Fig. 2.

Engrailed gain of function involves the paracrine signalling properties of En. (A) Description of the two En2 mutants defective for paracrine signalling. (B) Diencephalon shortening requires En2 transfer. Activation of the En2(5E) transfer-deficient mutant following mRNA injection did not affect the size of the diencephalon. epi, epiboly. (C) Transcriptional activity of wild-type En2 and En2(5E). MAP1B:luciferase reporter plasmid was transfected into HeLa cells with an empty vector (ctrl) or together with the indicated constructs and analysed for luciferase activity after 24 h. a.u., arbitrary units. (D) In vivo ectopic expression of En proteins. Zebrafish embryos were injected at the one-cell stage with the indicated constructs. Following CYC addition at 50% epiboly, cell extracts from 90% epiboly embryos were prepared and analysed by western blotting with polyclonal (anti-En) or monoclonal (anti-tub) antibodies. (E) Inhibition of En2 transfer rescued the phenotype of En2 activation. Zebrafish embryos were injected at the one-cell stage with mRNA encoding En2ER^T2 and injected again in the extracellular space at the blastula stage with two different anti-En antibodies. CYC was added at 50% epiboly in the water bath to activate the protein. Eye phenotypes were scored at 2 dpf. The error bars represent statistical errors. ***P<0.001.