Fig. 4. Molecular genetic and vital dye lineage marking of serosal EMT. (A-F) Localization of Wt1 (green) and ß-Gal (red) proteins during gut development. Wt1 expression is largely confined to the serosal mesothelium in the embryo (A-C) and newborn (NB0; D-F). The anti-ß-Gal antibody marks cells in the subserosal space of the developing gut tube (arrows in B,C,E,F). Both Wt1 and ß-Gal are expressed in most cells of the serosal mesothelium (C,F, arrowheads). (G-K) CCSFE (green) was used to vitally label surface cells of gut explants from E12.5 Wt1-Cre; Rosa26R embryos. (G) At day 0 of culture, surface cells were positive for CCFSE (area framed by arrowheads), but the subjacent mesenchyme is negative. (H,I) After two days of culture, CCFSE-positive cells (arrowheads) at the gut surface co-label with Wt1. CCFSE-marked cells in the subserosal space are not positive for anti-Wt1 antibody (arrows). Also, many mesenchymal cells label with both anti-ß-Gal antibody and CCFSE (arrows, J,K). ß-Gal-positive cells unmarked by CCFSE are also seen in the mesenchyme (open arrowheads). Note a small number of CCFSE-labeled cells without the ß-Gal marker (filled arrowheads). Scale bars: 20 µm.