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First published online 24 October 2006
doi: 10.1242/jcs.03279

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Methylation of histone H3 at Lys4 differs between paternal and maternal chromosomes in Sciara ocellaris germline development

Departamento de Biología Celular y del Desarrollo, Centro de Investigaciones Biológicas, CSIC, 28040 Madrid, Spain

View larger version (22K): [in a new window]   Fig. 1. Scheme summarizing the most relevant chromosomal events and the distribution of acetylated histones H3 (H3K9ac and H3K14ac) and H4 (H4K8ac and H4K12ac) in early germline nuclei at the resting stage and male meiosis in S. ocellaris. Maternally derived chromosomes are in red and paternally derived chromosomes in blue. Early germ nuclei contain three maternal and three paternal autosomes (A) plus one maternal and two paternal X chromosomes (X). Chromosomes containing highly acetylated histones H3 and H4 are marked in yellow, whereas chromosomes containing under-acetylated histones H3 and H4 are unmarked. E, chromosomal elimination events. The figure has been modified from Goday and Ruiz (Goday and Ruiz, 2002).

View larger version (41K): [in a new window]   Fig. 2. Distribution of histone H3K4me2 in S. ocellaris embryonic germ cell nuclei at the resting stage. (A-C) Chromosome staining with DAPI, (A'-C') indirect immunolabelling with anti-H3K4me2 antibody and (A"-C") superimposed images with antibody staining in red. (A) S. ocellaris germ nuclei before, (B) during and (C) after the occurrence of Xp-chromosome elimination. In all cases only four chromosomes of the complement are strongly labelled (A"-C"). (B-B") A S. ocellaris germ nucleus undergoing elimination; the Xp chromosome is expelled from the nucleus (asterisk in B) and is not stained (asterisks in B' and B"). Bar, 10 µm.

View larger version (35K): [in a new window]   Fig. 3. Distribution of histone H3K4me3 in S. ocellaris embryonic germ nuclei at the resting stage. (A-C) Chromosome DAPI staining, (A'-C') indirect immunolabelling with anti-H3K4me3 antibody and (A"-C") superimposed images where antibody staining is in red. (A-C) S. ocellaris germ nuclei (A) prior to, (B) during and (C) after the occurrence of Xp chromosome elimination. In all cases only four chromosomes of the complement are highly labelled (A'-C' and A"-C"). (A-A") Somatic nuclei exhibit some H3K4me3 labelling at discrete nuclear sites (arrow). (B-B") A S. ocellaris germ nucleus undergoing elimination where it is seen that the Xp chromosome expelled from the nucleus (asterisk in B) is not stained (asterisks in B' and B"). Somatic chromosomes at mitosis are highly H3K4me3 labelled (arrow in B-B"). Bar, 10 µm.

View larger version (39K): [in a new window]   Fig. 4. Distribution of histone H3K4me2 in germ cells of S. ocellaris male larvae at 1 day (1d), 3 days (3d), 5 days (5d) and 10 days (10d) from hatching. (A-D) Chromatin DAPI staining, (A'-D') indirect immunolabelling with anti-H3K4me2 antibody and (A"-D") superimposed images where antibody staining is in red. (A,B) Maternal chromosomes appear condensed (long arrows) whereas paternal chromosomes, less stained with DAPI, initiate decondensation (short arrows and arrowhead); H3K4me2 staining is restricted to the maternal chromosome set. Arrowhead in (B) denotes the paternal chromosome corresponding to the longest autosomes pair. (C) Maternal chromosomes remain condensed (long arrows) whereas paternal ones appear totally decondensed (short arrows); (C',C") H3K4me2 labelling is restricted to the chromatin corresponding to the maternal chromosome set. (D) All chromosomes appear equally condensed and exhibit similar levels and distribution of H3K4me2 labelling (D',D"). Bar, 10 µm.

View larger version (40K): [in a new window]   Fig. 5. Distribution of histone H3K4me2 in S. ocellaris embryos at the early syncitial stage. (A-C) chromatin DAPI staining, (A'-C') indirect immunolabelling with anti-H3K4me2 antibody and (B",C") superimposed images where antibody staining is in red. (A) A young whole embryo showing that the germ nuclei at the polar region of the embryo (arrows in A) are devoid of labelling (arrows in A'). (B-B") Enlarged images of the same embryo where it is seen that the antibody labelling is restricted to the somatic mitotic chromosomes whereas germ nuclei at interphase (arrowheads in B) remain unstained. (C-C") Partial view of the polar region of an embryo showing dividing germ cells (arrows in C) lacking antibody staining in contrast with somatic nuclei at interphase that display H3K4me2 staining. Bars, 10 µm.

View larger version (38K): [in a new window]   Fig. 6. Distribution of methylated histones in S. ocellaris spermatocytes undergoing paternal chromosomes elimination at first meiotic division (A-C) and in spermatids (D,E). (A-C) DAPI-staining of anaphase-like stage figures. m, maternal group of chromosomes; p, paternal chromosomes; * indicates position of the single spindle pole. (D,E) DAPI staining of (D) early and (E) late spermatids. (A'-E') Indirect immunolabelling with antibodies against histones H3 and H4 as indicated, and (A"-E") superimposed images where the antibody staining is in red. At first meiosis (A"-C"), the four maternal chromosomes that face the pole are devoid of staining whereas the opposed paternal group of chromosomes, closely grouped, show antibody labelling. During spermiogenesis (D,E), antibody labelling is observed in the eliminated paternal chromatin. Bars, 10 µm.

View larger version (43K): [in a new window]   Fig. 7. Distribution of RNA pol II in S. ocellaris embryonic germ nuclei at the resting stage (A,C) and male larva germ nucleus at 5 days from hatching (B). (A-C) Chromosome DAPI staining. (A'-C') Indirect immunolabelling with antibody against RNA pol II (red in C') and double staining with antibody against H3K4me2 (green in C"). (A",B") superimposed images where RNA pol II staining is in red and (C"') merged image where RNA pol II is in red and H3K4me2 in green. (A-A") Prior to the occurrence of Xp chromosome elimination (nine chromosomes), RNA pol II is observed in four chromosomes of the complement. (B-B") RNA pol II staining is restricted to the condensed maternal chromosomes (arrows in B) whereas decondensed paternal chromosomes are devoid of staining. (C-C"') A germ cell nucleus after the occurrence of Xp chromosome elimination (8 chromosomes), showing that labelling of RNA pol II and H3K4me2 is present in the same chromosomes of the complement. Bar, 10 µm.

View larger version (28K): [in a new window]   Fig. 8. Distribution of RNA pol II CTD phosphorylated on Ser2 in S. ocellaris somatic and germline nuclei of an embryo at 72 hours of development (A) and in male premeiotic nuclei and spermatocytes at first meiotic division undergoing paternal chromosomes elimination. m, maternal group of chromosomes; p, paternal chromosomes (B). (A,B) Chromatin DAPI staining, (A',B') indirect immunolabelling with anti-pSer2 CTD RNA pol II antibody and (A",B") superimposed images where antibody staining is in red. (A) A somatic nucleus (arrow) staining positive whereas a germ nucleus at the resting stage is devoid of staining. (B) Partial view of a testis cyst showing that premeiotic germ nuclei (arrows) are clearly stained whereas no staining is detected during meiosis I on both maternal and paternal chromosomes. Bars, 10 µm.

View larger version (23K): [in a new window]   Fig. 9. Scheme summarizing the distribution of acetylated and methylated histones in early germline nuclei at the resting stage and male meiosis in S. ocellaris. Maternally derived chromosomes are red and paternally derived chromosomes blue. E, chromosomal elimination events.