers 3′RACE: 5′-ATC TGA GCG TCT CGC CAA AT-3′, and 5′RACE: 5′-ATG ATG GCT GGC CCG AGA GTG T-3′. Products were cloned in pGEM-T (Promega, Madison, WI, USA) and sequenced. Multiple sequences were aligned and compared using AlignX
An EcoRI/AflII restriction fragment containing eGFP-poly(A) was obtained by digestion of pEGFP-1 (Clontech, Mountain View, CA, USA), and ligated into the EcoRI/PmeI sites of pBluescript SK-I-Sce1 (a kind gift from Dr Michael Tsang, University of Pittsburgh) to make pBS-I-Sce1-GFP. Homologous zebrafish eno2 arms were amplified from BAC zC51M24 (CHORI21 library) by PCR using primers 5′-GGA ATT CCA TCA CAA TGT ATC AGC-3′ and 5′-GTA CCC ATG GCA ATG ATG CTT ACA AC-3′ (3′arm; eno2 exon 2) and 5′-GGG GTA CCT GTT AGT AAA GGC AGA T-3′ and 5′-A ATT CAA TGG CAC GTT TGA TCG-3′ (5′arm; eno2 5′ genomic flanking sequence). The homologous arms were inserted into the EcoRI/NcoI sites (3′ arm) or KpnI/EcoRI sites (5′ arm) of pBS-I-Sce1-GFP, such that the eGFP ORF was in frame with exon 2 from the zebrafish eno2 gene. The resulting plasmid was verified by restriction digest and direct DNA sequencing, and then linearized by EcoRI digest. A 12 kb fragment of the eno2 gene was captured from BAC zC51M24 using gap repair recombination (the inducible bacterial strains for carrying out the recombination were a kind gift from Dr Neal Copeland, NCI). DY380 cells were transformed with BAC zC51M24 by electroporation. DY380-BACzC51M24 cells were grown at 32°C to OD590 = 0.5 then induced at 42°C for 15 min before being transformed with linearized plasmid by electroporation. Recombinants were identified by ampicillin resistance, and verified by restriction digest and direct DNA sequencing. A similar strategy was used for cloning the eno2:4R-Tau gene cassette, except that the Tau ATG replaced the zebrafish eno2 ATG in order to preserve the N-terminus of the resulting protein. The homologous arms were generated by PCR amplification from BAC zC51M24, using the following primers: 5′-GGG GTA CCT GTT AGT AAA GGC AGA TTC-3′ and 5′-CCG CTC GAG TCC ACA TTA CCT TCT GTT G-3′ (5′ arm; eno2 5′genomic flanking sequence), and 5′-CCG CTC GAG TCC ATC ACA ATG TAT CAG C-3′ and 5′-CTC ACC ATG GCA ATG ATG CTT ACA ACA GAA GGC CTT AAA GAC AAG-3′ (eno2 intron 1), and cloned into the XhoI/NcoI sites (5′ arm) or KpnI/XhoI sites of pBS-I-Sce1-GFP. The open reading frame of human 4-repeat Tau was amplified from plasmid t2 (a kind gift from Dr Matthew Farrer, Mayo Clinic, Jacksonville, Florida) by PCR using primers 5′-ATG GCT GAA CCC CGA CAG GAG-3′ and 5′-GAC CCA TGG ATC CTC ACA AAC CCT GCT TGG C-3′, and cloned into the StuI/NcoI sites of pBS-I-Sce1-eno2-GFP. The IRES sequence derived from pIRES2-eGFP (Clontech) was inserted into the BamHI/NcoI sites of pBS-I-Sce1-eno2:Tau-GFP. The resulting plasmid was linearized using XhoI prior to transformation and recombination, as described above.
