1-Mice and isolation of embryo
All animal experiments were conducted in compliance with French and European regulations. Balb/c or C57BL/6 males are crossed with females to generate embryos and the morning of the vaginal plug observation is considered as 0.5 dpc. Pregnant females are sacrificed by cervical dislocation. Embryos at 8 dpc are staged by somite counting. Presomitic embryos are staged according to the development of the allantois bud: OB (No Bud), EB (Early Bud) and LB (Late Bud) .
2-Isolation and organ culture of 7 and 8 dpc Yolk Sacs
Embryos from OB to EB stages, used for in situ electroporation, are collected in Hanks balanced salt solution (HBSS: Invitrogen), and after removal of the Reichert membrane (the ectoplacental cone being preserved), they are injected with the plasmid solution as described below. After injection and pulse application, the extra-embryonic compartment is removed with ultra-fine forceps and maintained in organ culture as described below. From 8 dpc, the YS used for retroviral infection, are directly explanted from the embryos.
YS explants are transferred into 6-well plates (TPP) containing "Complemented OptiMEM medium", i.e. OptiMEM with Glutamax (Invitrogen), 10% Fetal Calf Serum (Hyclone), 1% Penicillin-steptomycin (Invitrogen) and 0.1% β-mercaptoethanol (Invitrogen), and are kept in culture at 37°C, 5% CO2 for 1 (8 dpc) or 3 (OB and EB stages) days, before analyse (OB and EB stages) or retroviral infection (8 dpc). Prior to further processing, YS-explants are dissociated by gentle pipeting and filtered to obtain a single cell suspension.
3-In situ Electoporation
For in situ electroporation, we use the peGFP-C1 vector (Clontech), in which the cytomegalovirus (CMV) promoter drives the expression of the GFP reporter protein.
To visualise plasmid solution delivery, 0.01% Fast Green is added to 1 μg/μl peGFP-C1 plasmid DNA. Borosilicate standard wall (1.0 mm O.D./0.58 mm I.D.) glass capillaries (Harvard Apparatus) are pulled and filled with DNA/Fast Green solution for injection.
After dissection, the plasmid solution is injected in the YS cavity as described in Fig. 2, and embryos are placed in a Petri dish, in HBSS. To accurately target the presumptive blood islands, OB-EB embryos are oriented so that the caudal half part of the visceral YS is facing the anode. The two gold genetrodes (BTX Instrument Division Ref 512) are positioned parallel to the presumptive blood islands, and pulses are applied using a square-wave pulse generator (Electro Square Porator ECM 830, BTX Instrument Division; San Diego, CA, USA).
4-Retroviral infection of 8 dpc YS
Production of ecotropic retroviral supernatants
We used for retroviral transduction a MSCV-based retroviral vector, called MPI . HEK 293T cells (4 × 106 cells; 30–40% confluence) are seeded in DMEM supplemented with 10% FCS and 1% Penicillin-steptomycin (Invitrogen), in 150 mm culture dishes. The following day, the MPI expression vector (6.4 μg) is co-transfected with 3.2 μg phCMV-intron GagPol and 3.2 μg FB-MO-SALF  helper plasmids, encoding the MLV gag-pol and ecotropic env proteins, respectively, using Fugene 6 (Roche), as transfection reagent, to produce retroviral particles. Viral supernatants are collected twice a day, from 24 h to 72 h post-transfection, filtered (0.45 μm) and concentrated on Centrifugal Filter Devices (Centricon®Plus-20 from MICON Bioseparation). Concentrated supernatants are distributed into 30 μl aliquots, kept frozen at -80°C. Titration is performed by transducing mouse 3T3 fibroblasts (plated at 105 cells per well in 6-wells plates) in exponential growth phase with 5 μl concentrated supernatant in the presence of 4 μg/ml polybrene. Cells are analysed for GFP expression 4 days post exposition to the viral supernatant, by flow cytometry using a Facscan (Becton Dickinson). Typically, the titre of concentrated supernatants is 7 × 106-107 viral particles per ml.
