Supplementary materials and methods (doc 112K)

Supplementary materials and methods
Whole Mount Immunofluorescence
Immunostaining for l-plastin and phosphohistone 3 (Ser10, PH3) was performed according to the
published protocol (1). In brief 2-3dpf larvae were fixed in 4% PFA at room temperature for 2h and
than rinsed, blocked, and incubated with rabbit polyclonal anti-l-plastin antibody (a gift of P.
Martin, Bristol, UK,) or anti PH3 (Millipore, cat. no.06-570) overnight at 4°C. Subsequently Alexa
Fluor 633 secondary antibody (Molecular Probes) was used to reveal l-plastin or PH3 positive cells.
The expression of the transgenes, GFP and GFP-RAS, was still visible as green fluorescence.
Cell proliferation analysis by BrdU labelling
For BrdU labelling, 30hpf larvae were manually dechorionated and incubated in 10mM BrdU in E3
fish water for 6 hours. Following fixation in 4% PFA, larvae were treated with 2N HCl for 40min
and processed for immunofluorescence with mouse anti-BrdU antibody (1:500, Sigma) and rabbit
anti-GFP antibody (1:1000, Torrey Pines, CA). Alexa 546 and Alexa 633-conjugated secondary
antibodies (1:200) were used to reveal BrdU in GFP+ cells either in fli-RAS or fli.-GFP control
Cytology and staining
Wild-type and injected juvenile zebrafish were anaesthetized with 0.02% tricaine and blood and
kidney collection was performed as previously described (2). Kidney marrow single-cell
suspensions were generated by pipetting and filtering the dissociated organ through a 40-μm nylon
mesh filter (Cell strainer, BD). Cytospin preparations were made by centrifugation of the kidney
cells at 300 r.p.m. for 3 min onto glass slides in a Cytospin4 cytocentrifuge (Shandon). Larva and
adult blood smears and cytospin preparations were processed through May Grunwald-Giemsa
staining (Sigma Diagnostics) for morphological analyses and differential cell counts.
RNA extraction, qPCR and data analysis
For RNA preparation we used phenol-chloroform extraction (TRIZol, Invitrogen). cDNA template
was obtained using SuperScript VILO cDNA synthesis kit (Invitrogen). RNA was prepared from
3dpf fli-RAS, and fli-NICD whereas 3dpf fli-GFP larvae were used as control. Quantitative Real
Time PCR was performed with LightCycler 480 II thermocycler (Roche) using the SYBR Green
Master kit (Roche). For RT-qPCR of the hematopoietic genes, primers used were as described
before (3). All the other primers were designed using Primer 3 and are shown in supplementary
Table III. For all samples the expression level was analysed using Q-Gene software, which
expresses data as mean normalized expression. Relative genes expression in fli-RAS or fli-NICD
embryos compared to whole fli-GFP age match embryos, was determined by calculating ∆∆ CT
values after normalization to beta-actin.
RNA-Seq and statistical analysis
Total RNA from 3dpf wild type, fli-RAS and fli-NICD embryos was prepared by TRIzol reagent
(Invitrogen) and RNAeasy kit (Qiagen) and quantified via Nanodrop (Thermo Fisher Scientific
Inc.). 10 μg of total RNA was used to construct sequencing libraries using the Illumina TruSeq
RNA Sample Prep Kit. Libraries were sequenced with on an Illumina HiSeq 2000 with read lengths
of 2×51 nt. The Illumina pipeline was used for image analysis, demupliplexing and base-calling.
Raw sequencing reads were analysed using the CLC bio Assembly Cell version 3.2 (CLC bio,
Aarhus, Denmark). Low quality basec-calls were removed, and reads were aligned to cDNA
sequences predicted by Ensembl based on the Zv9 zebrafish genome assembly (
Read counts were quantified and normalized to yield RPKM expression values (4) Log2 fold
changes were calculated using the DESeq package for Bioconductor using the variance stabilization
option (5).
Imaging of zebrafish embryos and larvae
For both live imaging studies and fixed samples, larvae were mounted on slides in 1.5% low
melting agarose (Sigma). Images were collected using a Leica SP2 Confocal Microscope or Nikon
S100 stereomicroscope equipped with epi-fluorescence and multiple filters. For time-lapse imaging
zebrafish were prepared as previously described(6). Imaging was carried out also using a Leica SP5
confocal microscope.
Conversion of Kaede
Kaede was converted from green to red using a Leica SP5 confocal microscope and a 20x objective.
Using the ROI function the respective tissue was illuminated with UV light (405nm) for 2min.
Images of converted embryos were taken at 73hpf and 5dpf using a Leica DMI 6000B microscope.
Image rendering
Images were rendered using Volocity 5, Adobe Photoshop CS4, Image J and Quicktime Pro.
Histology and immunofluorescence
Fli-RAS 5dpf larva and juvenile fish and age-matched control were killed by anaesthetic overdose
and fixed for 2h at room temperature in 4% paraformaldehyde. After over night incubation in 10%
and 20% sucrose, sample were transferred to optimum cutting temperature (OCT; Sakura Finetek,
Torrance, CA). Cryostat sections (16 um thick) were air-dried and incubated over night with rabbit
polyclonal anti l-plastin (1:500) or PH3 (1:1000) and mouse anti GFP (1:1000). Secondary
antibodies conjugated with Alexa 488, Alexa 546 and Alexa 633 were from Molecular Probes
(Carlsbad, CA). 4,6-Diamidino-2-phenylindole, DAPI (1:10000) counter stain was carried out on all
Apoptotic assay by TUNEL assay
For TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay, PFA-fixed
embryos were permeabilized by treatment with
at –20°C and then stained using In situ Cell Death Detection Kit, POD (Roche) according to the
manufacturer’s instructions.
Whole Mount In situ Hybridization
Whole-mount in situ hybridization for c-myb (ZFIN ID: ZDB-GENE-991110-14) was performed as
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