Nestin-Cre activity and Nestin expression.

<p>(A, B) Immunofluorescence for GFP and Nestin in 13.5dpc NestinCre; R26RYFP embryo. Ventral diencephalon (VD) (A) and Ratkhe's pouch (RP) (B). Note that EYFP expression always correlates with that of Nestin within the CNS. (C, D) Immunofluorescence for Nestin on 16.5dpc (C) and 18.5dpc (D) wild-type pituitaries. (E, F) Immunofluorescence for GFP (E) and for GFP and Nestin (F) on Nestin-Cre; R26ReporterEYFP eleven month-old pituitaries. The arrows point to Nestin^+ve, EYFP^+ve cells. Scale bar: 50 µm; ant.: anterior; int.: intermediate; pos.: posterior; inf: infundibulum</p

RPA localization in chicken oocytes.

<p>Oocytes stained for RPA (red) and SYCP1/SYCP3 (green). RPA and SYCP1 co-staining was performed first (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002560#pgen.1002560.s001" target="_blank">Figure S1</a>). After imaging, the slides were then stained for SYCP3 and new pictures were taken. (A) Zygotene, RPA foci are on synapsed and unsynapsed chromosomes (indicated with arrows and arrowheads, respectively). (B–D) Pachytene; RPA foci on autosomal synaptic axes are still abundant at early pachytene (B), but rapidly decrease and then disappear by late pachytene (C–D). Pachytene cells were classified as late if they contained fewer than five foci on synaptic autosomes. (C) Late pachytene oocyte containing a synapsed ZW bivalent RPA negative. (D) Late pachytene oocyte containing an unsynapsed Z and W carrying chains of RPA foci on the Z. Z and W were identified by hybridisation to Z and W chromosome paints (data not shown). Drawings of ZW synaptic configurations are shown to the right of panel C and D. Scale bar = 10 µm.</p

Chick oocytes undergo a wave of γH2AFX coincident with the beginning of meiosis.

<p>(A–B) Ovarian section from E14 embryos injected with BrdU two hours before dissection and double-stained for BrdU (green) and γH2AFX (red). Many germ cells are positive for both markers (e.g. white arrows). (C–D) Oocyte nuclei from E14 ovarian spreads double-stained for γH2AFX (red) and SYCP3 (green). SYCP3 is only present in some of the γH2AFX positive cells. (E–H) γH2AFX timecourse on ovarian spreads. The cells were double-stained with SYCP3 for staging. γH2AFX is downregulated by mid-pachytene. (I–J), Pachytene cells triple-stained for SYCP3 (green), RPA (green) and γH2AFX (red): γH2AFX is only present in the cell containing asynaptic RPA+ve chromosomes (left nucleus), where it colocalises with some RPA foci (white arrows). Scale bar = 10 µm.</p

A global downregulation of transcription occurs at leptotene-zygotene and is maintained into pachytene and early diplotene.

<p>(A) Schematic of the Z chromosome showing the location of the Z probes used in the analysis. (B) RNA-FISH analysis using Z (Z1–Z9) and autosomal (A1–A3) probes on oocytes from E14 (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002560#s4" target="_blank">Materials and Methods</a> for probe identity). The oocytes were identified by subsequent staining for γH2AFX. The bars represent the percentage of expressing oocytes. (C) (Top) Timecourse results using the probes screened at E14and subsequently analysed at E19, 1 dph (D1), 6 dph (D6); the oocytes were identified by staining for SYCP3. The Y axis indicates the percentage of expressing oocytes. (C) (Bottom) Charts representing the percentage of leptotene/zygotene (L/Z), pachytene (P) and diplotene (D) cells in the ovaries analysed at E19, D1 and D6; the counting was carried out on spreads generated from the cell dispersions used for RNA-FISH, after staining for SYCP3 and RPA. (D) Oocyte nuclei from ovarian spreads subject to RNA-FISH using the probe Z5 (cyan) and stained for SYCP3 (red) and RPA (green); (Top) Positive oocyte containing ZW^RPA+; (Bottom) Positive oocyte containing a fully synapsed ZW. The positive signal (cyan) is also indicated by a white arrow. Scale bar = 10 µm. (E) Plots summarising the results from the RNA-FISH on spreads using probes Z4, Z5, Z7, Z8. (Top) Relative percentages of synaptic and asynaptic ZW within the positive late pachytene oocytes. (Bottom) Chart representing the relative percentages of synaptic and asynaptic ZW within the total population of late pachytene oocytes. The activity of the pachytene ZW pair is not biased by its synaptic status.</p

The chick sex chromosome meiotic synaptic behaviour: a new model.

