Fig. 4. FCM and FC specific expression of genes from array validates genetic and microarray strategy. (A-F) Simultaneous fluorescent detection of dei and gol, two genes predicted to be enriched in FCMs. dei (A,C) and gol (D,F) transcripts detected by in situ hybridization and anti-ß-Gal staining (B,E) in stage 12 rP298 embryos. The enhancer trap insertion rP298 marks FCs in somatic (Ruiz-Gómez et al., 2000) and visceral mesoderm (Klapper et al., 2002; SanMartin et al., 2001). All panels are confocal images with C and F showing the red and green channels merged. dei transcripts in somatic (arrowhead, A,C) and visceral (arrow, C) mesoderm cells were detected in non ß-Gal-expressing FCs (red nuclei; B,C), which were therefore putative FCMs. gol transcripts in somatic (bent arrow, D,F) and visceral (arrowhead, D,F) mesoderm did not overlap with ß-Gal-expressing FCs (E,F). A similar situation was detected in visceral mesoderm: ß-Gal-expressing nuclei (arrow, E) were not observed in gol-expressing cells (arrowhead, F). (G-I) Confocal images of wild-type embryos at mid-stage 12 immunostained for Kr (G) and Ase (H); the corresponding merged image is shown in I. Ase was detected transiently in Kr-positive FCs in close proximity to Kr, suggesting that Ase was expressed in FCs immediately after the progenitor cell had divided (arrows). (J-O) Confocal images of rP298 embryos at late stage 12, showing immunoreactivity to Ubx (J), Trn (M) and ß-Gal (K,N). The corresponding merged images are shown in L,O. The yellow color (bent arrow, L) indicates expression of Ubx in FC. Trn was expressed in a punctate pattern in close proximity to nuclei expressing the rP298 reporter (arrowheads, O), suggesting that FCs express Trn. (P,R) nidogen transcripts detected by fluorescent in situ hybridization in a late stage 11 embryo (green, arrows in P) tightly surrounded ß-Gal expressing FCs (arrows, Q), as shown in the merged image (R).