Retroviral transduction conditions
Ten dissociated YS explants (about 105 cells) are seeded in 48 well-plates containing 1 ml "Complemented OptiMEM medium", further supplemented with Flt3-ligand (20 ng/ml), Stem Cell Factor (100 ng/ml), EPO (103 U/ml) and 4 μg polybrene. Viral supernatant is added to obtain one retroviral particle per cell (MOI = 1) during 12 hours. After infection, transduced cells are transferred on OP9 stromal cells and cultured as described below. Interestingly, a 25–30% transduction efficiency is achieved on OrgD1-YS, either with a 24 hours or a shorter (4 or 6 hours) exposure to the retroviral supernatant.
In situ hybridization
YS explants, fixed in 4% paraformaldehyde in PBS overnight at 4°C and rinsed with PBS, are hybridized with riboprobes at 65°C, as previously described . Digoxigenin (DIG)-labelled antisense RNA probes are obtained from PCR fragments subcloned into expression vectors (βH1, provided by I. Max-Audit: 250 bp subcloned into pCR™; Lmo-2 constructed by V. Lemarchandel: 450 bp subcloned into pBSKII and α-foeto-protein (AFP) provided by L. Robb: 845 bp subcloned into pBSKII) and synthesized using the DIG RNA Labelling Kit (Roche Diagnostics, Indianapolis, IN). To obtain cryostat sections of hybridized samples, YS explants are fixed (4% paraformaldehyde, 4% sucrose, 0.12 M CaCl2, 0.2 M Na2HPO4, 0.2 M NaH2PO4, H2O), embedded in 0.12 M phosphate buffer, 7.5% gelatin, 15% saccharose and frozen in liquid nitrogen. Embedded embryos are sectioned at 10–15 μm and transferred to Superfrost slides.
Cryostat sections of YS explants, prepared as described above, are incubated first with 0.3% H2O2 to block endogenous peroxydase activity, then in PBS, 10% fetal calf serum (2 hours at room temperature) to prevent non specific antibody binding. Samples are incubated with purified Rat anti-Mouse CD31 (clone MEC13.3, Pharmingen) overnight at 4°C, followed by incubation with biotinylated-Goat anti-Rat (DAKO), as secondary antibody for 1 hour at room temperature and Streptavidine-horseradish peroxydase complex (VECTOR) for 30 Min. at 37°. Peroxydase activity is revealed by exposure to its diaminobenzidine substrate. Sections are mounted in Glycergel (DAKO) and photographed using DIC optics.
6-Microscopy and photography
Samples are observed either on a Fluorescence-equipped Olympus SZX12 stereomicroscope (wholemount in situ hybridized or electroporated YS) or on a Zeiss Axioplan microscope (YS-explants sections). Images are acquired respectively with the DP50 Olympus digital camera (Analysis software) or Zeiss AxioCam MRc (Axiovision 4.3 software). Images are further processed with the Adobe Photoshop CS software.
7-Analysis of hematopoietic cell production
Culture on OP9 stromal layer
Cells are seeded on OP9 stromal cells (from S.-I. Nishikawa and T. Nakano, Riken Center for Developmental Biology, Kobe, Japan), in "Complemented OptiMEM medium", further supplemented with Flt3-ligand, Stem Cell Factor and EPO, and cultured as described in . At various culture time points, cells are analysed by flow cytometry or submitted to clonogenic assays.
Cells recovered after 1 or 4 days from the culture on OP9 stroma, are assayed for the presence of colony-forming units, by seeding 3000 cells per 30 mm culture dish, in triplicate, in 1 ml methylcellulose medium (M3234; Stem Cell Technologies, Vancouver, BC, Canada) supplemented with cytokines (50 ng/ml Stem Cell Factor, 10 ng/ml Il-3, 10 ng/ml Il-6, 3 U/ml EPO and 10 ng/ml TPO). After 3 to 7 days at 37°C, colonies are scored based on their morphology on an inverted microscope.
Flow Cytometry Analyses and cell sorting
Cells are analyzed on a FACS Sort (Becton-Dickinson) as previously described , and FACS data are analyzed with CellQuestPro software (Becton-Dickinson).
The following antibodies (all from Pharmingen) are used to reveal the presence of lineage-specific surface markers: CD45 (Clone 30-F11), CD31 (Clone MEC 13.3) and Mac-1 (Clone M1/70) for myeloid cells, Ter119 (Clone Ly-76) for erythrocytes. Cell sorting was performed with a FACS Diva cell sorter (Becton-Dickinson).