Fig. 3

Zebrafish Engrailed 2a and 2b are able to transfer between cells in vivo and ex vivo. (A,B) In vivo intercellular transfer of En2 protein. (A) The En2ER^T2-P2A-mCherry plasmid was injected at the one-cell stage (10ng/µl). Embryos were fixed at 90% epiboly and immunostained for En2ER^T2 (green) and mCherry (red). En2 transfer was visualised by the presence of En2ER^T2 staining (green) in non-injected cells (negative for mCherry staining). (B) Cells expressing mCherry (red) were transplanted into 30% epiboly embryos ubiquitously expressing En2ER^T2. En2ER^T2 transfer was revealed by the detection of EnER^T2 signal (green) in mCherry-positive cells (yellow cells, arrow). Scale bars: 20µm. (C,D) Internalisation of Eng2a and 2b in HEK293 cells. Following extracellular addition, internalisation of fluorescein-labelled En2, Eng2a or Eng2b protein (green) was visualised (C) and quantified (D) after 1h incubation at 37°C in the presence of 0.4% Trypan Blue (red) to quench extracellular fluorescence. MFI, mean fluorescence intensity. (E) Secretion of Eng2a and 2b. HEK293 cells expressing the indicated proteins under the control of doxycycline were cultured for 24h and cell surface accumulation of the secreted protein was monitored by flow cytometry. ***P<0.001. (F) Paracrine activity of Eng2a and Eng2b proteins. mRNA encoding En2ER^T2, Eng2aER^T2 or Eng2bER^T2 were injected at the one-cell stage, the protein was activated with CYC at 50% epiboly and eye defects were scored at 30hpf. The error bars represent statistical errors (F) or s.e.m. (D,E).