Topoisomerase II is required for the proper separation of heterochromatic regions during Drosophila melanogaster female meiosis

Abstract

Heterochromatic homology ensures the segregation of achiasmate chromosomes during meiosis I in Drosophila melanogaster females, perhaps as a consequence of the heterochromatic threads that connect achiasmate homologs during prometaphase I. Here, we ask how these threads, and other possible heterochromatic entanglements, are resolved prior to anaphase I. We show that the knockdown of Topoisomerase II (Top2) by RNAi in the later stages of meiosis results in a specific defect in the separation of heterochromatic regions after spindle assembly. In Top2 RNAi-expressing oocytes, heterochromatic regions of both achiasmate and chiasmate chromosomes often failed to separate during prometaphase I and metaphase I. Heterochromatic regions were stretched into long, abnormal projections with centromeres localizing near the tips of the projections in some oocytes. Despite these anomalies, we observed bipolar spindles in most Top2 RNAi-expressing oocytes, although the obligately achiasmate 4th chromosomes exhibited a near complete failure to move toward the spindle poles during prometaphase I. Both achiasmate and chiasmate chromosomes displayed defects in biorientation. Given that euchromatic regions separate much earlier in prophase, no defects were expected or observed in the ability of euchromatic regions to separate during late prophase upon knockdown of Top2 at mid-prophase. Finally, embryos from Top2 RNAi-expressing females frequently failed to initiate mitotic divisions. These data suggest both that Topoisomerase II is involved in the resolution of heterochromatic DNA entanglements during meiosis I and that these entanglements must be resolved in order to complete meiosis.

Figure 1. Expression of a Top2 RNAi construct in the ovary leads to abnormal chromosomal projections during meiosis I.

DNA is labeled with DAPI (blue), chromatin is labeled with an antibody recognizing H3S10p (green), and the spindle is labeled with an antibody recognizing α-tubulin (red). (A) Top2 RNAi/+ control oocyte with achiasmate 4^th chromosomes that have moved towards the spindle poles. Arrowhead points to the H3S10p-labeled heterochromatic DNA thread emanating from the achiasmate 4^th chromosome. (B–D) Shown are examples of Top2 RNAi/matαGAL oocytes containing abnormal DNA projections that do not appear to connect chromosomes (arrowheads). Images are projections of partial Z-stacks. Scale bars are 5 microns.

Figure 2. Centromeres are present within and near the tips of the DNA projections in Top2 RNAi/matαGAL oocytes.

DNA is labeled with DAPI (blue), chromatin is labeled with an antibody recognizing H3S10p (green), and centromeres are labeled with an antibody recognizing CID (red). (A) Top2 RNAi/+ control oocyte with achiasmate 4^th chromosomes that have moved towards the spindle poles with all eight centromeres properly bioriented. (B–C) Examples of Top2 RNAi/matαGAL oocytes with abnormal DNA projections containing CID foci at or near the tips of the projections, indicating that the centromeres of multiple chromosomes have moved towards the spindle poles. Images are projections of partial Z-stacks. Scale bars are 5 microns.

Figure 3. Aberrant DNA projections are composed of heterochromatin.

DNA is labeled with DAPI (blue), heterochromatin is labeled with an antibody recognizing histone 3 trimethylated on lysine 9 (H3K9me3) (green), and the spindle is labeled with an antibody recognizing α-tubulin (red). (A) Top2 RNAi/+ control oocyte with achiasmate 4^th chromosomes that have moved towards the spindle poles. The 4^th chromosomes and the centromeric regions of the chromosomes are labeled with H3K9me3 and are oriented towards opposite spindle poles. (B–C) Top2 RNAi/matαGAL oocytes containing abnormal DNA projections that are labeled with the H3K9me3 antibody. Additionally, the H3K9me3 antibody localization in the chromosome mass of both oocytes is not oriented towards opposite spindle poles. Images are projections of partial Z-stacks. Scale bars are 5 microns.

Figure 4. Top2 RNAi/matαGAL oocytes show defects in chromosome biorientation and in the separation of heterochromatic regions of the X and 4^th chromosomes.

