Mutations in <i>mxc</i> Tumor-Suppressor Gene Induce Chromosome Instability in <i>Drosophila</i> Male Meiosis

Abstract

Drosophila Mxc protein is a component of the histone locus body (HLB), which is required for the expression of canonical histone genes, and severe mxc mutations generate tumors in larval hematopoietic tissues. A common characteristic of cancer cells is chromosomal instability (CIN), but whether mxc mutants exhibit this feature is unknown. Here, examination of post-meiotic spermatids created after male meiosis revealed that a fraction of the spermatids in hypomorphic mxc G46 mutants contained extra micronuclei or abnormally sized nuclei, corresponding to CIN. Moreover, we observed that the so-called lagging chromosomes retained between chromosomal masses separated toward spindle poles at telophase I. Time-lapse recordings show that micronuclei were generated from lagging chromosomes, and the abnormal chromosomes in mxc G46 mutants lacked centromeres. In normal spermatocyte nuclei, the HLB component FLASH colocalized with Mxc, whereas FLASH was dispersed in mxc G46 spermatocyte nuclei. Furthermore, we observed genetic interactions between Mxc and other HLB components in meiotic chromosome segregation, which suggests that inhibition of HLB formation is responsible for aberrant chromosome segregation in mxc G46. Quantitative real-time PCR revealed that canonical histone mRNA levels were decreased in mxc G46. Lastly, similar meiotic phenotypes appeared in the spermatids of histone H4 mutants and in the spermatids in testes depleted for chromosome-construction factors. Considering these genetic data, we propose that abnormal chromosome segregation leading to CIN development results from a loss of chromosome integrity caused by diminished canonical histone levels in mxc mutants.

Nuclear Export of Cyclin B Mediated by the Nup62 Complex Is Required for Meiotic Initiation in Drosophila Males

“…To judge whether two consecutive meiotic divisions were properly performed, we observed nuclei in post-meiotic spermatids under phase contrast microscopy, as previously described [36,37]. A pair of testes from pharate adults or newly eclosed adult flies (0-1 day old) were dissected to isolate spermatocyte cysts in Testis buffer (183 mM KCl, 47 mM NaCl, 10 mM EDTA, pH 6.8) and covered with 18 × 18 mm-coverslip (Matsunami, Osaka, Japan) to flatten the cysts.…”

“…A pair of testes from pharate adults or newly eclosed adult flies (0-1 day old) were dissected to isolate spermatocyte cysts in Testis buffer (183 mM KCl, 47 mM NaCl, 10 mM EDTA, pH 6.8) and covered with 18 × 18 mm-coverslip (Matsunami, Osaka, Japan) to flatten the cysts. For observation of nuclear organization in onion stage spermatids, thecysts of spermatids collected from adult testes in Testis buffer supplemented with DAPI were mildly flattened under a cover slip, and phase contrast micrographs and fluorescence images were successively acquired [36,37]. To observe fixed spermatid samples, we removed the coverslips after freezing the slides, and transferred them into 100% methanol for 5 min.…”

Loss of Histone Locus Bodies in the Mature Hemocytes of Larval Lymph Gland Result in Hyperplasia of the Tissue in mxc Mutants of Drosophila

“…We induced the ectopic expression of dsRNA against mxc mRNA in the three regions of LG, namely, CZ, MZ, and PSC, while using region-specific Gal4 drivers. The UAS-mxcRNAi HMS00444 used in this study can efficiently deplete endogenous mxc mRNA in the testes while using the testis-specific Gal4 driver [25]. In this study, we used the Hml-Gal4 driver to deplete the endogenous mRNA in CZ, the upd3-Gal4 driver to deplete it in MZ, and the col-Gal4 driver for depletion in PSC (for control see Figure 2a-c; for mxc depletion see Figure 2d-f).…”

“…Although the mechanism underlying the changes in histone gene expression has not yet been clarified, it can be interpreted, as follows; the alterations in the expression of the five histone mRNAs resulted in DNA replication stress and the accumulation of DNA damage [24,68]. However, another study showed that the mRNA levels of the five canonical histones were constantly reduced in the testes of the hypomorphic mutants for mxc [25]. In addition, DNA damage foci that were examined by immunostaining using anti-H2Av antibody were not evident in the mutant cells.…”

“…Conversely, the larval imaginal discs were underdeveloped in the mxc mutants. The development of germ line cells, including the progression of meiotic divisions, was also compromised in the mxc mutants [19,22,24,25].…”