Figure 7

Expression of some selected genes downregulated by high-dose X-ray treatment and neoblast marker DjMCM2 by double in situ hybridization and real-time RT-PCR in neoblast-enriched cell fractions. (a) Expression of the neoblast marker DjMCM2 in an intact untreated planarian, as visualized by whole mount in situ hybridization. Scale bar: 500 μm. (b) Expression of the neoblast marker DjMCM2 in a transverse section of an intact untreated planarian, as visualized by section in situ hybridization. Arrowheads indicate the clustered neoblasts along the midline and lateral lines; arrows indicate the dispersed neoblasts. Scale bar: 50 μm. (c-g) Double in situ hybridization. Panels c-e depict the expression of DjMCM2 (red fluorescence) and TAF-Ibeta1 (NBT/BCIP blue precipitation) in the same planarian body region. Yellow arrowheads indicate some lateral patches where the co-localization of the two transcripts is particularly evident. Scale bars: 260 μm in panels c and d, and 100 μm in panel e. Panel f depicts the expression of DjMCM2 (red fluorescence) and H2Az (NBT/BCIP blue precipitation) in the same planarian body region. Cyan arrowheads indicate some lateral patches where the two transcripts do not co-localize. Yellow arrowheads indicate some regions were the co-localization is particularly evident. Scale bar: 260 μm. Panel g depicts the expression of DjMCM2 (red fluorescence) and Sam68 (NBT/BCIP blue precipitation) in the same planarian body region. Yellow arrowheads indicate some lateral patches where the two transcripts co-localize. Scarce co-localization can be observed along the midline. Scale bar: 220 μm. (h) Real-time polymerase chain reaction (PCR) analysis of the expression of some selected genes that were downregulated as a consequence of high-dose X-ray treatment in neoblast-enriched cell fractions. RNA was extracted from cell fractions obtained by filtration through nylon meshes of 50 μm (light gray), 20 μm (gray), and 8 μm (dark gray) pore size. Expression levels are indicated in relative folds, assuming a value of 1 for 50 μm nylon meshes. Values are expressed as mean ± standard deviation of three independent samples conducted in duplicate. The expression values of the 8 μm nylon mesh samples were compared with those of 50 μm nylon mesh samples using the unpaired t-test. P < 0.05 was considered statistically significant. *P < 0.05.