Materials and Methods
Animals
The mice used were of the CD1 strain (Charles River Laboratories International) and were housed in the pathogen-free facility at the San Raffaele Scientific Institute (Milano, Italy). Zebrafish (Danio rerio) embryos, collected by natural spawning, were raised and maintained according to established techniques. Embryos were staged according to Kimmel and colleagues, and raised at 28°C in fish water (Instant Ocean, 0.1% Methylene Blue) in Petri dishes. Beginning from 24 hpf, embryos were cultured in fish water containing 0.003% PTU (1-phenyl-2-thiourea; SIGMA) to prevent pigmentation. The following lines were used: AB (obtained from the Wilson lab, University College London, London, UK); tg(flk1:EGFP) (from the Stainier lab, University of California at San Francisco, USA), tg(gata1:dsRed); tg(flk1:EGFP)S843 (from the Santoro lab, Molecular Biotechnology Center, Università di Torino, Torino, Italy).
RT-PCR
RT-PCR was performed on total RNA extracted from oocytes, embryos (about 30 embryos per sample) at different developmental stages and adult organs using the TOTALLY RNA isolation kit (Ambion), treated with RQ1 RNase-Free DNase (Promega) and oligo(dT)-reverse transcribed using Super-Script II RT (Invitrogen), according to the manufacturer's instructions. The following primers were used for PCR reactions: adap2_fw 5'-GCTTAGACTTCTGGGATG-3', adap2_rev 5'-CGAGATAACGGTTTTCAAGGC-3'. PCR products were loaded and resolved onto 2% agarose gels.
In Situ Hybridisation
Probes were isolated by RT-PCR using specific primers (see online supplementary table S3 http://jmg.bmj.com/content/51/7/436/suppl/DC1) and cloned into the pCRII-TOPO vector (Invitrogen). Antisense and sense riboprobes were in vitro labelled with modified nucleotides (digoxigenin-UTP, Roche). WISH was performed on mouse embryos as described. At least eight embryos per stage were analysed. Prehybridisation was performed in a formamide-tween20 solution, after which the DIG-labelled riboprobes were added to the embryos and incubated at 65°. In situ hybridisation on mouse cryostat sections was performed as described.
WISH on zebrafish embryos was substantially carried out as described, on embryos fixed for 2 h at room temperature in 4% paraformaldehyde/phosphate buffered saline, then rinsed with PBS-Tween, dehydrated in 100% methanol and stored at −20°C until processed for WISH. A minimum of 20 embryos/time point were analysed.
The following probes were synthesised as described in the corresponding papers: cmlc2 and vmhc,notch1b and bmp4.
Images of stained embryos were taken with a Leica MZFLIII epifluorescence stereomicroscope equipped with a DFC 480 digital camera and IM50 Leica imaging software (Leica).
For histological sections, stained embryos were refixed in 4% paraformaldehyde, dehydrated, wax embedded, sectioned (8 μm) by a microtome (Leitz 1516) and stained with eosin. Images were taken with an Olympus BH2 microscope, equipped with a Leica DFC 320 digital camera and the IM50 software (Leica).
Morpholino Injections and Phenotype Analysis
Antisense morpholinos (MOs; Gene Tools) were designed against the AUG translation start site region and the coding sequence, adap2-MO (5'-TTGTTCTTTTCCCGATTTGCCATAG-3') and against the 5'-UTR region, UTR-adap2-MO (5'-AAAACACTCCTGTCGCGTCAGAATC-3'). As a control for unspecific effects, each experiment was performed in parallel with a std-MO (standard control oligo) with no target in zebrafish.
All morpholinos were diluted in Danieau solution and injected at the 1–2-cells stage. Rhodamine dextran (Molecular Probes) was usually coinjected as a tracer. After injection, embryos were raised in fish water at 28°C and observed up to the stage of interest. For a better observation, the injected embryos were anaesthetised using 0.016% tricaine (Ethyl 3-aminobenzoate methanesulfonate salt, SIGMA) in fish water.
Images were acquired by using a Leica MZ FLIII epifluorescence microscope equipped with a Leica DCF 480 digital camera and the IM50 software (Leica). Confocal microscopy was performed with a Leica TCSNT confocal microscope equipped with an Ar/Kr laser (blocking filter BP 530/30 for EGFP and blocking filter LP 590 for ds Red).
For histological analysis 3 and 5 dpf zebrafish early larvae were fixed overnight at 4°C with bouin fixative. The samples were then dehydrated in a graded ethanol series, wax embedded, sectioned (8 μm) by a microtome (Leitz 1516) and stained with haematoxylin/eosin. Images were taken with a Leica DM6000 B microscope equipped with a Leica DCF480 digital camera and the LAS software.