Figure 4

Cytological analysis of electroporated OrgD3 YS-explants. Abbreviations: AFP: α-foeto-protein; Control non-electroporated YS-explants (A), maintained in organ culture, organise into a "bubble-like" structure that contains a compacted part, at the site adhering to the culture dish (asterisk). Erythroid cells (arrow), differentiated from explanted YS mesoderm, are located close to this adhering site (A). The "bubble-like" part harbours clusters similar to YS-blood islands (B: Enlargement of the square in A), which also contains erythroid cells (arrow). CD31⁺ cells are present in the both in the "bubble-like" structure and adhering site (asterisk) (C). Whereas in the adhering site, the nature of labelled cells (arrows) is unclear, CD31⁺ endothelial cells (D) are clearly present cells in the bubble. In electroporated YS explants, both without (E, F) or with (G, H) plasmid, the endoderm, revealed by AFP wholemount in situ hybridization, remains external, but does not cover the whole mesoderm, as shown on the sections of wholemount embryos (F, H) made along the axis shown in E, G. β-H1⁺ erythroid cells (I, control YS and J, electroporated YS) are mostly located at the adhering site (asterisk), while lmo2-expressing cells (K, control YS) are distributed either in the adhering site (asterisk) or in close contact with the endoderm (arrow). At the end of the organ culture step, both the blood islands-like clusters and the adhering sites (L, N) display GFP⁺ cells (M, O: same explants as L, N), often in area containing erythroid cells (arrows). Scale bars: 100 μm, except D: 20 μm.