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. 2011 Aug 18;7(8):e1002231. doi: 10.1371/journal.pgen.1002231

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Nabeshima et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) Mid-pachytene nuclei in wild type show one major focus of HIM-8 (white) staining corresponding to the paired X PCs, as well additional fainter HIM-8 speckles elsewhere in the nucleus. Images on the left are projections of deconvolved optical sections spanning the full nuclear depth. Scale bar, 2 µm. Images on the right are 3-D surface renderings comprising half the depths of nuclei 1–4. These images show HIM-8 speckles localizing to discrete sites along the lengths of the X chromosome and autosomes, with paths of the chromosomes visualized by immunostaining of chromosome axis marker HTP-3 (pink). While there is generally at least one speckle adjacent to each chromosome axis, the most prominent HIM-8 speckles are found along the axis of the X chromosome and at one end of an autosome. Nuclei 1–3 show the typical localization of two prominent HIM-8 speckles (white arrowheads) along the axis of the X chromosome (white dotted trace); 3 or more prominent speckles are sometimes detected on the X. Nucleus 4 shows a cluster of HIM-8 speckles (grey triangles) characteristic of one end of an autosomal axis (grey dotted trace). (B) Analysis of HIM-8 localization in the him-8(me4) mutant. Mid-pachytene nuclei show two major foci of HIM-8 marking the unpaired X PCs; in this field, the HIM-8 foci are well separated in the Z dimension in all nuclei. Scale bar, 2 µm. The indicated nucleus is depicted in 3-D as in (A). HIM-8 speckles localize adjacent to the axes of the unpaired X chromosomes, which are highlighted by immunostaining of H3K9me2 (orange), which concentrates on unsynapsed X chromosomes [48]. (C) Analysis of HIM-8 localization in the meDf2 homozygote. Mid-pachytene nuclei show one major focus of HIM-8 staining corresponding to the paired mnDp66 chromosomes, which contain a copy of the X-PC region fused to chromosome I. The meDf2 X chromosomes, which lack the X-PC and thus do not have an associated major HIM-8 focus, are unpaired. Scale bar, 2 µm. The indicated nucleus is depicted in 3-D as in (A). (Note: the major HIM-8 focus on the mnDp66 chromosome is in the half of the nucleus not depicted in the 3-D rendering.) The axes of the unpaired X chromosomes are identified by lack of associated immunostaining for SC central region protein SYP-1 (green). Although these X chromosomes lack PCs and the associated major HIM-8 foci, HIM-8 speckles still localize along the unpaired X chromosome axes. In addition, the paths of the X chromosome axes in this nucleus are coiled (left) or folded (right), although they appear comparable in length to those seen in the him-8(me4) mutant. This type of organization is common for X chromosomes in meDf2 homozygotes and may account for the fact that meDf2 X chromosomes have relatively compact ovoid territories despite exhibiting increases in the number of discernable painted chromosome segments.