Fig. 4

Intercellular transfer of Eng2b but not 2a is involved in DMB positioning. (A,B) Co-detection of Eng2 proteins with FITC-labelled 4D9 and TRITC-labelled 4G11 antibodies by immunohistochemistry (A) or western blot analysis on HeLa cells (B). Using both techniques, 4D9 recognised Eng2a and Eng2b, whereas 4G11 recognised only Eng2a. (C-H) Paracrine activity of endogenous En2 proteins. Zebrafish embryos were injected in the intercellular space at blastula stage with anti-myc or anti-Engrailed (4G11 or 4D9) antibody. Mesencephalon length was quantified by pax6 (C,E,G) or wnt1 (D,F,H) in situ hybridisation. Representative in situ hybridisation staining of pax6 (dorsal view) (C) and wnt1 (lateral view) (D). Measurements were distributed into seven size classes (smallest size, class 1) and plotted as cumulative frequency index. 4D9 extracellular injection induced mesencephalon shortening (E,F) in a dose-dependent manner (supplementary material Fig. S4), which was the case for neither anti-myc (E-H) nor 4G11 injection (G,H). Co-injection of the 4D9 antibody with its epitope peptide (pep) significantly reduced this effect (E,F). ***P<0.0001, Mann–Whitney tests. (I) Inhibition of homeoprotein transfer rescued the phenotype of Eng2b activation but not of Eng2a. Zebrafish embryos were injected at the one-cell stage with the indicated mRNAs and injected again in the extracellular space at the blastula stage with 4D9 anti-En antibody. CYC was added at 50% epiboly in the water bath to activate the protein. Eye phenotypes were scored at 2dpf. The error bars represent statistical errors. (J) Transcriptional activity of En2 and the two zebrafish Eng2. The MAP1B:luciferase reporter plasmid was transfected into HeLa cells along with an empty vector (Ctrl) or together with the indicated constructs, and cells were analysed for luciferase activity after 24h. a.u., arbitrary units. **P<0.01, ***P<0.001.