A restriction digest containing the following components was prepared on ice: plasmid DNA (pBS-I-Sce1-eno2:GFP, or pBS-I-Sce1-eno2:Tau-IRES-GFP) 0.6 µg, injection dye (0.5% phenol red, 240 mM KCl, 40 mM HEPES pH 7.4) 1 µl, 10× I-Sce1 buffer (100 mM Tris–HCl, 100 mM MgCl2, 10 mM Dithiothreitol, pH 8.8) 0.5 µl, I-Sce1 (New England Biolabs) 1 µl (5 U), ddH2O to total 10 µl. Single cell embryos were microinjected with 0.5 nl of the restriction digest reaction, containing 30 pg DNA, using a glass micropipette. All surviving fish were raised to sexual maturity and crossed in pairs to identify germline chimeras. The genotype of progeny embryos of F0 pBS-I-Sce1-eno2:GFP and pBS-I-Sce1-eno2:Tau-IRES-GFP microinjected fish was determined by PCR of pooled embryo lysate genomic DNA using primers 5′-CGT AAA CGG CCA CAA GTT CAG C-3′ and 5′-CGA TGT TGT GGC GGA TCT TGA AG-3′. Pairs of fish that transmitted the transgene to progeny embryos were then outcrossed to wild-type fish and embryo genotyping repeated, in order to determine which one of each pair was the germline chimera. F1 progeny of each germline F0 fish were then raised to adulthood. Tg(eno2:GFP) F1 fish were identified by epifluorescence microscopy of F2 embryos after outcross to wild-type fish. Tg(eno2:4R-Tau) fish were identified by PCR of genomic DNA extracted from tail fin. Two- to three-month-old fish were anaesthetized in 4% tricaine (Acros). A small segment of caudal fin was excised and incubated in 100 µl of DNA extraction buffer [10 mM Tris pH 8.0, 10 mM EDTA, 200 mM NaCl, 0.5% SDS and 200 ug/ml proteinase K (Invitrogen)] at 55°C for 3 h. The sample was then heated to 100°C for 5 min to inactivate proteinase K, and 0.6 µl of each sample was used as a template for PCR using the GFP primers shown above. Each line of transgenic fish was derived from a single F1 founder, and each F1 founder was derived from a single F0 germline chimera. Stocks were maintained by genotyping as above. The data reported here are derived from F2, F3 and F4 generation zebrafish.
Immunohistochemistry (IHC) and immunofluorescence
Adult fish brains were dissected and fixed in 4% PFA, followed by cryoprotection in PBS-sucrose. Cryosections measuring 20 µm were incubated overnight at 4°C with primary antibody, diluted: 1:500 (GFP; cat# MAB3580 Chemicon, Temecula, CA, USA), 1:250 (ChAT; cat# AB144P, Chemicon; this antibody was incubated with sections for 72 h at 4°C), 1:100 (IP3R1; cat#ACC-019, Alomone Labs, Jerusalem, Israel), 1:1000 (GABA; cat# A2052, Sigma), 1:500 (Human Tau; cat#AHB0042, Biosource, Camarillo, CA, USA) or 1:500 (TH; cat# AB152, Chemicon), in carrier buffer (PBS, 1% goat or donkey serum, 1% BSA). For IHC, a biotinylated anti-mouse secondary antibody (2 µg/ml; cat# BA-2000, Vector Laboratories, Burlingame, CA, USA) was diluted 1:200 in carrier buffer, followed by staining in NovaRed (Vector Laboratories) and counterstaining in Mayer's haematoxylin (Sigma, St Louis, MO, USA). For immunofluorescence, primary antibodies were detected using Alexa-488 (anti mouse) and Alexa-555 (anti-rabbit or goat) conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA), diluted 1:2000 in carrier buffer.
Whole brains from 3-month-old zebrafish were sonicated in RIPA lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% NP-40, 5 mM EDTA, 0.5% sodium deoxycholate and 0.1% SDS), in the presence of protease inhibitors (complete mini, Roche, Indianapolis, IN, USA) on ice. The homogenate was centrifuged at 20 000g for 20 min at 4°C. The protein concentration of each brain extract was determined by Bradford assay and 10 µg was loaded onto 12% SDS-PAGE after boiling in SDS sample buffer, and electroblotted onto PVDF membrane (Bio-Rad). The membrane was blocked in 150 mM NaCl, 100 mM Tris–HCl pH 7.5, 0.1% Tween-20 (TBST), 10% dried non-fat milk for 60 min at room temperature, probed with mouse monoclonal anti-Tau antibody (1:500, cat#AHB0042, Biosource, Camarillo, CA, USA) or polyclonal anti-actin antibody (1:1000; cat#A2066, Sigma, St Louis, MO, USA) overnight at 4°C in TBST, 10% milk, washed in TBST, and bound antibody detected using an HRP conjugated secondary antibody (goat anti-mouse, cat# 31430, Pierce, Rockford, IL; goat anti-rabbit, cat# 4010-05, Southern Biotech, Birmingham, AL) diluted 1:10,000 in TBST and chemiluminescent substrate (ECL, Amersham Biosciences, Chalfont, UK).