Control of meiotic chromosomal bouquet and germ cell morphogenesis by the zygotene cilium

A hallmark of meiosis is chromosomal pairing, which requires telomere tethering and rotation on the nuclear envelope through microtubules, driving chromosome homology searches. Telomere pulling toward the centrosome forms the “zygotene chromosomal bouquet. ” Here, we identified the “zygotene cilium” in oocytes. This cilium provides a cable system for the bouquet machinery and extends throughout the germline cyst. Using zebrafish mutants and live manipulations, we demonstrate that the cilium anchors the centrosome to counterbalance telomere pulling. The cilium is essential for bouquet and synaptonemal complex formation, oogenesis, ovarian development, and fertility. Thus, a cilium represents a conserved player in zebrafish and mouse meiosis, which sheds light on reproductive aspects in ciliopathies and suggests that cilia can control chromosomal dynamics. Description Making a chromosomal bouquet During meiosis, chromosomal pairing requires microtubule-dependent telomere tethering and rotation on the nuclear envelope, a process that also drives chromosome homology searches. Formation of the structure called the “zygotene chromosomal bouquet” involves telomere pulling toward the centrosome. Mytlis et al. describe the so-called “zygotene cilium, ” which helps to ensure chromosomal bouquet formation and germ cell morphogenesis in zebrafish and mouse oocytes. The cilium anchors the centrosome, allowing it to resist telomere pulling, and is essential for bouquet and synaptonemal complex formation and successful oogenesis. —SMH and BAP The zygotene cilium is required in zebrafish and mouse meiosis for chromosomal pairing, oocyte development, and fertility. INTRODUCTION Meiosis is a cellular program that is essential for producing haploid gametes and is conserved in sexually reproducing organisms. A hallmark of meiosis is chromosomal pairing through synaptonemal complexes and meiotic cohesins, which is required for the execution of homologous recombination. However, nuclear events of meiosis occur in the cellular context of a differentiating gamete, where chromosomal pairing depends on cytoplasmic counterparts. In pairing, telomeres perform a unique function: They slide on perinuclear microtubules, shuffling chromosomes and mechanically driving their homology searches. Telomeres tether to Sun/Kash complexes on the nuclear envelope (NE), which associate with perinuclear microtubules (MTs) through dynein. This facilitates telomere rotations around the NE, which in turn shuffle chromosomes, driving their search for homologs. The perinuclear MTs at these stages emanate from the centrosome MT organizing center. Ultimately, rotating telomeres are pulled toward the centrosome and cluster on this side of the NE while looping their chromosomes to the other side, a configuration called the zygotene chromosomal bouquet. These telomere dynamics are essential for pairing and fertility. The bouquet, discovered in 1900, is universally conserved and was proposed as a hub for chromosomal pairing. Nevertheless, how cytoplasmic counterparts of bouquet formation are mechanically regulated has remained enigmatic. RATIONALE Here, we report the “zygotene cilium,” a previously unrecognized cilium, in oocytes. Using quantitative three-dimensional confocal image analysis, serial block-face scanning electron microscopy, and live microscopy, we characterized in zebrafish the intracellular and intercellular organization of the zygotene cilium, as well as its developmental stage–specific formation during oogenesis. We show that the zygotene cilium specifically connects to the bouquet centrosome, constituting a cable system of the cytoplasmic bouquet machinery. Furthermore, zygotene cilia extend throughout the germline cyst, a conserved germ cell organization. We reasoned that the zygotene cilium could regulate bouquet formation and germline cyst cellular organization. RESULTS We analyzed multiple viable loss-of-function mutations in zebrafish ciliary genes that in humans cause ciliopathies, genetic disorders that are caused by ciliary defects, including cep290, kif7, and cc2d2a. Using these mutants, as well as laser-induced excision of the zygotene cilium and live time-lapse imaging, we demonstrate that the organelle is essential for chromosomal bouquet and synaptonemal complex formation, germline cyst morphogenesis, ovarian development, and fertility. Mechanistically, we provide evidence that the cilium functions by anchoring the bouquet centrosome to counterbalance telomere rotation and pulling. We further show that the bouquet configuration is required for proper synaptonemal complex formation. Loss of the zygotene cilium resulted in bouquet and germline cyst perturbations that in turn induced oocyte apoptosis. Crossing the ciliary mutants to tp53 mutant fish demonstrated that they induce P53-dependent apoptosis, likely by activating a meiotic checkpoint. Ultimately, loss of the zygotene cilium leads to ovarian dysgenesis and deficient fertility in adult females. We also show that the zygotene cilium is conserved in both male and female meiosis in zebrafish, as well as in mammals. CONCLUSION Our work uncovers the concept of the cilium as a critical player in meiosis and provides evidence that the bouquet is functionally required for synaptonemal complex formation. Our findings further shed new light on reproduction phenotypes in ciliopathies and propose a cellular paradigm that cilia can control chromosomal dynamics. Zygotene bouquet–stage oocyte. Shown is an oocyte at the zygotene bouquet stage, where chromosomes (green) are tethered on the NE through their telomeres, which cluster apposing the centrosome (yellow circle), with their free looping ends facing the other side. The bouquet cytoplasmic machinery is shown as a cable system composed of the perinuclear MTs (blue cables) that are organized from the centrosome, which connects to the zygotene cilium (purple cable). CREDIT: ILLUSTRATION BY ELLA MARU STUDIO