Animals and maintenance
Adult male and female zebrafish (mind bomb
, line #hi904; mind bomb
, line #ta52b; t-complex polypeptide 1
, line #hi3564; histone deacetylase 1
, line #hi1618; denticleless homolog
, line #hi447) were obtained from the Zebrafish International Resource Center (Eugene, OR; http://zebrafish.org/zirc/fish/lineAll.php
). Adult zebrafish were maintained according to standard procedures (Westerfield, 1993
), and following guidelines approved by the University of California, San Francisco Institutional Animal Care and Use Committee. Zebrafish embryos and larvae were maintained in egg water (0.03% Instant Ocean) unless otherwise stated.
To obtain stable physiological recordings, zebrafish larvae at 3 days post-fertilization were immobilized in 1.2% low-melting temperature agarose in zebrafish egg water. Larvae were embedded so that the dorsal aspect of the brain was accessible for electrode placement. Embedded larvae were bathed in egg water and visualized using an Olympus BX50 microscope (Olympus America Inc., Center Valley, PA). Under direct visual guidance, a glass microelectrode (<1.2 mm tip diameter, 2-7 MΩ) was placed in the forebrain or optic tectum. Electrodes were filled with 2 M NaCl and electrical activity was recorded using an Axopatch 1D amplifier (Molecular Devices, Sunnyvale, CA). Voltage records were low-pass filtered at 1-2 kHz (-3 dB, 8-pole Bessel), high-pass filtered at 0.1-0.2 Hz, digitized at 5-10 kHz using a Digidata 1300 A/D interface, and stored on a PC computer running pClamp software (Molecular Devices).
Electrophysiological recordings were analyzed post hoc using Clampfit software (Molecular Devices). Spontaneous gap-free recordings, 15-30 minutes in duration, were analyzed for all fish (n = 75). A threshold for detection of spontaneous events was set at 3x noise (peak-to-peak amplitude) and 100 msec (duration); all events exceeding these thresholds were analyzed.
For locomotion tracking, single zebrafish larvae were placed individually in 96-well Falcon culture dishes (BD Biosciences, Franklin Lakes, NJ, U.S.A.). Each well contained 70 μl embryo medium. Swimming behavior was monitored at 3 dpf for 10 minutes in WT larvae (n = 22) and mib mutant larvae (n = 28) using a CCD camera (Hamamatsu C-2400, Hamamatsu City, Japan) and EthoVision 3.1 locomotion tracking software (Noldus Information, Inc., Leesburg, VA). Locomotion plots were categorized by two observers blind to larvae phenotype as displaying class 0 (no movement, baseline activity), class 1 (small increase in movement), or class 3 (considerable movement, convulsive behavior) activity (Baraban et al., 2005
). Using Ethovision software (Noldus) the percentage of time spent swimming/moving was analyzed for individual locomotion plots.
RNA extraction, hybridization, scanning, analysis and gene categorization
A total of six microarrays were hybridized to compare gene expression patterns between mib mutants (n = 3) and age-matched WT sibling controls (n = 3), including a dye swap for each tissue; each microarray used RNA pooled from 10 zebrafish larvae. At 3 dpf, larvae were sorted by morphology and total RNA was extracted using Trizol® Reagent (Invitrogen, Carlsbad, CA), treated with DNAse (Ambion/Applie Biosystems, Austin, TX) and quantified with GeneQuant® spectrophotometer. An Agilent Bioanalyser 2100 (Agilent Technologies Inc., Santa Clara, CA) was used to assess the integrity/quality of mRNA.
