The AB zebrafish strain was maintained at 28.5°C as described by Westerfield (5). Embryos were staged as described by Kimmel et al (6). Developmental stages refer to hpf or dpf. This study was approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Research Center for Model Organisms (Shanghai, China) with approval ID 2012-0008.

Various restriction enzymes and the TOP10 Escherichia coli strain were purchased from Takara Bio Inc. (Japan). T4-DNA ligase was purchased from Promega Corporation (Madison, WI, USA) and DNA high fidelity polymerase KOD-Plus was purchased from Toyobo Co., Ltd. (Japan). The DIG-deoxyribonucleotide triphosphate (dNTP) labeling kit, blocking reagents, anti-DIG-AP and BM purple were purchased from Roche Diagnostics (Indianapolis, IN, USA). RNase inhibitor was purchased from Ambion (USA), levamisole, heparin and yeast RNA were purchased from Sigma (St. Louis, MO, USA). TRIzol reagent was purchased from Invitrogen Life Technologies (Carlsbad, CA, USA) and the plasmid Maxiprep kit was purchased from Qiagen (Hilden, Germany). The first-strand cDNA Quantscript RT kit was purchased from Tiangen Biotech Co., Ltd. (Beijing, China). A fluorescence microscope (SMZ-1500; Nikon, Japan), ultra-violet spectrophotometer (ASP-3700; ACT Gene, Piscataway, NJ, USA), Biometra T personal polymerase chain reaction (PCR) amplification instrument (Goettingen, Germany), gel imaging analysis system (Tanon 3500, Shanghai, China) and SPX biochemical incubator (GNP-9160; Shanghai Jinghong Laboratory Instrument Co., Ltd., China) were also used.

Total RNA was extracted from 40 embryos at 48 hpf, using TRIzol reagent according to the manufacturer’s instructions. The cDNA was synthesized from 1 μg total RNA using the first-strand cDNA Quantscript RT kit. The specific primers were designed according to the zebrafish mpo (zmpo) genomic sequence on the National Center for Biotechnology Information (NCBI) web site (gene ID, 337514); forward primer: 5’-TTCAAGTCCAGAACCAGTGAGCCT-3’ and reverse primer: 5’-TTTAGCAGTGGCAGGAAGGATGGA-3’. The length of the amplified PCR product was 2442 bp, including two restriction enzyme cutting sites (Xho I at 216 bp and EcoRI at 2384 bp) and the probe sequence (at 737 bp). The probe sequence was designed to span exon borders of the gene. PCR was performed as follows: 95°C for 10 min; then 35 cycles at 95°C for 30 sec, 58°C for 30 sec and 72°C for 60 sec. The integrity of the PCR product was examined by 1% agarose gel electrophoresis. The purity was analyzed based on the absorbance ratio at 260 and 280 nm (A260/280). Then the zmpo fragment and the pBK-CML vector were digested with XhoI and EcoRI enzymes and connected at the same sticky end with the T4 ligase, which resulted in the construct of an mpo/pBK-CML plasmid.

The mpo/pBK-CML plasmid was linearized with the SalI restriction enzyme. DIG-antisense RNA probes were generated by T7 in vitro transcription (1 μg linearization template DNA, 1 μl DIG-dNTP mix, 2 μl 100 mM dithiothreitol (DTT), 4 μl 5X transcription buffer, 1 μl RNase inhibitor, 1 μl T7 RNA polymerase and diethylpyrocarbonate (DEPC) H₂O to make 20 μl, incubated for 2 h at 37°C). Then, 1 μl DNaseI was added and incubated at 37°C for 20 min to purify the product. The final precipitation was stored at −20°C.

The required developmental stage of the embryos was selected according to the somite. Embryos younger than 48 hpf were dechorionated. The embryos were digested in 1 mg/ml pronase for ∼2–10 min. The digestion was stopped when >10% embryos were free from their chorions. The chorions were broken with air from a pipe. If the chorions were difficult to remove, they were manually broken with a pair of tweezers. Embryos older than 24 hpf were decolored in 5% hydrogen peroxide and 5% potassium peroxide fish water for 15 min. Embryos (up to 40 embryos per 1.5 ml eppendorf tube) were fixed in fresh 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) and agitated overnight at 4°C. Then, the embryos were washed twice in PBS with Tween-20 (PBST) for 5 min each and dehydrated with gradient (25, 50, 75 and 100%) methanal/PBST for 10 min each at room temperature (RT), then stored at −20°C.

The embryos were removed from the −20°C storage and rehydrated with gradient (75, 50, 25 and 0%) methanal/PBST. The embryos were fixed with 4% PFA for 20 min and rinsed twice with PBST for 5 min at RT. The embryos were digested with an appropriate concentration of proteinase K (10 μg/ml in PBST) at 37°C for 3 min and then fixed and rinsed as above. They were then incubated at 68°C in negative hybrid liquid (HYB⁻, 50% formamide in 5X SSC buffer) for 15 min and prehybridized at 68°C in 300 μl positive hybrid liquid (HYB⁺, 0.5 mg/ml yeast RNA and 50 μg/ml heparin in HYB⁻) for 4 h. Following this, the HYB⁺ was replaced with fresh HYB⁺ containing the DIG-labeled probe (concentration, ∼1 ng/μl) and incubated overnight at 68°C.

