Fig. 10

EnR-pitx2 RNA blocks expression of sox-17β, goosecoid, and Xbra in response to Xnr-1. (A, B) Stage 11 embryos injected in two blastomeres at the four-cell stage with 100 pg of Xnr-1 RNA alone or together with 200 pg of EnR-pitx2 RNA. (A) Ectopic sox-17β (purple) induced by Xnr-1 (animal view). (B) Siblings in which 200 pg of EnR-pitx2 RNA was co-injected. Ectopic sox-17β transcripts are seen surrounding sharply delimited patches of unstained cells (arrows). (C, D) Lateral views of embryos from a similar experiment but β-Gal mRNA was included in the RNA mixture to allow lineage tracing (blue). (C) Injection of Xnr-1 alone induced sox-17β expression both in the progeny of injected cells and in surrounding cells. (D) EnR-pitx2 mRNA blocked cell-autonomously the induction of sox-17β. (E, F) Rescue of sox-17β expression by wild-type pitx2 RNA. (E) Cell-autonomous inhibition of induction caused by co-injecting 25 pg of EnR-pitx2 RNA with 100 pg of Xnr-1 into one blastomere at the four-cell stage. (F) Reversal of the effect by co-injection of 25 pg of EnR-pitx2 RNA, 100 pg of Xnr-1, and 200 pg of wild-type pitx2 RNA. Expression of sox-17β in the clone of injected cells is rescued by co-injection of wild-type pitx2 RNA. (G) Ectopic goosecoid induced by Xnr-1 mRNA. (H) Sibling in which 200 pg of EnR-pitx2 RNA was co-injected. Lineage tracing of co-injected β-Gal RNA (blue) shows that ectopic goosecoid is not induced in the progeny of cells overexpressing Xnr-1 when the EnR-pitx2 RNA is present. (I) Ectopic Xbra induced at the periphery of the clone after injection of 50 pg of Xnr-1 into one blastomere at the four-cell stage. The same dose caused induction of sox-17β in the center of the clone (not shown). (J) Almost complete inhibition of Xbra induction at the border of the marked clone after co-injection of 50 pg of Xnr-1 and 200 pg of EnR-pitx2 mRNA.