Autorepressive and cross-repressive regulatory interactions between sFRP1/5 and Dkk3 refine the size of the ANE. (A,D) ANE expression of dkk3 is expanded towards the posterior pole (Ab,e) and is upregulated (D) in sFRP1/5 morpholino-injected embryos. dkk3 expression is severely downregulated in embryos injected with sfrp1/5 mRNA (Ac,f). (B,E) Embryos injected with Dkk3 morpholinos fail to completely restrict the sfrp1/5 expression domain to the anterior pole (Bb,e) and the level of sfrp1/5 is strongly upregulated (E). Expression of sfrp1/5 is eliminated in embryos injected with dkk3 mRNA (Bc,f). (C,D) The anterior sfrp1/5 expression domain is expanded in sFRP1/5 morphants (Ca,b) and is strongly upregulated (D). Similarly, Dkk3 morpholino-injected embryos show expanded dkk3 expression (Cc,d) and severe upregulation of dkk3 transcripts (E). (D,E) qPCR analysis showing that sfrp1/5 and dkk3 expression is strongly upregulated in sFRP1/5 (D) and Dkk3 (E) morpholino-injected embryos. MO, morpholino. Scale bars: 20 µm.