Hybridization, scanning and analysis were completed by the NIH Neuroscience Microarray Consortium (http://arrayconsortium.tgen.org/np2/home.do
) using an Affymetrix (Santa Clara, CA) zebrafish genome array with ~14,900 Danio rerio
transcripts. Different internal quality controls were used, including hybridization controls (BioB, BioC, BioD and cre), Poly A controls (dap, lys, phe and thr) and “housekeeping” control genes (GAPDH, alpha 1 Actin). Sequence information for this array was selected from the following public data sources: RefSeq (July 2003), GenBank (release 136.0, June 2003), dbEST (July 2003), and UniGene (Build 54, June 2003). Probe sets on the array were designed with 16 oligonucleotide pairs to detect each transcript. Affymetrix Gene Chip Operation Software (GCOS) Version 1.4 was used to analyze the resulting image files. The global scaling technique was used to scale the fluorescence intensity of each chip to a target signal of 150. The data files (CEL files), resulting from analysis with the Affymetrix GCOS software, were imported into GeneSpring GX 7.3.1 software (Agilent Technologies Inc.) for further data analysis.
Transcripts were considered as differentially expressed using multiple non-parametric two-tailed unpaired Student's t-test with a Benjamin Hochberg multi-test correction (GeneSpring GX 7.3.1). A P value ≤ 0.05 was considered to be significant. Hierarchical clustering (GeneTree, Salt Lake City, UT) was applied to the data files. Categorization of genes identified by microarray analysis was carried out using GO (Gene Ontology) Slim terms.
PCR, cloning and sequencing
Several genes of interest (GOI) were chosen for further analysis. 1 μg of DNAse-treated total RNA from whole zebrafish (10 fish/pool) was reversed transcribed (SuperScript™III First-Strand Synthesis System, Invitrogen) using a mix of oligo dT20
and random hexamers. Two sets of primers pairs, forward and reverse, were specifically designed using Primer 3 web software (http://frodo.wi.mit.edu/primer3/
) for each investigated gene to obtain a longer sequence (primers sequences are available in Supplemental Table 1
). The most conserved regions were identified by sequence alignment (ClustalW) (Thompson et al., 1994
) of all available gene sequences from GeneBank including other fish species (Cyprinus carpio, Carassius auratus, Dicentrarchus labrax, Oncorhynchus mykiss, Salmo salar, etc.). Investigation of primer cross-specificity was done using BLAST software. The predicted secondary structure of the entire DNA sequence was checked using Mfold software (http://frontend.bioinfo.rpi.edu/applications/mfold
; (Zuker, 2003
). Each reaction cycle (32 loops) consisted of incubations at 94°C (30 sec), 60°C (30 sec), and 72°C (60 sec) with Taq DNA Polymerase (Taq PCR Core kit, Qiagen). PCR products were separated by agarose (2%) gel electrophoresis stained with ethidium bromide and cloned in pCR®II-TOPO® plasmid vector (TOPO TA Cloning System, Invitrogen). DNA sequencing was performed by Elim Biopharmaceuticals, Inc. (Hayward, CA).
Whole-mount in situ hybridization (WISH)
For antisense and sense RNA probes, the plasmids corresponding to each gene were linearized with appropriate restriction enzymes (HindIII, SpeI or BamHI and Not I, XbaI or ApaI, New England Biolabs, UK). The linearized DNA template (1 μg) was purified (QIAquick®, Qiagen) and incubated for 3 hour at 37°C in a solution containing 10X transcription buffer, dithiothreitol (DTT; 100mM), 10X Dig NTP Mix (Roche), RNAse inhibitor (20U/μl), and RNA polymerase (20U/μl) T7 or SP6. The DNA template was digested with DNase (10U/μl) for 15 minutes at 37°C. After incubation, 30 μl of RNAse-free water and 30 μl of LiCl were added for purification and left overnight at -20°C. After centrifugation at 4°C, the pellet was rinsed with 70% ethanol (RNAse free), dried and stored in hybridization mix at -20°C until hybridization.
Embryos (3 dpf) were fixed in 4% paraformaldehyde (PFA) and then stored in 100% methanol at -20°C. In situ RNA hybridization was performed, as described (Hauptmann and Gerster, 1994
). Once developed, the embryos were mounted in 70% glycerol for whole-mount imaging. All images were captured using a Zeiss Axioskop microscope equipped with a Nikon E995 or Optronics MicroFire digital camera. Raw images were imported into Adobe Photoshop and adjusted regarding the level of brightness, contrast and cropping.
