All zebrafish work was performed in the Zebrafish Research Facility (ZRF) of the University of Alabama at Birmingham (UAB). Adult fish and Pumaoutletmujerembryos are maintained as described by Westerfield M (1995) [76] by the ZRF Animal Resources Program which maintains full AAALAC accreditation and is assured with OLAW. All knockout lines are generated on the AB stain. All animal studies have UAB IACUC approval.
For the homology analysis, we used the following transcripts: hPUMA (ENST00000439096.3), hNOXA (ENST00000316660.7), hBMF (ENST00000397573.5), hBid (ENST00000622694.5), hBim (ENST00000308659.12), hHrk (ENST00000257572.5), hBik (ENST00000216115.3), hBad (ENST00000309032.8), zPuma (ENSDART00000137918.3), zNoxa (ENSDART00000123131.3), zBmf (ENSDART00000060713.5), zBid (ENSDART00000100716.7), zBim (ENSDART00000114318.3), zHrk (ENSDART00000132567.2), zBad (Bada: ENSDART00000125349.3 and Badb: ENSDART00000077219.5), tp53 (ENSDART00000051549.5), tp63 (TA: ENSDART00000163541.2 and ΔN: ENSDART00000065135.5), and tp73 (ENSDART00000124737.3).
Gene knockouts were generated as described previously [77]. Zinc Finger, TALEN, or gRNA target sites were identified using the ZiFiT Targeter software developed by the Zinc Finger Consortium (http://zifit.partners.org/ZiFiT/), TAL Effector Nucleotide Targeter 2.0 (https://tale-nt.cac.cornell.edu/), and the Zhang lab gRNA design tool (http://crispr.mit.edu); respectively (target sites listed in figures). OPEN Pool ZFN were assembled into the pH3U3-mcs plasmid and selected using the omega knockout bacterial hybrid selection strain USO hisB-pyrF-rpoZ (Addgene #18049) [78]. TALENs were assembled using The Golden Gate TALEN and TAL effector kit (Addgene #1000000016) [79]. The CRISPR gRNA sequences were clones into pDR274 (Addgene 42250). The cas9 mRNA was transcribed from pT3TS-nCas9n (Addgene 46757) [80]. After cloning specific target plasmids/guides into pCS2 variant vector, mRNA was generated by in vitro transcription off NotI linearized DNA using the Invitrogen mMESSAGE mMACHINE™ SP6 Transcription Kit (Fisher Scientific AM1340) and purified with the MEGAclearTM Transcription Clean Up Kit (Fisher Scientific AM1908). Approximately 1–2 nl of nuclease mRNA (or sgRNA/Cas9 mRNA) were microinjected into the yolk of one-cell-stage zebrafish embryos. For indel efficiency evaluation, genomic DNA was extracted from ~24 3 dpf-injected embryos and evaluated with HRM (see below). The remaining embryos (F0s) from the clutches were raised. Out of frame indels identified in F1 progeny were maintained and propagated. To “cleanup” genetic background all lines were breed at least two generation to the wild-type strain AB.
To determine if the mutated allele is undergoing nonsense-mediated decay or alternative splicing, a small piece of tail was cut from a single heterozygous fish (of each allele). RNA was extracted from each tail using Trizol Reagent (Life Technologies, 15596026), and cDNA was synthesized from each RNA sample using the High-Capacity cDNA Reverse Transcription Kit (Life Technologies, 4368814). The cDNA was PCR amplified using Takara Ex Taq DNA Polymerase (Takara Bio, RR001A), purified with the Promega Wizard SV Gel and PCR Cleanup System (Promega, A9282), and examined on a 1% agarose gel (for examining alternative splicing) and sequenced by the UAB Heflin Center for Genomic Sciences Sanger Sequencing Core. The mutated allele was determined to have undergone complete nonsense-mediated decay if only the wild-type sequence was detected in the sequence chromatogram.
To isolate genomic DNA from adults, tail clippings from each fish were incubated at 98 °C for 20 min in 40 µl 25 mM NaOH in a 96-well plate; then neutralized with 40 µl of 40 mM Tris-HCl. Early-stage or stained embryos were incubated at 55 °C 2 h in 25 µl ELB (10 mM Tris pH 8.3, 50 mM KCl, 0.3% Tween 20, 0.3% NP40, 1 mg/ml Proteinase K) in 96-well plates; then incubated at 95 °C for 15 min to inactivate the Proteinase K. PCR reactions contained 1 µl of LC Green Plus Melting Dye (Biofire Defense, BCHM-ASY-0005), 1 µl of 10x enzyme buffer, 0.2 µl of dNTP Mixture (10 mM each), 0.3 µl of MgCl2, 0.3 µl of each primer (10 µM), 1 µl of genomic DNA, 0.05 µl of Genscript Taq (E00101), and water up to 10 µl. The PCR reaction protocol was 98 °C for 30 s, then 45 cycles of 98 °C for 10 s, 59 °C for 20 s, and 72 °C for 15 s, followed by 95 °C for 30 s and then rapid cooling to 4 °C. Following PCR, melting curves were generated and analyzed using the LightScanner instrument (Idaho Technology) over a 65–95 °C range. Primers used for identifying zebrafish knockout lines are listed in Supplementary Table 1.
