The Hybrid Incompatibility Genes Lhr and Hmr Are Required for Sister Chromatid Detachment During Anaphase but Not for Centromere Function

Abstract

Crosses between Drosophila melanogaster females and Drosophila simulans males produce hybrid sons that die at the larval stage. This hybrid lethality is suppressed by loss-of-function mutations in the D. melanogaster Hybrid male rescue (Hmr) or in the D. simulans Lethal hybrid rescue (Lhr) genes. Previous studies have shown that Hmr and Lhr interact with heterochromatin proteins and suppress expression of transposable elements within D. melanogaster It also has been proposed that Hmr and Lhr function at the centromere. We examined mitotic divisions in larval brains from Hmr and Lhr single mutants and Hmr; Lhr double mutants in D. melanogaster In none of the mutants did we observe defects in metaphase chromosome alignment or hyperploid cells, which are hallmarks of centromere or kinetochore dysfunction. In addition, we found that Hmr-HA and Lhr-HA do not colocalize with centromeres either during interphase or mitotic division. However, all mutants displayed anaphase bridges and chromosome aberrations resulting from the breakage of these bridges, predominantly at the euchromatin-heterochromatin junction. The few dividing cells present in hybrid males showed fuzzy and irregularly condensed chromosomes with unresolved sister chromatids. Despite this defect in condensation, chromosomes in hybrids managed to align on the metaphase plate and undergo anaphase. We conclude that there is no evidence for a centromeric function of Hmr and Lhr within D. melanogaster nor for a centromere defect causing hybrid lethality. Instead, we find that Hmr and Lhr are required in D. melanogaster for detachment of sister chromatids during anaphase.

Keywords: Drosophila; Hmr; Lhr; anaphase; chromosome aberrations; interspecific hybrids; sister chromatid separation.

Figure 1

Lhr^KO mutants exhibit a minor difference in the most distal fluorescent bands of the 3R heterochromatin (arrows) compared to wild type Oregon R flies. The cells shown are male (left) and female (right) late prophases from F1 third instar larvae generated by crosses between Oregon R females and Lhr^KO mutant males. Bar, 5 μm.

Figure 2

Examples of CABs observed in colchicine-treated metaphases from Lhr and Hmr mutants. (A) Male control metaphase; the third chromosomes are easily distinguished from the second chromosomes by the higher fluorescence of their pericentric heterochromatin. (B) Chromatid deletion (arrows). (C) Second chromosome ISOB in centric heterochromatin, probably at the euchromatin–heterochromatin (eu-het) junction (arrows). (D) Third chromosome ISOB at the eu-het junction (arrows). (E) Incomplete ISOB; a second chromosome (arrow) is broken within the heterochromatin or at the eu-het junction, but lacks the corresponding acentric fragment. (F) Incomplete ISOB; a third chromosome broken at the eu-het junction (arrow) lacking the corresponding acentric fragment. (G–I) Metaphases with complete chromosome complements and an additional euchromatic fragment (G; arrow), an additional autosomal arm broken in the heterochromatin (H, arrow), or an additional Y fragment (I, arrow). (J) TF involving single chromatids of the X and the second chromosome (arrow). (K) Double TF involving both sister chromatids of the two third chromosomes (arrow). (L) TF leading to a ring Y chromosome (arrow). Bar, 5 μm.

Figure 3

A model for the formation of ISOBs. The primary event leading to an incomplete ISOB is the formation of a chromatin bridge generated by a transient sister chromatid association during anaphase, represented by an ellipse. This association can be generated by failure to resolve aberrant sister chromatid cohesion or by tangles, such as those caused by mutations in the condensin genes (see Discussion for details on the possible origins of anaphase bridges). The anaphase drawing refers to the mitosis in which the anaphase bridge forms (M1), and depicts a single chromosome composed of a pair of sister chromatids; centromeres are represented by circles. G1 and metaphase 2 (M2) refer to the subsequent cell cycle, and show both homologous chromosomes, one of which segregated normally during the previous anaphase. The metaphase configurations are those observed in colchicine-treated cells of mutants that are shown in Figure 2. Stretching of the bridge during anaphase would result in the resolution of the sister chromatid association, and a rupture at the eu-het junction. This situation could have three possible outcomes: (i) the acentric fragment (F) segregates with the chromatid to which it was originally attached, giving rise to a telophase nucleus containing both homologous chromosomes plus an acentric element (at cell pole 1, P1), and to another nucleus containing only the centric element and no corresponding acentric fragment (at cell pole 2, P2). (ii) The acentric fragment segregates with its corresponding centric element producing a normal nucleus at P1 and a nucleus bearing a complete ISOB at P2. (iii) The acentric fragment is lost, leading to a normal telophase nucleus at P1 and to a nucleus containing a broken chromosome without the corresponding fragment at P2. This model describes the possible outcomes of a rupture at the eu-het junction but could also be extended to ruptures in the euchromatin.

