Fig. 5

ASF1 is required for HIRA-dependent assembly of paternal chromatin. a Confocal images of eggs at pronuclear apposition stained for acetylated histone H4 (H4act) and DNA. Female genotypes are indicated. In asf1 KD eggs, limited de novo histone deposition is observed in the male nucleus (arrowhead). In Hira KD or yem KD eggs, histones are barely detectable in the male nucleus. Scale bar: 10 μm. b Metaphase of the first zygotic mitosis. In control embryos (left), paternal chromosomes (arrowhead) stain brightly with anti-H4act antibodies and appear greenish in the merged image. In asf1 KD embryos, the male nucleus (arrowhead) fails to condense into mitotic chromosomes and stains weakly for H4act. Scale bar: 10 μm. c Anaphase of the first zygotic mitosis in control (left) and asf1 KD embryos (right). In the control, paternal chromosomes are still distinguished by the anti-H4act staining and appear yellowish. In asf1 KD embryos, paternal chromatin (arrowhead) lags behind migrating maternal chromosomes. Scale bar: 10 μm. d Second mitotic division. In asf1 KD embryos, the male nucleus (arrowhead) typically appears partially decondensed and stains weakly with anti-H4act antibodies (orange color). In contrast, the male nucleus in yem KD embryos remains condensed and free of H4act (it thus appears red). Scale bar: 10 μm. e HIRA localizes and persists in the male nucleus in the absence of ASF1. Left: Anti-HIRA antibodies normally stain the male nucleus in asf1 KD egg during female meiosis II. Right: HIRA remains abundant in the male nucleus in asf1 KD eggs during pronuclear apposition and the first zygotic mitosis. HIRA was detected in all eggs or cycle 1 embryos analyzed (100%; n = 15). Color panels show merged stainings. Scale bar: 10 μm