Chromatin immunoprecipitation (ChIP) assay and map of genomic regions examined in this study. (A) Post-translational histone modifications in MBT⁺ stage embryos, ZF4 cells and MBT⁺ embryo-derived cultured cells were examined by ChIP-qPCR. (B) Genomic regions examined by ChIP on indicated genes. Numbers indicate the position of amplicons relative to the TSS (+1).

Expression pattern of selected genes in zebrafish embryos. (A) Data extracted from Agilent microarray analyses of gene expression in unfertilized eggs and MBT embryos. Data was generated from 3 biological replicates for both stages, with 3 and 4 arrays for unfertilized egg and MBT stages, respectively. Signals were quantile-normalized, multiple probes for each gene were aggregated using the median, and mean±SD calculated based on log₂-transformed values. Negative controls are the mean of the replicates of the median of 150 different negative control probes scattered throughout the array. (B) RT-PCR analysis of expression of indicated genes in MBT⁺ embryos; -RT, PCR without reverse transcription.

Embryonic genes are repressed and trimethylated on H3K9 and H3K27 in somatic tissue. (A) RT-PCR analysis of expression of indicated genes in muscle biopsies from three different fish; -RT, PCR without reverse transcription. (B,C) ChIP analysis of indicated histone modifications on the promoters of indicated genes. In (C), insets highlight H3K9ac and H4ac occupancy on mlc2 and bactin2.

Histone modification and gene expression patterns in cultured embryo- derived cells. (A) Cells isolated from MBT⁺ embryos were cultured for 24 h and histone PTMs examined as in Figure 3. (B) RT-PCR analysis of gene expression in embryo-derived cells cultured for 1, 2 or 5 days. -RT, PCR without reverse transcription on RNA isolated from Day 1 cultured cells.

Whole-mount in situ hybridization analysis of expression of indicated genes in MBT⁺ stage embryos. Enlargement of klf4 hybridization pattern (boxed area) shows mosaic expression between blastomeres. A negative control otx1b hybridization using a sense otx1b probe is also shown.

Histone modification patterns on promoters of developmentally-regulated genes in the ZF4 cell line. (A) ChIP-qPCR analysis of histone PTMs on indicated genomic sites (relative to the TSS). (B) RT-PCR analysis of expression of indicated genes; “-“ refers to a control PCR without reverse transcription.