Figure 4

Examples of aberrant anaphases observed in Lhr and Hmr mutants. (A) Wild-type control anaphase. (B) Anaphase with bridge. (C and D) Anaphases with a broken bridge and a fragment. (E) Anaphase with a fragment. (F) Anaphase with fragments (arrows). Bar, 5μm.

Figure 5

Examples of well-aligned metaphases observed in brains from wild type (wt, Oregon R) and Hmr; Lhr double homozygous mutants. NB, neuroblast; GMC, ganglion mother cell. Bar, 10 μm.

Figure 6

Localization of Hmr in male brain cells. Note the dynamic behavior of the bulk of the protein; in interphase, it is nuclear and does not colocalize with the Cid signals (A, A’, A’’, A’’’), it dissociates from the chromosomes during prophase (B, B’, B’’, B’’’) and metaphase (C, C’, C’’, C’’’, and D, D’, D’’, D’’’) and reassociates with the chromosomes during late anaphase (E, E’, E’’, E’’’). A fraction of metaphases showed Hmr accumulations on the 2R heterochromatin (arrowhead) (D). See text for a detailed description of the dynamic localization of Hmr. Bar, 2.5 μm.

Figure 7

Localization of Lhr in male brain cells. Lhr does not colocalize with the Cid signals in interphase nuclei (A, A’, A’’, A’’’). Note that Lhr exhibits a dynamic behavior similar to that of Hmr (shown in Figure 6). In very early prophase, in which only heterochromatin is condensed, Lhr is still associated with the heterochromatin (B, B’, B’’, B’’’), but dissociates from the chromosomes in both late prophase (C, C’, C’’, C’’’) and metaphase (D, D’, D’’, D’’’) cells. Also note that, in the early anaphase shown (E, E’, E’’, E’’’), Lhr localizes in regions distal to the Cid signals that probably correspond to the spindle poles; in the late anaphase (F, F’, F’’, F’’’), Lhr is instead localized proximally to the Cid signals and is incorporated into the heterochromatin. See text for a detailed description of the dynamic localization of Lhr. Bar, 2.5 μm.

Figure 8

Hybrid males exhibit defects in chromosome condensation and integrity. Larval brains were fixed with formaldehyde, but not treated with colchicine or hypotonically swollen (see Materials and Methods), and then stained for DNA (DAPI) and the mitotic phospho histone H3 (PH3). (A) Male prometaphases/metaphases from wild type D. melanogaster and D. simulans; note the differences in the X chromosome heterochromatin staining and in the size of the Y chromosome. (B) Prophase-like figures from wild-type D. melanogaster and hybrid males. (C) Prometaphases/metaphases from hybrid males with poorly condensed and broken (arrows) chromosomes. Note that two of the cells shown (the one on the left and the central one) exhibit fuzzy chromosomes with unresolved sister chromatids. (D) Anaphases observed in wild type D. melanogaster and in hybrid males; note that the anaphase chromosomes in the hybrids are more condensed than in the control; one of them also exhibits a broken bridge (arrow). Bar, 5 μm.

Figure 9

Hybrid males form a mitotic spindle and do not exhibit defects in chromosome alignment at metaphase. A prometaphase and two well-aligned metaphases observed in hybrid males. For control metaphases see Figure 5; NB, neuroblast; GMC, ganglion mother cell. Bar, 10 μm.