FIGURE SUMMARY
Title

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

Authors
Mytlis, A., Kumar, V., Qiu, T., Deis, R., Hart, N., Levy, K., Masek, M., Shawahny, A., Ahmad, A., Eitan, H., Nather, F., Adar-Levor, S., Birnbaum, R.Y., Elia, N., Bachmann-Gagescu, R., Roy, S., Elkouby, Y.M.
Source
Full text @ Science

Fig. 1. A zygotene-specific oocyte cilium.
(A) AcTub and Arl13b labeling shown at the indicated stages (n = 28 cysts in n = 8 ovaries). Scale bars for cyst panels, 5 μm. Scale bars for pachytene panels, 10 μm. (B) GluTub labeling shown as in (A) (n = 82 cysts in n = 7 ovaries). Scale bars are as in (A). (C) Pixelwise colocalization test for AcTub and Arl13b and for AcTub and gluTub. n, number of cilia. Bars are mean ± standard deviation (SD). (D) TEM images of cilia in leptotene-zygotene oocytes. Top left panel: leptotene-zygotene oocytes (dark-blue arrowhead indicates presumptive synapsed chromosomes interfacing the NE) within a germline cyst (light-blue arrowheads - cytoplasmic bridges. Right panels show cross-section of a ciliary axoneme, in higher magnifications (color-coded boxes), including ciliary membrane (yellow arrow) and MT doublets (red arrow), between oocyte membranes (light-blue arrowheads). Bottom panels: ciliary structures adjacent to a cytoplasmic bridge (light-blue arrows), including centriole (white), linker (gray), basal body (orange) transition zone (green), ciliary pockets (pink), axoneme (yellow), shown in higher magnifications (color-coded boxes in right panels). Scale bars are indicated. n = 23 oocytes in n = 5 ovaries. (E) AcTub and Sycp3 labeling shown in a zygotene cyst. n = 24 cysts in n = 2 ovaries. Scale bar, 10 μm. (F) The developmental distribution of the zygotene cilia as detected by AcTub (n > 100 ovaries), Arl13b (n = 18 ovaries), and GluTub (n = 11 ovaries). n = number of cysts. (G) 3D rendering of a zygotene bouquet cyst from SBF-SEM data, showing: cytoplasm of cyst oocytes (different transparent colors), nuclei (gray), cilia (maroon; maroon-gray arrowhead), and basal body centrosomes (green). Bottom panel: same cyst render stripped from cytoplasm to show the cilia. Scale bars are 5 μm. (H to J) Zygotene bouquet oocytes from ovaries labeled for (Scale bars are 5 μm): (H) Telomeres (Telo-FISH) and AcTub showing that the cilium emanates from the cytoplasm apposing the telomere cluster. n = 3 ovaries. A snapshot from 3D construction in movie S7. (I) γTub and AcTub showing that the cilium emanates from the centrosome, which was shown to localize apposing the telomere cluster (10). n = 5 ovaries. (J) Microtubules [Tg(βact:EMTB-3XGFP)] showing perinuclear cables (nucleus is counterstained with DAPI). n = 7 ovaries. A snapshot from 3D construction in movie S8. Another example is shown in movie S9. (K) Co-labeling of microtubules (EMTB-GFP), γTub, AcTub, and DAPI, reveals that the zygotene cilium emanate from the centrosome, which is embedded in a perinuclear microtubule network. A merged image and all individual channels are shown. n = 5 ovaries. γTub (mIgG1) and AcTub (mIgG2b) antibodies are both isoforms of mIgG. We used mIgG secondary antibody that binds both primary γTub and AcTuB antibodies, and a mIgG2b secondary antibody that specifically binds to the AcTuB antibody and not γTub antibody (arrowheads in the three bottom panels). (L) Scheme showing the bouquet cytoplasmic cable system.

Fig. 2. The zygotene cilium is required for bouquet formation.
(A and B) 3D measurements of ciliary lengths and average ciliary length per cyst. n, number of cilia. ND, not detected. Bars are mean ± SD. (C) Distribution of cysts with given ciliary length or absence. (D) Images of cysts from ciliary (AcTub) length categories as in (B) to (D). Percentage of ovaries with indicated categories are plotted. n, number of ovaries. Scale bars, 10 μm. (E) Images of “leptotene-zygotene-like” oocytes of average and large sizes. Scale bars, 5 μm. (F) “Leptotene-zygotene-like” oocyte sizes. n, number of oocytes. Bars are mean ± SD. 9.1 μm represents the largest wt leptotene oocytes. (G) Left panel: images of mid-bouquet-stage oocytes with tight, expanded, and dispersed telomeres (categories shown in top right panel). ND, nondetected. Scale bars, 5 μm. Right bottom panel: the percentage of each category in all genotypes. n, number of oocytes.

