Observation of Extensive Chromosome Axis Remodeling during the "Diffuse-Phase" of Meiosis in Large Genome Cereals

Abstract

The production of balanced fertile haploid gametes requires the faithful separation of paired (synapsed) chromosomes toward the end of meiotic prophase I (desynapsis). This involves the timely dissolution of the synaptonemal complex during the pachytene-diplotene transition, a stage traditionally referred to as the "diffuse stage." In species with large genomes such as, barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.) we know most about the early stages of meiotic prophase I. There, synapsis initiates at the telomeric ends of chromosomes and progresses toward the centromeric regions through the ordered assembly of the synaptonemal complex (SC). Synapsis is impacted by recombination (crossing over, CO) which locally modifies the extent of chromatin compaction and extension. CO is uneven along the chromosomes, occurring mainly toward the telomeric regions resulting in a highly skewed distribution of recombination events. However, we know very little about the process of desynapsis which occurs during the "diffuse stage," where the synapsed and recombined chromosomes faithfully desynapse and separate into daughter cells. Here, using 3D-SIM super-resolution immuno-cytology combined with the use of antibodies directed against two crucial SC proteins, ASY1 and ZYP1, we followed the whole of meiosis I (i.e., both synapsis and desynapsis) in both barley and wheat. We showed that synapsis forms a characteristic tri-partite SC structure in zygotene (more clearly seen in barley). Toward the end of meiosis I, as the SC starts to disassemble, we show that extensive chromosome axis remodeling results in the formation of characteristic "tinsel-like" structures in both wheat and barley. By using a mutant (des10) that is severely compromised in polymerization of ZYP1during synapsis, we show that tinsel structure formation during SC dissolution is not dependant on full synapsis and may relate instead to changes in expansion stress. Our observations highlight a potentially new role for ASYNAPSIS1 (ASY1) in desynapsis, in addition to chromosome synapsis and cohesion.

Keywords: ASY1; cereal; chromatin; meiosis; synapsis.

Figure 1

Super resolution microscopy of synapsis in large genome cereals. (i) Cartoon of cycle of chromatin expansion and contraction as described in Kleckner et al. (2004) with indication of the diffuse stage. (ii) Synapsis was monitored using ASY1 (green) and ZYP1 (magenta) by 3D-SIM for barley Bowman (a–d) and wheat Chinese Spring (e–h). Synapsis starts in leptotene at one end of the nucleus (a,e) and ZYP1 polymerizes to bring the chromosomes together during zygotene (b,f) though the tripartite structure of the synaptonemal complex is only visible in barley wt (b, arrow). At pachytene, synapsis is complete in barley (c) and wheat (g) with ectopic ASY1 signals (c,g, arrows). During diplotene, ASY1 remodels in both species (d,h) to form tinsel structures. Scale bar 5 μm.

Figure 2

Modeling of the tinsel structure in wt and des10. Modeling the tinsel structure in wt (a) and des10 (b) by creating “surfaces” for ASY1 and ZYP1 using Imaris surface tool (a,b). This revealed areas of axes with no ZYP1 (white arrow) in both wild type (a) and des10 (b), suggesting dissolution of the SC. ZYP1 aggregates were visible in wild type (a, yellow arrow), with abundant surrounding ASY1, that could suggest contraction of local chromatin. Individual bivalents are manually tracked using the Imaris measurement tool (c,d) revealing different thickness of bivalent areas in des10 (d, white arrow), that may suggest differences in the thickness of the remnant ASY1 axes. Using the tool surface on DAPI channel (with 50% transparent effect to view inside the DAPI signal) revealed that ASY1 remodeling remains within the chromatin (e,f).

Figure 3

ASY1 re-organization during desynapsis. De-synapsis was monitored with 3D-SIM using ASY1 (green) and ZYP1 (magenta) in barley wild type (a–f) and des10 (g–l). Enlarged areas of wild type (d–f) and des10 (j–l) slides show the organization of ASY1 along the ZYP1 axes (yellow lines). White circles highlight isolated ASY1 signals (d–f) that may represent the process of dissolution of ASY1 after the tinsel structure organization. In des10 (j–l), ZYP1 axes are not linear (dashed yellow lines), but ASY1 displays a similar behavior to wild type.

Figure 4

ASY1 re-organization during desynapsis and chromosome segregation. Strong ASY1 signal are detected after prophase I during chromosome segregation in both wild type (a–f) and des10 (g–m). Discrete ASY1 foci (green) are visible in wild type diplotene (a). At metaphase I, ASY1 signal does not seems specific and is located in the cytoplasm (b). During anaphase I, ASY1 signal is either linear (c) or globular (d,e). Discrete ASY1 signals are also found in tetrads (f). ASY1 behaves the same in des10, but the delay in prophase enables more details to be studied. ASY1 signal is present on diplotene chromosomes (g), and it is possible to visualize the last ASY1 signal near the end of the chromosomes (h, arrow). During metaphase I, ASY1 is mainly in the cytoplasm (I) but during anaphase I, it is possible to see the ASY1 signal relocated around the lagging chromosomes (k,l arrows). Discrete ASY1 signal is also found in tetrads (m). Scale bar 5 μm.