(A) Quantification of the phenotypes observed for the 359-bp and AATAT heterochromatic probes. Bioriented represents oocytes with two FISH probe foci oriented in opposite directions. Maloriented represents two FISH probe foci oriented in the same direction. Stretched represents FISH signal that is highly elongated rather than in discrete foci. Associated represents oocytes with only a single FISH probe focus. (B–E) DNA is labeled with DAPI (blue), a FISH probe targeting the 359-bp repeat predominantly on the X chromosome and a minor region on the 3^rd chromosome is in green, and a FISH probe to the AATAT heterochromatic repeat on the 4^th chromosome and a minor repeat on the X chromosome is shown in red. (B) Top2 RNAi/+ control oocyte with two foci for both probes oriented in opposite directions, indicating proper biorientation of the X and 4^th chromosomes. (C–E) Shown are examples of Top2 RNAi/matαGAL oocytes displaying aberrant separation of heterochromatic regions. (C) Probes indicate that both heterochromatic regions have failed to separate. (D) The X chromosomes have failed to separate. The 4^th chromosomes have separated and bioriented, but one 4^th chromosome appears to be highly stretched out and present in an abnormal DNA projection. (E) Both heterochromatic regions have failed to separate, but the AATAT repeat and, to a small degree, the 359-bp region have stretched into the abnormal DNA projection. For the 359-bp probe 96.3% (79/82) of Top2 RNAi/matαGAL oocytes displayed a complete failure of the probe to separate. In two (2.4%) Top2 RNAi/matαGAL oocytes the 359-bp probe appeared stretched out and one oocyte (1.2%) had 2 maloriented foci. Images are projections of partial Z-stacks. Scale bars are 5 microns.

Figure 5. Top2 RNAi/matαGAL oocytes show defects in chromosome biorientation and in the separation of heterochromatic regions of the 2^nd and 3^rd chromosomes.

(A) Quantification of the phenotypes observed for the AACAC and Dodeca heterochromatic probes. Bioriented represents oocytes with two FISH probe foci oriented in opposite directions. Maloriented represents two FISH probe foci oriented in the same direction. Stretched represents FISH signal that is highly elongated rather than discreet foci. Associated represents oocytes with only a single FISH probe focus. (B–E) DNA is labeled with DAPI (blue), a FISH probe targeting the AACAC repeat on the right arm of the 2^nd chromosome is in green, and a FISH probe to the Dodeca heterochromatic repeat on the right arm of the 3^rd chromosome is shown in red. (B) Top2 RNAi/+ control oocyte with two foci for both probes oriented in opposite directions, indicating proper biorientation of the 2^nd and 3^rd chromosomes. (C–E) Examples of Top2 RNAi/matαGAL oocytes displaying aberrant separation of heterochromatic regions. (C) Probes indicate that both heterochromatic regions have failed to separate. (D) The AACAC heterochromatic region of the 2^nd chromosomes has failed to separate. The Dodeca heterochromatic region of the 3^rd chromosome has separated but the heterochromatic region is highly stretched out and present in the abnormal DNA projections. (E) Both heterochromatic regions appear abnormal and the 2^nd and 3^rd chromosomes are improperly oriented so as to segregate away from each other. Images are projections of partial Z-stacks. Scale bars are 5 microns.

Figure 6. Embryos from Top2 RNAi/matαGAL mothers appear to be arrested in meiosis I.

DNA is labeled with DAPI (blue) and spindles are labeled with an antibody recognizing α-tubulin (yellow). (A and B) Top2 RNAi/+ embryos displaying normal development. Boxed regions are shown magnified below each image. (C and D) Embryos from Top2 RNAi/matαGAL mothers with two nuclei indicating proper fertilization but an arrest in meiosis I. 17/20 embryos from Top2 RNAi/matαGAL mothers contained only 2 nuclei. Of the remaining 3 embryos, one contained only a single nucleus and 2 appeared to undergo an aberrant division. Arrowheads indicate regions magnified below both images. Images are projections of partial Z-stacks. Scale bars are 20 microns.