Nitrocellulose membranes, mouse anti-HPC-1, anti-goat peroxidase-conjugated secondary antibodies, propidium iodide (PI), and bromodeoxyuridine (BrdU) were purchased from Sigma (St. Louis, MO, USA). Anti-zpr1, anti-zpr3, anti-zn12, and anti-zn5 antibodies were purchased from the Zebrafish International Resource Center (ZIRC, Eugene, OR, USA). TOPRO3, Alexa Fluor 555 goat anti-rabbit IgG antibody, Alexa Fluor 555 goat anti-mouse IgG antibody, Alexa Fluor 488 goat anti-rabbit IgG antibody, the MultiSite Gateway Three-Fragment Vector Construction Kit, Click-it EdU Alexa Fluor 555 Imaging Kit, and trypsin were purchased from Invitrogen (Carlsbad, CA, USA). Rabbit anti-phosphohistone 3 (pH3) antibodies were purchased from Millpore (Billerica, MA, USA). Mouse anti-BrdU antibody was purchased from BD Biosciences (Franklin Lakes, NJ, USA).
AB strain zebrafish were purchased from Aquatic Tropicals Inc. (Bonita Springs, FL, USA) and housed at 28.5°C on a 14/10-h light/dark cycle. Embryos were collected after natural spawns, incubated at 28.5°C and staged by hpf.
Morpholino oligo and mRNA injections
For loss-of-function experiments, two morpholino oligos (Gene Tools, LLC, Cowallis, OR, USA) were used that were targeted to non-overlapping sequences in proximity to the translation start site of the zebrafish mdka mRNA (NCBI Reference Sequence: 131070.2). Control morpholinos contained a 5-nucleotide mismatch. Morpholinos were diluted in 1× Danieau buffer at 1 mg/mL, and embryos were injected with 2 ng to 5ng of morpholinos at the 1- to 8-cell stage.
For the mRNA rescue injections, 80 pg of id2a mRNA in a pCS2-id2a vector was co-injected with 5 ng of Mdka morpholino (see).
Western blot analysis
Mdka and GFP were detected in western blots using techniques described previously. Proteins were extracted from pools of 15 to 20 embryos by lysing the embryos in buffer with protease inhibitors (Complete Mini, Roche, Germany). Proteins were separated in a 12% SDS-PAGE gel and transferred to a nitrocellulose membrane. The membrane was blocked in 5% non-fat dry milk in PBS for 2 h and incubated with rabbit anti-Mdka antibody (1:300;) or rabbit anti-GFP antibody (1:500, Abcam, Cambridge, MA, USA). Blots were rinsed with PBS and incubated with goat horseradish peroxidase-conjugated secondary IgG (1:5,000) for 1 h. Bound antibodies were visualized using the enhanced chemiluminescence assay (ECL detection system, Amersham Biosciences, Arlington Heights, IL, USA). As loading controls, blots were stripped and incubated with anti-actin (1:1,000, Calbiochem, Germany).
Embryos were fixed overnight in 4% paraformaldehyde dissolved in 100 mM phosphate buffer, cryoprotected by infiltration in 20% sucrose in phosphate buffer, and frozen in Optimal Cutting Temperature (OCT) media. Cryosections at 7 to 10 μm in thickness were mounted on glass slides and processed for immunohistochemistry using standard procedures. Briefly, after drying, sections were incubated with 20% normal sheep serum in phosphate buffered saline and 0.5% triton X-100 (PBST), followed by overnight incubation at 4°C with primary antibodies. After washing with PBST, sections were incubated in secondary antibodies for 1 h at room temperature, washed extensively in PBST. Sections were counterstained with 1:1,000 dilution of DAPI to label nuclei and sealed with mounting media and glass coverslips.
Systemic labeling with BrdU or EdU
Proliferating cells were labeled with either BrdU or EdU by soaking embryos for 20 min in ice-cold 5 mM BrdU or 1.5 mM EdU dissolved in embryo rearing solution containing 15% DMSO. For BrdU staining, sections were incubated in 4 N HCl and immunolabeled using a mouse anti-BrdU antibody that was visualized with goat anti-mouse IgG antibodies conjugated to Alexa Fluor 555. EdU was visualized using the Click-iT™ EdU imaging kit with Alex Fluor 555 according to the manufacturer’s protocol.
In situ hybridization
In situ hybridization on retinal sections was performed using digoxigenin (DIG)-labeled riboprobes, synthesized as previously described. Briefly, sections were hybridized with probes overnight at 55°C. The next day, the sections were washed and incubated with an alkaline-phosphatase-conjugated anti-DIG antibody overnight at 4°C. After washing, riboprobes were visualized using 4-nitrobluetetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP; Roche, Indianapolis, IN, USA) solution as the enzymatic substrate (Roche).
For whole mount in situ hybridizations, embryos were fixed in 4% paraformaldehyde, dehydrated in methanol and stored at -20°C for a minimum of 12 h. Embryos were then returned to room temperature, rehydrated, fixed again in 4% paraformaldehyde, permeabilized with 0.1 M proteinase K, fixed a third time in 4% paraformaldehyde, treated with acetic anhydride, and washed in PBS with 0.1% Tween. Embryos were next washed in pre-hybridization buffer, which was removed and replaced with 500 μL of hybridization solution containing 200 ng of probe. Embryos were hybridized overnight at 55°C. The next day, embryos were washed and the digoxigenin was detected using antibodies conjugated to alkaline-phosphatase and a colorimetric reaction with NBT/BCIP as the enzymatic substrate. Embryos were transferred to single concavity slides for inspection and photomicroscopy.
