Fig 1.
mtm99-14 and mtm00-03 are allelic mutants with defective meiosis.
(A) Projected images of WT and mutant meiocytes stained with DAPI during early prophase I. Initially spherical heterochromatic knobs (arrows) become elongated at the leptotene-zygotene transition that accompanies a positional change of the nucleolus (n) from the nuclear interior to the periphery. At zygotene, paired knobs (arrowhead) and chromosomes (dashed line) were observed in WT. An obvious aberrant phenotype of both mutants was observed during the pachytene stage, manifested as fluffy chromosomes. Scale bar represents 5 μm. (B) Acetocarmine staining of male meiocytes during the metaphase I (upper panel) and tetrad stages (lower panel) of WT, mtm00-03, mtm99-14, and heteroallelic mtm99-14/mtm00-03 plants. In WT male meiocytes, ten bivalents are aligned on a metaphase I plate and later produce normal tetrad cells. Mutant cells mainly present univalents and rare interconnected chromosomes counted as bivalents (arrows). Scale bar represents 10 μm. (C) Quantitative results of meiotic phenotypes during the metaphase I and tetrad stages. Numbers of cells analyzed are shown in parentheses. The spo11-1-1 is another mutant allele (see next section).
Fig 2.
Structure and expression of the SPO11-1 gene.
(A) Schematic diagram of the maize genomic region. Both mtm99-14 and mtm00-03 mutants contain remnants of Mu sequences (purple boxes) that are associated with deletions (grey boxes) spanning PhyC2 and Spo11-1 genes. Coding regions and introns/UTRs are represented as orange and yellow boxes, respectively. The additional Mu insertion allele (spo11-1-1) is indicated by a red triangle. Green boxes represent transposable elements (Tourist and CACTA-like elements). (B) RT-PCR analyses to amplify the Spo11-1 at 5’ and 3’ regions exhibited splicing variants (asterisks) in WT and confirmed that Spo11-1 transcripts are absent in all mutant alleles. Positions of primer sets are indicated by red arrows in (D). Afd1/Rec8 expression was used as a control. 0RT represents a negative control lacking cDNA. (C) Quantitative PCR showed differential expression of the Spo11-1α and Spo11-1β transcripts using specific primers indicated by black arrows in (D). (D, E) Gene models (D) and predicted proteins (E) of SPO11-1α and SPO11-1β are shown. The additional region of SPO11-1β (amino acid 73–115) is indicated by a pink box.
Fig 3.
SPO11-1 deletion affects DSB formation, chromosome pairing and homologous recombination.
(A-D) Zygotene-stage meiocytes of WT (A, C) and the spo11-1-Δ03 mutant (B, D) showing TUNEL (A, B), γH2AX signals (C, D) in green and DAPI stained chromatin in red. Most mutant cells exhibited no signal, but ~10% of cells exhibited a few signals (arrows). (E-H) Meiocytes at zygotene and pachytene stages from WT and spo11-1-Δ03 hybridized with 5S rDNA (green, arrows) and telomere probes (white signals in E and G) demonstrate impaired homologous pairing in the spo11-1 mutant, although telomeres cluster normally. (I-L) Meiocytes at diakinesis and metaphase I from WT and spo11-1-Δ03 hybridized with 5S rDNA (green, arrows) and 180bp-knob probes (white signals) showing that normal crossover formation is revoked in spo11-1 meiocytes. Note that a bivalent detected in Fig 3L represents non-homologous chromosome association. All scale bars represent 5 μm.
Fig 4.
Numerous SPO11-1 signals are distributed in nuclei during early prophase I.
(A-B) Immunostaining of SPO11-1 (green) and DAPI-stained chromosomes (red) in WT meiocytes at different stages (A) and in a spo11-1 mutant meiocyte (B). SPO11-1 signals are shown as gray-scale in the lower panels. Scale bars represent 5 μm. (C) Dot blot analysis of SPO11-1 antibody against SPO11-1, SPO11-2 and SPO11-3 peptides (see S7 Fig for details). (D) Numbers of SPO11-1 foci detected using the affinity-purified antibody in WT nuclei at different stages and in spo11-1-Δ03 nuclei at early zygotene. Numbers of foci detected in WT nuclei using pre-immune serum (S7 Fig) are also shown (prebleed).
Fig 5.
SPO11-1 localization revealed by super-resolution microscopy in wild-type.
