Vivit

For local treatment with chemicals or proteins, AG1X2-formate beads (for chemicals) or Affigel Blue beads (BIO-RAD, 1537302; for BMP4) were soaked in different concentrations of the desired protein or chemical overnight at 4 °C. Beads were washed in PCS before grafting. Dimethyl sulfoxide (DMSO, 0.2%) or BSA (0.1%) was used to dilute the chemicals or proteins, respectively, and for soaking the control beads. Final concentrations used for microbead-soaking: 50 ng/μl recombinant human BMP4 (R&D systems, 312-BP), 200 µM dorsomorphin dihydrochloride (Tocris, 3093), 2 µM ionomycin (Sigma, I9657). For chemical treatment to the whole embryo, the chemical was diluted first in PBS (1:10 v:v) and then in egg albumen (9:10 v:v), which was used to culture the embryos (under the vitelline membrane). For treatments with VIVIT (Tocris, 3930) and BAY 11-7821 (Tocris, 1744), embryos were first soaked in the chemical diluted in PCS for 1 h, prior to culture with albumen containing the same concentration of the chemical. Final concentrations used for treatment of whole embryos: 20 µM dorsomorphin, 200 µM flufenamic acid (Sigma, F9005), 2 µM ionomycin, 50 µM, nicardipine (Sigma, N7510), 20 µM U73122 (Sigma, U6756), 20 µM U73343 (Sigma, U6881), 12 µM VIVIT (Tocris, 3930), 12.5 µM BAY 11-7821 (Tocris, 1744).