Functional tests for presence in Et of known regulatory linkages of the Sp skeletogenic GRN. Test for global HesC repression of skeletogenic regulatory state. (A) The 28-h embryos injected with hesC MASO exhibit an expanded domain of the skeletogenic lineage marker alx1. (B) The 20-h embryos injected with hesC MASO showing no global expression of dominant-negative gate genes. Alx1 expands locally only, whereas ets1 and tbrain are unaffected. (C) Quantitative effects of hesC MASO on mRNA abundance at 18 h on expression of skeletogenic genes alx1, delta, ets1, hesc, and tbrain. Alx1 and hesC mRNAs are significantly up-regulated. The difference in cycle number (ddCt) with respect to an uninjected control group is shown on the ordinate. Error bars represent the SD of two independent experiments. (D) Spatially nonrestricted expression of Sp pmar1 expression construct on injection into Et eggs. (E) Quantitation of multiple expression domains observed in (D).