Fig. 3. Centrosome anchoring by the zygotene cilium is required for telomere clustering in bouquet formation and for proper synaptonemal complex formation.
(A) Live time-lapse imaging of chromosomal bouquet rotations in Tg(h2a:h2a-gfp);cep290+/+ and sibling Tg(h2a:h2a-gfp);cep290–/– ovaries. Chromosomal tracks and their sum at Tf are shown. Individual track velocities (n, number of tracks) and average track velocity per cell (n, number of cells) are plotted. Oocytes with mean and low outlier (below the SD) values of average track velocities are shown for cep290–/–. Images are snapshots from movie S12. Scale bars, 5 μm. (B) Laser ablation of the zygotene cilium. The targeted cilium (teal arrowhead) is shown “pre-ablation” and the ablation region at its base (S1:1) is indicated throughout the time-lapse. The ablated ciliary associated centrosome (yellow circle) dislocates upon ablation in both XY and Z (“centrosome out of focus”) axes (see cartoon, right). Time is indicated in mili-seconds. Scale bar, 5 μm. n = 17 cilia (oocytes) from n = 11 ovaries. Images are snapshots from movie S13; another example is shown in movie S14. (C) Images of mid-bouquet stage oocytes from wt and cep290–/–;kif7+/– ovaries co-labeled for the centrosome (γTub) and telomeres (Telo-FISH). The distance between the centrosome and the nucleus (white arrow) in the wt is significantly decreased in mutant oocytes with defected telomere clustering (yellow arrowheads); see cartoons below. The normalized distances are plotted and bouquet phenotype categories are color-coded. Scale bar, 5 μm. n, number of oocytes. (D) The normalized centrosome-nucleus distance in oocytes pooled from all genotypes in (C), categorized based on their bouquet phenotype. n, number of oocytes. (E) Images of Sycp3 localization patterns in early and late zygotene stages. Note normal Sycp3 patterns in wt oocytes (yellow arrowheads) and their absence in mutant oocytes (scattered foci, magenta arrowheads). Sycp3 signal accumulates in nucleoli (empty red circle) in all wt and mutant oocytes. Scale bars, 5 μm. (F) The distribution of normal and abnormal Sycp3 oocytes from (E). n, number of oocytes. (G) Images of “leptotene-like” oocytes shown in scale. Scale bars, 5 μm. (H) “Leptotene -like” oocyte sizes per genotype. n, number of oocytes. Bars are mean ± SD.

Fig. 4. The zygotene cilium is required for germline cyst morphogenesis.
(A, D, and E) Images of cyst (white outlines) phenotype categories labeled for the cytoplasmic marker DiOC6 and AcTub (A), showing gaps between oocytes (magenta arrows), elongated cytoplasmic bridges (yellow arrows) and isolated oocytes (white arrows). Scale bars, 10 μm. The percentage of cyst phenotype categories and number of isolated oocytes are shown in (D) (n, number of cysts) and (E) (n, number of ovaries), respectively. (B) Images of 3D cyst morphology analysis per category. Normalized distances are plotted. n = 5 to 7 cysts from n = 2 to 3 ovaries per genotype. Bars are mean ± SD. (C) Vasa and AcTub labeling shows normal cysts in wt ovaries, and disintegrated cysts (elongated CBs, yellow arrows) as well as isolated zygotene oocytes (white arrows) in cep290–/– ovaries. Phenotype distribution is plotted in fig. S11. Scale bars, 10 μm. (F) Severity of cyst phenotypes and gonad conversions plotted for three categories of ciliary defects pooled from all genotypes. n, number of gonads.

Fig. 5. Ciliary mutants show defective ovarian development and fertility.
(A) cCaspase3 apoptosis labeling (white arrowheads) in juvenile ovaries. DiOC6 is a cytoplasmic marker. Scale bars are 50 μm. The number of cCaspase3-positive oocytes per gonad are plotted. Each dot represents a gonad, n = 5 to 10 gonads per genotype. Bars are mean ± SD. (B) Rates of juvenile gonad conversion. n, number of gonads. (C) Images of eggs obtained by squeezing for IVF experiments. Scale bar, 1 mm. (D) Adult females and ovaries showing: ovaries within the peritoneal cavity (black outline), st.III premature oocytes (green arrows and outlines), young transparent st.I oocytes (blue arrows), st.II oocytes (pink arrows) and degenerated tissue masses (red arrow and outline). Ruler graduations and scale bars, 1 mm. Mutant females exhibit scoliosis.

Fig. 6. The zygotene cilium is conserved in zebrafish male meiosis and mouse oogenesis.
(A to C) Seminiferous tubules of adult zebrafish testes labeled with AcTub and Sycp3 [(A), n = 35 tubules in n = 3 testes] or Telo-FISH [(B), n = 29 tubules in n = 2 testes] or γH2Ax [(C), n = 34 tubules in n = 3 testes]. Left panels: optical sections of entire tubules (white outline; scale bars, 20 μm). Right panels: magnified images of zygotene bouquet spermatocytes (early zygotene bouquet in top panels, and late zygotene bouquet in bottom panels; scale bars, 10 μm). L/Z, leptotene/zygotene primary spermatocytes; P, pachytene spermatocytes; E1, initial spermatids; E2, intermediate spermatids; E3, final (mature) spermatids. (D) TEM images of adult testis showing ciliary structures (see arrowheads in key) within (left panels) prophase spermatocytes (synapsed chromosomes, pink arrowheads). Colored panels are magnifications of color-coded boxes. Magnifications and scale bars are as indicated. n = 25 spermatocytes in n = 2 testes. (E) Mouse E14.5 ovaries labeled with Vasa and Sycp3. Scale bar, 20 μm. (F) Mouse E14.5 ovaries labeled with Vasa and AcTub showing AcTub-positive CBs (pink arrows) and cilia (white arrows) in Vasa-labeled cysts. n = 4 ovaries. Scale bar, 15 μm. (G to J) E14.5 ovaries labeled with AcTub, Vasa, and γTub [(G), n = 2 ovaries], or Arl13b (H), n = 2 ovaries], or GluTub [(I), n = 2 ovaries], or Adcy3 [(J), n = 2 ovaries]. Scale bars, 5 μm. (K) E14.5 ovaries co-labeled with Vasa, Sycp3, GluTub, and DAPI. Individual channel images are shown in fig. S18 (n = 2 ovaries). Scale bars, 5 μm. In (E) to (J), cilia (white arrows) and CB-like structures (pink arrows) are indicated.

Acknowledgments
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