Erythroid gene expression in the pVBI is suppressed by FGF signaling. (A) The anterior/posterior boundary within the VBI is unchanged by SU5402 treatment, but Scl expression is expanded into posterior territory. Embryos were injected at the 4-cell stage with 200 pg β-galactosidase mRNA into (i) both DMZ blastomeres or (ii) both VMZ blastomeres. Embryos were treated from stage 8 with increasing concentrations of SU5402 (25-100 μM) and grown along with controls (con) to stage 18/19 then stained for β-galactosidase. (iii,iv) Embryos injected with 200 pg β-galactosidase RNA per DMZ blastomere were treated with 25μM SU5402 from stages 8-18. Embryos were stained for βgal and subsequently probed for Scl by whole-mount in situ hybridization. Black dashed line in subpanel iv delineates the limit of β-gal staining (DMZ territory) at stage 18. Subpanels i and ii are ventral views; and subpanels iii and iv, anterior views. Embryo numbers are in the bottom right corner of each subpanel. (B) Injection of a dominant-negative FGF receptor induces precocious erythroid gene expression in VMZ explants; 200 to 400 pg XFD mRNA (levels that strongly block MAPK activity, Figure S3b) was injected into both VMZ blastomeres of 4-cell stage embryos. Embryos were grown to stage 10 when VMZ explants were excised and cultured until sibling embryos reached stages 18 and 30. Explants were processed for real time RT-PCR probing for Scl (i), Runx1 (ii), and Fli1 (iii). Precocious expression of Scl and Runx1 at stage 18 was observed in XFD injected explants compared with uninjected (Ui) control VMZ explants, whereas expression of the endothelial gene Fli1 was not precociously induced. Values represent the average of 3 experiments; error bars represent SD. (C) FGF antagonists Spred 1 and 2 mimic the effects of SU5402. Embryos at the 2-cell stage were injected in the marginal zone with 1ng of Xt Spred 1 (ii) or 2ng Xt Spred 2 (iv) mRNAs, levels previously shown to block MAPK activation efficiently,34 and grown alongside uninjected controls (i,iii) to neurula stages when they were probed for expression of Scl by whole-mount in situ hybridization. Expansion of Scl expression into posterior and lateral regions was seen as for SU5402 treatment. Embryo numbers are in the bottom right corner of each subpanel.