The probe was removed and the embryos were washed twice for 30 min each with 50% formamide in 2X SSC buffer, then for 15 min in 2X SSC buffer and twice for 30 min each in 0.2X SSC buffer at 68°C. Then, the embryos were rinsed three times for 5 min each at RT in MABT (100 mM maleic acid, 150 mM NaCl and 0.1% Tween-20; pH 7.5) and blocked for 1 h at RT with blocking solution (10% blocking reagent and 50% lamb serum in MAB). Anti-DIG-AP (1:4000 dilution in blocking solution) was added and agitated overnight at 4°C.

The embryos were washed with blocking solution for 30 min and MABT for 1 h at RT. Then, the embryos were soaked three times for 5 min each in staining buffer (100 mM Tris, 50 mM MgCl₂, 100 mM NaCl, 0.1% Tween-20 and 1 mM levamisole; pH 9.5). The embryos were transferred to a 12-well plate, incubated in 1 ml/well BM purple stain for 30 min, monitored with a dissecting microscope every 30 min. The reaction was terminated by being washed twice in PBST and fixed with 4% PFA at 4°C to store. Images were taken in 3% methyl cellulose.

The cDNA of 48 hpf embryos was used as a template to amplify the mpo gemonic fragment by PCR. The PCR product was verified by 1% agarose gel electrophoresis and the single band at 2400 bp was observed as expected. The PCR product was cloned into the pBK-CML carrier and the mpo/pBK-CML recombinant plasmid was constructed. The plasmid was linearized by SalI and the single band at 7300 bp was observed. The plasmid was digested with two enzymes (XhoI and EcoRI), producing two bands at 5200 and 2100 bp, respectively, which were the pBK-CML vector and mpo gene segment. This demonstrated that plasmid construction was successful (Fig. 1A). The DIG-labeled antisense mpo mRNA probe was generated by T7 in vitro transcription and confirmed in the electrophoresis tank soaked with DEPC H₂O overnight. There was a single band near 2100 bp (Fig. 1B).

The expression patterns of mpo were investigated in zebrafish embryos from 12 to 72 hpf by in situ hybridization using the DIG-labeled antisense RNA probe. As shown in Fig. 2, the earliest expression of zebrafish mpo was detected in cells of the ICM at 18 hpf and 1 to 2 h later, it was detected in cells in the rostral blood island (RBI). Strong signals were observed in the anterior ICM, then it spread over the yolk sac. By 72 hpf the mpo-expressing cells were in the circulation and distributed throughout the embryo. In our previous study, we established the transgenic enhanced green fluorescent protein [Tg(zlyz:EGFP)] zebrafish line (7), which expresses EGFP in primitive neutrophils (mpo⁺). The EGFP distribution coincided with the result of in situ hybridization of mpo (Fig. 2).

To test whether mpo expression detected by in situ hybridization at an early stage could predict the myelopoiesis in zebrafish development, we compared the peripheral blood smear between wild type (WT) and Tg zebrafish. In our previous study, we established the Tg(MYCN:HSE:EGFP) zebrafish line, which suggested that MYCN converted erythropoiesis to myelopoiesis (Shen et al, the influence of MYCN gene on the transcriptional regulation of hematopoiesis. abs. 751, 9–12 June, 2011). As demonstrated by in situ hybridization, the MYCN gene increases the expression of mpo in embryos at 22 hpf (Fig. 3A and C). For cytological analyses, blood cells collected from the zebrafish at 60 dpf were transferred onto glass slides using Cytospin, stained with Wright-Giemsa stain and examined under oil immersion by light microscopy (8). This revealed that the blood cells from WT fish were predominantly erythrocytes, with myeloid cells occasionally observed. By contrast, erythrocytes were significantly inhibited in Tg fish, which were enriched with granulocytes (Fig. 3B and D).

In order to avoid background staining, unincorporated nucleotides were removed from the probe preparation. We routinely used Ambion NucAway Spin Columns to purify the RNA probe according to the manufacturer’s instructions (Cat. AM10070). Following the final precipitation, the hydrolyzed probe was placed in HYB⁺ (final concentration, ∼1 ng/μl) and stored at −20°C. Each time the probe was used, it was degenerated in advance by placing it at 95°C for 5 min then on ice for 5 min to eliminate single RNA spontaneous formation of hairpin structures. Probes were reused for 4–5 times. When 24 hpf embryos were collected, the conventional decolorization method was used and they were placed in 0.03% phenylthiourea (PTU) in fish water at 12–36 hpf. We used the improved decolorizing liquid of 5% hydrogen peroxide and 5% potassium peroxide in fish water. As a result, the embryos only required soaking at 24 hpf for 10–15 min. The effect of decolorization was good without damaging the integrity of the embryo. Prior to hybridization, the embryos were permeabilized by digesting with proteinase K (10 μg/ml in PBST) at RT for 5–30 min, depending on the developmental stage. The permeability of the probe was appropriate and the staining background was low. In addition, the traditional permeabilization method included digesting the embryos at RT for 5–30 min, depending on the developmental stage. This is difficult to control. We improved the digestion temperature to 37°C and the process was shorted to 5 min.