Quantitative real-time PCR (qPCR)
Gene expression levels of seven chosen genes were examined using RNA pooled from 10 WT sibling larvae (n = 6) or 10 mib mutant larvae (n = 6). RNA was extracted in the same manner as for microarray experimentation (above) and reverse-transcription reactions (RT-PCR) were performed in the same manner as for cloning and sequencing using SuperScript™III First-Strand Synthesis System (Invitrogen). The cDNA templates were diluted 1:2 with DEPC (Diethyl pyrocarbonate) sterile water before qPCR applications to minimize the presence of potential inhibitors.
The qPCR reactions were performed using SybrGreen® fluorescent master mix on an ABI Prism® 7700 Sequence Detection System driven by ABI prism SDS v9.1 (Applied Biosystems). Primers were designed to produce amplicons ranging in size between 81bp and 130bp () using Primer Express v3.0 (Applied Biosystems). All primers were synthesized by Invitrogen. Samples were run in triplicate and contained 1× SYBR green master mix, 10 μM of each primer and RNAse free water for a final volume of 10 μl. Samples without reverse transcriptase and samples without RNAs were run for each reaction as negative controls. Cycling parameters were as follows: 50°C × 2min, 95°C × 10min, then 40 cycles of the following 95°C × 15s, 60°C × 1min. For each sample a dissociation step was performed at 95°C × 15s, 60°C × 20s, and 95°C × 15 s at the end of the amplification phase to check for the presence of primer dimmers or non specific products (Suppl. Fig. 1
Real-time qPCR primer's sequences, GeneBank accession number and amplicon size for the 7 genes of interest and the 4 endogenous genes used in SybrGreen assay.
Triplicate quantification values (CT; cycle threshold), provided from real-time qPCR instrumentation, were imported into a Microsoft Excel spreadsheet for further analysis. Raw data was analyzed using qCalculator software (programmed by Ralf Gilsbach) which estimates qPCR efficiency E= 10(– 1/slope) and the relative gene expression between samples after normalization with the most reliable EndG basing on both the Comparative ΔΔCT (Livak and Schmittgen, 2001
) and the Efficiency Based (Pfaffl, 2001
) methods. Similar results were obtained with both types of analyses.
For all genes, qPCR efficiencies, detection limits and dynamic ranges were assessed by mean of 4-fold serial dilutions of pooled cDNA (5 standards assayed in triplicate: 1/1; 1/4; 1/16; 1/64; 1/256). Serially diluted cDNAs were used to construct standard curves and estimates of efficiencies, slope of the curves and the correlation coefficient (Suppl. Table 2
Given that there is no reason to expect a single gene to be the most stably expressed endogenous gene (EndG) in all samples, the reliability of one or more reference genes was determined before proceeding with quantitative mRNA expression studies. 12 samples (6 samples of WT and 6 samples of mibhi904
) were used to verify expression stability of four different commonly used EndGs (Tang et al., 2007
; Chen et al., 2008
; Lin et al., 2009
): small subunit ribosomal RNA (18S), protein elongation factor 1 alpha subunit (EF1α), beta-actin (β-act) and beta-2 microglobulin (β2M). Bestkeeper Excel-based tool (Pfaffl et al., 2004
) and NormFinder (Andersen et al., 2004
) software were used to rank all the EndGs. Bestkeeper assesses candidate genes by pair-wise correlations based on cycle threshold values (CT) which are then combined into an index and calculates the standard deviation (SD) of the CT values between the whole data set. NormFinder uses a model-based approach to rank all reference gene candidates based on inter- and intra-group expression variations. Analyzed with both programs (BestKeeper and NormFinder), the β-act gene was the most stable gene followed by EF1α, β2M and 18S (data not shown); β-act was used in our studies for data normalization.
Student's t test was used to determine statistical significance between the normalized relative expression values in qPCR assay. A P value ≤ 0.05 was considered to be significant.