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Figure 6. xSuv4-20h double morphants fail to silence Oct-25 expression in deep-layer ectoderm due to reduced H4K20me3 enrichment at Oct-25 promoter.
RNA in situ hybridization analysis for Oct-25, Oct-60 and Oct-91 in embryos (A) and animal caps (B) for ctrl-morphants or xSuv4-20h double-morphant embryos, injected unilaterally at two-cell stage and fixed at midneurula stage (NF15). Injected sides were defined by coinjected Alexa-fluorescence prior to in situ hybridisation. (A) Dorsal views of stained embryos with anterior to the left. For Oct-25 Vibratome cross-sections, ctrl-MO and double-morphant embryos are shown. (B) Comparative expression analysis for Oct-25, Oct-60 and Oct-91 in animal caps from bilaterally injected embryos, fixed at midneurula stage (NF15). (C) qRT-PCR profiles for Oct-25 and Oct-91 in ctrl-MO and xSuv4-20h double-morphant embryos. Data represent normalized ratios of mRNA levels as means of four independent experiments, error bars indicate SEM. (D, E) Chromatin immunoprecipitation (ChIP) analysis on Oct-25, GAPDH, thra, thibz genes and major satellite repeat region 3 (MSAT3). (D) H4K20me3 levels on the indicated genes in normal embryos, and (E) in uninjected versus double-morphant embryos. Fold enrichment was calculated as the ratio between H4K20me3 precipitated material over negative control (No antibody sample). Data represent mean values of three to five independent experiments, error bars indicate SEM.
doi:10.1371/journal.pgen.1003188.g006 |