<p>Schematic representing the dynamics of synapsis and DSB repair of the ZW pair during early meiosis. Autosome axes in black; Z in green; W in red. RPA foci are shown as blue dots, γH2AX as circles. In about 80% of the oocytes that reach pachytene ZW undergoes synapsis and DSB repair by mid-late pachytene and desynapses at diplotene. In 20% of oocytes ZW remains asynapsed and maintains unrepaired DSBs, nevertheless the cells go to diplotene. Different shades of blu represent different levels of transcriptional activity (dark blue: high, light blue: low). The oocytes undergo a global downregulation of transcriptional activity at leptotene/zygotene. At pachytene, no Z specific wave of silencing is evident in any of its configurations. Autosomes and Z chromosomes behave alike; they maintain a low level of transcription at least into early diplotene. See Discussion for more details.</p

Oocytes with unresolved DNA DSBs on the Z chromosome progress to diplotene.

<p>(A–F) 6 dph oocytes stained for RPA (red) and SYCP3 (green). The diplotene cells were classified as early when desynapsis was partial and late when desynapsis was complete. (A–B) Late pachytene; (C–D) early diplotene; (E–F) late diplotene. (A, C, E) Cells RPA negative (RPA^−ve), and (B, D, F) cells carrying a chain of RPA foci (RPA^+ve). (G) Graph showing the percentage of RPA^+ve cells at each stage. Total number of cells analysed: late pachytene, 141; early diplotene, 223; late diplotene, 169. ZW chromosome painting performed after antibody staining showed that the Z chromosome accounted for 85% of the RPA^+ve cells identified at pachytene and 74% at early diplotene, making the percentage of late pachytene ZW^RPA+ = 21.7% and the percentage of early diplotene ZW^RPA+ = 19.6%. The percentage of ZW^RPA+ pairs at late diplotene was not calculated as in many of these cells it was not possible to unambiguosly identify the entire Z axis. The quantification was done on ovaries from the chicken line ISA Brown. (H) 14 dph oocytes stained for RPA (red) and SYCP3 (green). 90% of the cells are negative for SYCP3; no RPA was identified in these cells (number of cells analysed: 60). Scale bar = 10 µm.</p

The late pachytene ZW^RPA+ pair has no MLH1 focus.

<p>Oocytes triple stained for SYCP3 (green), RPA (blue) and MLH1 (red). (A) The ZW^RPA+ pair carrying a chain of RPA foci on the Z never has a MLH1 focus (white arrowhead indicates the expected location of the focus); total number of oocytes analysed: 62. (B) The fully synapsed ZW bivalent always carries a MLH1 focus at one end (white arrow); Total number of oocytes analysed: 138. Drawings of the ZW pair to the right of ech panel. ZW pairs were identified using ZW chromosome paints (data not shown). Scale bar = 10 µm.</p

ZW bivalent desynapsis occurs in synchrony with autosomal desynapsis.

<p>Oocytes double-stained for SYCP3 (green) and RPA (blue) and subsequently hybridised to Z and W chromosome paints (green and red clouds respectively in top right insets); only the late pachytene and early diplotene cells negative for RPA were considered. (A–C) Late pachytene cells (total number analysed: 134); (A) ZW is fully synapsed; (B) ZW is partially desynapsed; (C) Z and W are fully desynapsed. (D–F) Early diplotene cells (total number analysed: 54); (D) ZW is fully synapsed; (E) ZW is partially desynapsed; (F) Z and W are fully desynapsed. Relative percentages are indicated in the insets at bottom right corner of each panel. To the side of each panel is a schematic of the ZW pair (Thick black line, synapsed ZW; black and grey thin lines, desynapsed Z W, respectively). In most pachytene cells the ZW pair is synapsed, in most early diplotene cells ZW is partially desynapsed. Scale bar = 10 µm.</p

Many ZW^RPA+ pairs have PAR-PAR misalignment.

<p>Late pachytene oocytes triple-stained for SYCP3 (green), RPA (blue), MLH1 (red) and subsequently hybridised to Z and W chromosome paints (top right insets: Z, green cloud; W, red cloud). Total number of ZW^RPA+ oocytes analysed: 134. (A) Z and W axes are clearly separated; (B) only the q-end of the Z axis is close to the W; (C) both ends of the Z are close to the W (see text for percentages of each class). The ZW bivalents are also schematised to the right (black line = Z; grey line = W; green = Z paint; red = W paint). Scale bar = 10 µm.</p

Immunofluorescence and real-time quantitative RT-PCR analysis of mice carrying TES deletion on <i>Sox8</i>-null background.

<p>(A) Immunostaining of 14.5 dpc XY testis of wild type and TES^-/- embryos on a <i>Sox8</i>-null background. (B) Immunostaining of 8 week-old XY testes of wild type and TES^-/- mice on a <i>Sox8</i>-null background. Testes were stained for SOX9 (green), FOXL2 (red) and DAPI (blue). (C) Gene expression in XY TES deleted gonads with <i>Sox8</i>-null background at 14.5 dpc. Data are presented as mean 2^-ΔΔCt values normalized to <i>Hprt</i>. Sample size represents number of individuals and is indicated below each genotype. Error bars show SEM of 2^-ΔΔCt values. P value is presented above the relevant bars (unpaired, two-tailed t-test on 2^-ΔΔCt values). Dark grey bars: XY; light grey bars: XX.</p