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Nucleolus-associated telomere clustering and pairing precede meiotic chromosome synapsis in Arabidopsis thaliana

School of Biosciences, The University of Birmingham, Birmingham B15 2TT, UK

View larger version (194K): [in a new window]   Fig. 1. (A) Semi-thin section through a <0.3 mm flower bud, stained with toluidine blue, showing one complete anther locule. Each locule contains a group of PMCs in meiotic interphase with prominent nucleoli, surrounded by four cell layers. It can be seen that the tapetal cells, adjacent to PMCs, are much smaller than the PMCs. (B-D) Spread and silver stained PMCs to show the typical progression of nuclear and nucleolar morphology through early meiosis. (B) Leptotene; chromosome axes are fully developed and the nucleolus is slightly off-centre. (C) Zygotene; chromosome axes beginning to synapse and showing some aggregation; the nucleolus is shifted to a peripheral location. (D) Pachytene; the chromosomes are fully synapsed; the nucleolus remains peripherally located. Bar, 5 µm (B,C,D); 10 µm (A).

View larger version (78K): [in a new window]   Fig. 2. Sections through wax-embedded anthers following a 1 hour pulse label of BrdU and fixation 3 hours later. BrdU is detected by anti-BrdU antibody labeled with FITC (green). (A) BrdU labelled PMC nuclei at S-phase of meiotic interphase. (B) BrdU labeled tapetal cell nuclei; the PMCs in this anther are at leptotene/zygotene. Sections are counterstained with DAPI. Bar, 10 µm.

View larger version (108K): [in a new window]   Fig. 3. FISH of telomere, centromere (CEN=pAL1) and BAC probes to wild-type Arabidopsis nuclei. (A,B) Combined BrdU labelling and FISH following a 1 hour BrdU pulse +3 hours to show clustered telomere signals (A) and dispersed and extended CEN signals (B). BrdU is detected by anti-BrdU antibody labeled with FITC (green); both telomere and CEN signals are red. (C-I) Dual FISH of telomere (red) and CEN (green) probes to Arabidopsis PMC. (C) Nucleolus-associated clustering of telomeres and dispersal of CENs seen in meiotic interphase; the white dotted circle indicates the approximate boundary of the nucleolus (D). (D-I) Progressive changes in telomere and CEN signal numbers and distributions during meiotic prophase I; (D) early leptotene showing 15 clustered telomere signals and ten dispersed CEN signals, arrows here and elsewhere indicate the chromosome 1 CEN-associated telomere signals; (E,F) dual (E) and triple (F) filter images of the same leptotene nucleus showing ten dispersed CEN signals and eight semi-dispersed telomere signals, indicating colocalisation of some telomeres; (G) zygotene, showing nine telomere signals (two colocalised) and four CEN signals resulting from stage-specific chromatin clumping; (H) pachytene, showing nine telomere signals (two colocalised) and five CEN signals; (I) early diplotene showing early stage of desynapsis; note that telomeres are still paired at this stage. (J-L) Dual FISH with telomere (red) and BAC (green) probes to demonstrate that telomere pairing involves homologous chromosomes; (J) meiotic interphase showing clustered unpaired telomeres and BACs; (K,L) leptotenes, showing paired telomeres and BACs T9J23 (K) and F19K16 (L). All nuclei are counterstained with DAPI. Bar, 10 µm.

View larger version (92K): [in a new window]   Fig. 4. (A-F) FISH with telomere, CEN and BAC probes applied to meiotic prophases of the asy1 meiotic mutant. (A-D) FISH with telomere (red) and CEN (green) probes to show unpaired telomeres in meiotic interphase (A), paired telomeres in leptotene (B,C) and unpaired (dissociated) telomeres in the asynaptic post-leptotene stage (D). B and C show dual and triple filter images of the same leptotene nucleus. (E,F) Dual FISH with telomere (red) and BAC T9J23 (green) probes to show variable pairing (E) or unpairing (F) of the BAC in post-leptotene asynaptic nuclei. Bar, 10 µm.

View larger version (11K): [in a new window]   Fig. 5. Changes in telomere and CEN FISH signal number during meiotic interphase and prophase I of wild-type Arabidopsis. MI, premeiosis; L(I), early leptotene (clustered telomeres); L(II), late leptotene (dispersed telomeres); Z, zygotene; P, pachytene; D, diplotene.

View larger version (33K): [in a new window]   Fig. 6. The changes in telomere and CEN number and intranuclear distribution through meiotic interphase and prophase I. (A) Meiotic S-phase; telomeres (red) are clustered around the nucleolus and unpaired; pericentic heterochromatin (green) regions (CENs) are unpaired, extended and dispersed. (B) Meiotic interphase-G2; similar to preceding S-phase except that CENs are condensed. (C) Early leptotene; telomere number variable, between 10 and 20, indicating that telomere pairing is occurring; CENs remain unpaired and widely dispersed. (D) Leptotene; telomeres fully paired (10 signals) and widely dispersed; CENs unpaired and dispersed. (E) Zygotene; telomeres paired and loosely confined to one hemisphere of the nucleus; CEN signal number variable (1-5) due to homologous synapsis and aggregation. (F) Pachytene; telomeres and CENs fully paired and widely dispersed through nucleus.