Heparin acrylic beads

Surgical manipulations were performed with E2/HH13 embryos (about 20 sm). Transplantation of a donor tissue, cell aggregate or bead into a host embryo was performed using a tungsten needle. FGFs were delivered using heparin acrylic beads (Sigma) soaked overnight with 250 μg/ml human FGF4 (WAKO) or 250 μg/ml human FGF8B (WAKO) at 4°C. For chemical-soaked bead experiments, AG1X2 ion-exchange beads (formate form; BioRad) were incubated in 10 mM SU5402 (Calbiochem), 20 mM PD184352 (Sigma), 20 mM LY294002 (Sigma), 10 mM U73122 (Sigma), 20 mM SP600125 (Sigma) or 20 mM SB203580 (Sigma) for 2 h at room temperature before grafting.

Cell dissociation was performed by incubating in Ca²⁺Mg²⁺-free DFA for 3-5 min before transferring to normal DFA medium. Cells were tracked using the ImageJ Manual Tracking plugin (http://rsb.info.nih.gov/ij). Track speed and persistence were determined using the ImageJ Chemotaxis Tool plugin. Chemotaxis assay was performed as described previously (Theveneau et al., 2010 (link)). Heparin-acrylic beads (H5263, Sigma-Aldrich) were incubated overnight at 4°C in a 1 mg/ml SDF1 or 1 mg/ml PDGF-AA (AF-100-13A, PeproTech) solution in PBS. To measure dispersion, NC cells from embryos injected with H2B-mCherry (Carmona-Fontaine et al., 2008b (link)) were imaged for 12 h, and nuclei triangulation was analysed using the ImageJ Delaunay Triangulation plugin (Carmona-Fontaine et al., 2011 (link)). For small molecule inhibitor treatment, inhibitors were incubated 1 h before addition of PDGF-A protein (50 ng/ml, PeproTech). To study CIL, an explant confrontation assay was performed as described by Carmona-Fontaine et al. (2008a) (link). For the single-cell confrontation assay, single-cell CIL time was measured from the first frame of contact (t=0) until the last frame of contact (t=end). Protrusion area was analysed as previously described (Law et al., 2013 (link)).

Dissections were performed as described [12] , [13] (link) in ice-cold L-15 with 2% glucose. Dorsal forebrains from embryos were placed ventricular surface down on the dull surface of 8 µm pore polycarbonate membranes (Whatman, Clifton, NJ) floating on DMEM/F-12 with 20% calf serum, sodium pyruvate, nonessential amino acids, and penicillin/streptomycin. After 1 hr, 50 ng/ml BMP4 was added for three days, or 100 nM PD173074 was added for two days (Figure 1). Explants were processed for Ttr ISH or X-gal staining. For FGF8 bead studies (Figure S1), heparin acrylic beads (Sigma) were soaked in 10 µl of 100 ng/ml FGF8 or BSA, rinsed briefly in PBS, and placed on explants using pulled flame-polished microcapillary pipettes.

Recombinant human BMP4 (R&D Systems, 312-BP) was delivered using Affigel Blue beads (Bio-Rad, 1537302); recombinant human ACTIVIN A (R&D Systems; 338-AC) was delivered using Heparin-Acrylic beads (Sigma-Aldrich, H5236) and dorsomorphin hydrochloride (Tocris, 3093) was loaded onto AG1X2-formate beads. In each case the beads were incubated overnight at 4°C in the desired concentration of protein or chemical. Beads were washed in Pannett-Compton saline immediately before use.

Fifty micrograms per millilitre of mouse FGF8b (Sigma-Aldrich, F6926) in 0.1% bovine serum albumin in phosphate-buffered saline was delivered using heparin-acrylic beads (Sigma-Aldrich, H5236), while 250 μM SU5402 (Calbiochem, 572,630), 1 mM U0126 (Calbiochem, 662,005) and 1 mM U0124 (Calbiochem, 662,006) in DMSO were loaded onto AG1X2-formate beads (Streit et al. 2000 (link)). In each case, the beads were incubated overnight at 4 °C in the protein or chemical at the concentrations stated above. Beads were washed in Pannett-Compton saline just before use. Embryos were fixed at 3 h, 6 h or 9 h after the graft or after overnight incubation.