Embryos were placed at the closest position to the source of IR in a X-RAD 320 X-ray irradiator to expose the embryos to ~4.2 Gy/min. Apoptosis was assayed following IR treatment by AO or active Caspase-3 staining. For AO staining [74], embryos were incubated in 50 mg/ml of AO (Sigma) for 45 min followed by five consecutive 5-min washes. Images were immediately taken using dissecting microscopy with 488 nm wavelength filter.
Overall, 1.5 µl Thapsigargin (10 mM stock in DMSO, Sigma), 3 µl BFA (10 mM stock in DMSO, Sigma), and 1 µl PMA (10 mM stock in DMSO, Sigma) were added into 3 mL of sterile E3 egg water to prepare working solutions with a final concentration of 5 µM Thapsigargin [19], 10 µM BFA or 3.3 µM PMA. Approximately 30 24 hpf embryos (±15 min) were placed in each well of six-well tissue culture plates (BD Falcon). For immediate apoptosis induction, embryos were left for 4 h (Thapsigargin and PMA) or 6 h (BFA) in the dark at 28.5 °C, processed for active Caspase-3 whole-embryo staining and sorted based on phenotypic categories (described in figures) to define the degree of severity.
Embryos were fixed in 4% paraformaldehyde overnight at 4 °C and then permeabilized for at least 2 h in methanol (for anti-active Caspase-3 staining) or for 7 min in acetone (for anti-p63 staining). After 1 h blocking, embryos were incubated in primary antibody overnight at 4 °C. Anti-active Caspase-3 antibody (BD, 559565) was used at a dilution of 1:500 and anti-p63 primary antibody (Abcam, ab735) at 1:200. For Caspase-3 detection, the Alexa 488 goat anti-rabbit secondary antibody was used and for p63 detection, an Alexa 488 Donkey anti-mouse secondary antibody was used at a dilution of 1:200 for 2 h at room temperature or overnight at 4 °C. Subsequently, embryos were washed and stained in DAPI for 10 min (if nucleus measurement necessary) prior to imaging.
Embryos were dechorionated at described stages with incubation in 0.03% pronase (Sigma P5147) for 6 min and anesthetized using 0.4% tricaine. In a 60 × 15 mm Falcon petri dish (light and immunofluorescence imaging) and glass-coverslip-bottomed dish (confocal imaging), embryos are mounted in 1% low melting agarose. Gross images and images with AO or anti-active Caspase-3 staining were taken on a SMZ-18 Zoom Stereo Microscope. For quantification, all images were acquired at the same magnification, laser power, exposure time, and gain. Images with anti-p63 staining were taken on a Nikon A1 inverted confocal microscope and ~100-μm Z-stacks at 3.5-μm intervals were obtained. After each embryo was imaged, embryos were removed from the agarose to generate genomic DNA for genotyping. Further figure processing and analysis was performed using Nikon NIS Element and ImageJ.
RNA was isolated from at least 30 pooled whole embryos using the Qiagen RNeasy Mini Kit and subjected to cDNA preparation with High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher, 4368814). Quantitative PCR was performed using the CFX Connect Real Time System (Bio-Rad) with TaqMan™ Gene Expression Master Mix (Thermo Fisher, 4369016). Primers and probes are purchased from Thermo Fisher.
Approximately 30 pooled embryos at 24–30 hpf were homogenized in 60 µL protein cocktail (15 µL 4X sample buffer, 10 µL 6X protease inhibitor, 1.5 µL β-Me, and water up to 60 µL). The quantity of the protein loaded onto the western blots was assessed by hybridizing with anti-GAPDH primary antibody (Cell Signaling Technology, 2118) at a dilution of 1:2000. Subsequent SFS-PAGE gels were adjusted based on anti-GAPDH results. p53 was detected by hybridization with anti-p53 primary antibody (GeneTex, 128135) at a dilution of 1:1000, washed and incubated with peroxidase goat anti-rabbit IgG (1:2000; Jackson ImmunoResearch, 111-035-003) and developed with Clarity Western ECL Substrate (Bio-Rad, 1705061). Signal was detected by Bio-Rad ChemiDoc MP system. Western blot images were processed and quantified with Image Lab.
Our tumor cohorts were established by natural breeding of p53−/−x p53−/−parents. The cohort consisted of 96 fish and was derived from a single set of parents (a single male and female). At 4 months of age, all fish were separated into four tanks of 24 fish each. Adult fish were screened weekly or biweekly for tumors and/or missing/dead fish. Fish that were identified by eye to be tumor burdened were euthanized according to IACUC protocols. Kaplan-Meier analysis was performed using GraphPad Prism 8 software.
GraphPad Prism 8 was used in generation of all graphs and statistical tests. For phenotypic categories and qRT-PCR quantification, overall statistical significance was calculated using an unpaired t-test with error bars indicating SEM. Numbers of embryos and significance values are indicated in the figure legends.