Acridine orange labeling
Apoptotic nuclei were visualized by labeling live embryos with the vital exclusion dye, acridine orange. At 24 hpf, embryos were transferred to 0.003% 1-phenyl-2-thiourea (PTU) to block melanin synthesis. At various ages, embryos were then dechorionated and placed in acridine orange solution (5 μg/mL in embryo medium; Molecular Probes, Eugene, OR, USA) for 15 min followed by extensive washes in embryo medium. Embryos were lightly anesthetized and viewed under a fluorescence dissecting scope, and acridine orange-positive cells were counted.
The Gateway-based Tol2 kit system was used to generate transgenic fish. Mdka cDNA (pCS2p-mdk1; a gift from C. Winkler), was PCR-amplified with primers containing an attB1 site on the forward primer and a reverse attB2 site on the reverse primer. The sequence of these primers is as follows: Forward: 5′ GGGGACAAGTTTGTACAAAAAAGCAGGCTGTATGCGGGGCCTGTTTTCCACC 3′; Reverse: 5′ GGGGACCACTTTGTACAAGAAAGCTGGGTCGTTCCCTTTCCCCTTGCCTT 3′. The purified PCR products with added att sites were used immediately in BP reactions to generate middle entry clones. Multisite recombination reactions were performed to generate the pHSP70/4:mdka:egfp expression construct.
The expression construct and in vitro transcribed Tol2 transposase mRNA were co-injected into AB wild-type embryos at the 1- or 2-cell stage. Embryos positive for the gene construct were identified at 72 hpf by the HSP70/4-driven expression of EGFP in the lens (). Positive F0 fish were outcrossed to wild-type AB fish. F1 generation carriers were identified by GFP expression following heat shock and validated by PCR with genomic DNA, using primers for the sequence encoding the enhanced green fluorescent protein. Three lines, kec1001L3, kec1004L4, kec1004L6, were identified and used to propagate stable (Tg(pHSP70/4:mdka:egfp)) lines. These lines were characterized and found to yield approximately 50% GFP-positive progeny when mated to wild-type AB fish, suggesting a single Tol2 insertion. One transgenic line, Tg(pHSP70/4:mdka:egfp)kec1001L3, was used for all of the data presented here.
Embryos were collected from pairwise, hemizygous x hemizygous matings and treated by heat shock (37°C for 60 min) at 24 hpf. Wild-type and homozygous transgenic embryos were identified at 26 hpf by the absence or presence and relative intensity of GFP. At 30 hpf, 50 heads (forebrains and eyes) from each group were isolated and washed twice in ice-cold PBS and dissociated into single-cell suspensions in 5 mL of ice-cold 0.25 mg/mL trypsin solution for 10 min at room temperature. Cells were fixed in ice-cold 70% ethanol for 30 min and stained with propidium iodide. DNA content for the isolated cells was analyzed on a FACScan II (Becton-Dickinson), and histograms of DNA content were acquired by CellQuest (BD Biosciences) and MODFIT LT (Verity Software House, Topsham, ME, USA).
Cumulative BrdU labeling
To estimate the total length of the cell cycle and the length of the S-phase, a cumulative BrdU labeling approach was used[17, 40]. BrdU was injected into the yolk at 2-h intervals beginning at 26 hpf, which provides a sustained systemic dose of BrdU and labels all cells passing through the S-phase of the cell cycle. At 30 min after each injection, three to four embryos from each group were removed, dechorionated, and fixed for BrdU immunohistochemistry. At each time point, DAPI- and BrdU-labeled nuclei were counted in retinal sections, and the proportion of cells labeled with BrdU was determined and plotted as a function of time. A least-squares regression line was then fit to the data.
Percent labeled mitoses
The length of the G2-phase of the cell cycle was measured using the percentage labeled mitoses paradigm. At 24 hpf, embryos were placed in embryo solution containing PTU (see above). At 28 hpf, embryos were incubated in the EdU/DMSO solution for 20 min then removed and returned to embryo solution. Ten minutes following the EdU exposure, and at 1-h intervals, subsequently, four embryos from each group were removed, dechorionated, and fixed. Whole embryos were stained with antibodies against phosphohistone H3 (pH3), followed by EdU labeling chemistry. Nuclei were counterstained with 1:1,000 dilution of TOPRO3. Retinas from whole embryos were imaged using a Leica upright confocal microscope (Leica DM6000 CFS) with 25× water immersion objective. Approximately 40 to 80 optical sections, 1 μm in thickness, were acquired for each eye. By surveying each optical slice every pH3-positive cell was counted and scored for the presence/absence of EdU.
mRNA was isolated from the forebrains and eyes of embryos at 28 hpf following either morpholino injections (mismatch morpholinos vs. Mdka-targeted morpholinos) or heat shock treatment (wild type vs. Tg(pHSP70/4:mdka:egfp)kec1001L3 from a single clutch). Real-time PCR was performed using Power SYBER Green PCR Master Mix (Applied Biosystems) on the Applied Biosystems 7900HT Real Time PCR machine. Real-time PCR data were analyzed using the Comparative Ct method. Fold changes in expression are normalized to tubulin levels. The following primer pairs were used: id2a forward 5′ GCATCCTCTCACTACAGACACC 3′, id2a reverse 5′ CCTGATTAACGGTAAAGTGTCCT 3′; tubulin forward 5′ TGGAGCCCACTGTCATTGATG 3′, tubulin reverse 5′ CAGACAGTTTGCGAACCCTATCT 3′.
All quantitative data are represented as means and standard deviations. Statistical significance between groups was determined either by a one-way ANOVA or a Student’s t-test using GraphPad Prism software (GraphPad Software, La Jolla, CA, USA). P values <0.05 were considered statistically significant.
Images of sectioned retinas were captured using a Leica TCS SP5 confocal microscope (Vernon Hills, IL, USA).