Super-resolution images of SPO11-1 (green) and DSY2 (red) staining in WT meiocytes shown as projection images of nuclei (A, D, G, J), single Z sections (B, E, H, K), and magnified views of 2 μm2 regions (C, F, I, L). Scale bars represent 2 μm. Respective serial Z sections are shown in S8–S11 Figs.(A-C) A leptotene nucleus with long and intricate chromosome axes surrounding a nucleolus exhibiting most of its SPO11-1 foci around the nuclear periphery and less signal within the nucleus. (D-F) A late leptotene nucleus with an off-set nucleolus exhibiting numerous SPO11-1 foci distributed within the nucleus. No obvious pre-alignment of axes was observed (S9 Fig). Some SPO11-1 foci are located on DSY2-labeled AEs (arrow). (G-I) An early zygotene nucleus with telomere bouquet (asterisk) and pre-aligned axes (Z11-19 in S10 Fig) exhibiting numerous SPO11-1 foci. Note that the nucleolus exhibits less SPO11-1 signal. (J-L) A zygotene nucleus with progressive pairing and synapsed regions containing numerous SPO11-1 foci.
Fig 6.
Similar numbers of SPO11-1 foci load onto axial elements.
(A-C) Representative projection images of a WT meiocyte showing chromosome axes labeled by DSY2 (red) and SPO11-1 signals in green, captured by structured illumination microscopy. Only linear contrast adjustment was applied to enhance signals. Scale bars represent 5 μm. (D-F) Projection images of segmented SPO11-1 objects (E) and a skeletonized axis model (F) representing the medial lines along chromosome axes generated using our image processing pipelines (S12 and S14 Figs). (G-H) Surface rendering images of SPO11 and DSY2, visualized by the 3D Viewer tool in ImageJ. Scale bars represent 5 μm. (I) A magnified 3D view of the surface rendering image in G. Scale bars represent 1 μm. A respective rotation movie is presented in S1 Video. (J) Numbers of total SPO11-1 objects (dots) and axis-associated SPO11-1 objects (triangles) detected in meiocytes during early prophase I.
Fig 7.
An aberrant axial element structure in spo11-1 mutants.
Immunostaining of DSY2 (green) and DAPI staining (red) in WT (A, C, E, G) and spo11-1-Δ03 meiocytes (B, D, F, H). Maximal projection images are displayed for entire nuclei. Magnified images of 5 μm2 regions are shown in the middle panel. Scale bar represents 5 μm. (A, B) The morphology of chromosome axes is similar in WT and mutant meiocytes when DSY2 first appears in the nucleus at the early leptotene stage. (C, D) At late leptotene, AEs appear progressively as linear forms in WT (C). In contrast, abnormal AE structures are observed in mutant meiocytes (D). Note that DSY2 intensity is uneven in mutant meiocytes, with atypically bright fluorescent patches, unlike the uniform signals in the WT. The off-set nucleolus is marked with n. (E-F) Synapsis, manifested as paired doubled DSY2 stretches, is observed primarily near the telomere bouquet (*) of the WT (E), which is absent from mutant meiocytes (F).(G-H) A late zygotene WT meiocyte (G) showing consistent DSY2 signal along chromosomes, unlike the unevenly accumulated DSY2 (arrows) in spo11-1 meiocytes (H).(I) Dot plot graph showing total lengths (mm) of DSY2 signals from the leptotene to pachytene stages in WT and spo11-1 meiocytes. Each dot represents a measurement from one meiocyte. The linear plot represents the percentage of synapsed regions, as measured by ZYP1 signal length over total DSY2 signal length.(J) Distribution of kappa values representing AE curvature, as measured from five meiocytes for each stage in WT and spo11-1 mutants.
Fig 8.
Axial element proteins ASY1 and DSY2 exhibit abnormal localization in spo11-1 mutants.
(A, B) Zygotene meiocytes of the WT (A) and spo11-1 mutant (B) immunolabeled with ASY1 (red) and DSY2 (green), and observed by super-resolution microscopy. Note that synapsed regions in the WT (arrows) exhibit diminished ASY1 and enhanced DSY2 signals. In mutant meiocytes, both ASY1 and DSY2 accumulate abnormally (arrowheads). (C, D) Abnormal chromosome axes with curly and uneven ASY1 staining are observed in spo11-1 meiocytes. Scale bars represent 5 μm.
Fig 9.
Transverse filament loading is initiated but is not completed.
(A-D) Immunostaining of ZYP1 (green) with DAPI staining (red) in WT (A, B) and spo11-1-Δ03 (C, D) meiocytes at zygotene (A, C) and pachytene stages (B, D). Note the magnified region from the pachytene-like mutant meiocyte (D) showing discontinuous ZYP1 stretches (arrow). Scale bars represent 5 μm.(E-H) Transmission electron microscopy of synaptonemal complex spreads of WT (E) and spo11-1-Δ03 (F-H) meiocytes. Unlike the complete synapsis in WT, mutants exhibit abnormal synapsis (G and H). Note that synapsis occurs within one axis, forming “fold-back” structures (arrow), and promiscuous synapsis can also be seen as unequal synapsis with an overhang (arrowheads). In addition, unsynapsed AEs in mutants appear as curly and twisted structures (asterisk in H). Scale bars represent 1 μm.