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Fig. 4.
PRDM12 MO represses the trigeminal placode markers via trimethylation of histone H3K9 on the Foxd3 promoter. (A–E) WISH analysis using the indicated probes. Anterior views of mid-neurula embryos injected with Control MO (20 ng/embryo) or PRDM12 MO (20 ng/embryo) and lacZ mRNA (0.1 ng/embryo) with or without misPRDM12 mRNA (1 ng/embryo) into one side of each blastomere at the four-cell stage. (A–C) Expression of the presumptive trigeminal placode markers (Ath3, EBF3, and Islet1) decreased in the PRDM12 MO-injected embryo, whereas the reduction of these markers was rescued by injection of morpholino-resistant PRDM12 (misPRDM12). In contrast, (D and E) expression of the early pan-placodal marker (Six1) and the neural marker (Sox2) were unchanged. (F) Quantitation of the phenotype percentage in the embryos injected with MOs and mRNA. The injected embryos separated into two phenotypes (no effect or severe defect). Quantification of PRDM12 MO- and misPRDM12 mRNA-injected embryos in Ath3 (n=24), EBF3 (n=28), Islet1 (n=25) compared with PRDM12 MO-injected embryos at neurula stage. p<0.0005 by the chi-square test. (G and H) Control MO or PRDM12 MO and lacZ mRNA (0.1 ng/embryo) were sequentially injected into one side of the blastomere from the four-cell to the eight-cell stage. The Slug expression region was expanded anteriorly on the injected side (P, no effect in 100%, n=27). Scale bars represent 0.5 mm. (I–L) Reverse transcription–quantitative polymerase chain reaction (RT-qPCR) analysis of Ath3 (I), Foxd3 (J), Slug (K), and Sox2 (L) expression was performed on late neurula-stage embryos injected with PRDM12 MO into the animal blastomeres at the four-cell stage. (M and N) ChIP-qPCR analyses of the Foxd3 and Six1 promoter sites were performed using an anti-H3K9me3 antibody. Embryos injected with Control MO or PRDM12 MO were cultured until stage 15, and the pre-placodal ectoderm region was dissected. Error bars indicate ±S.E. (n=3). |
