A novel method to follow meiotic progression in Arabidopsis using confocal microscopy and 5-ethynyl-2′-deoxyuridine labeling

Abstract

BackgroundMeiosis progression in the more recent past has been investigated using 5-bromo-2′-deoxyuridine (BrdU) uptake by S-phase meiocytes undergoing DNA replication. BrdU uptake is detected by reaction with BrdU antibody followed by epifluorescent microscopy examination of chromosome spreads and/or squashes. We here report using confocal microscopic examination of intact meiocytes in conjunction with the new thymidine analog 5-ethynyl-2′-deoxyuridine (EdU). The simplicity of the EdU detection coupled with confocal examination of anthers provides a more exact temporal description of meiotic prophase I progression in Arabidopsis and opens up the possibility of examining the coordination of microsporocyte development with the other tissues of the anther.ResultsUsing our time course protocol, we have determined the duration of wild type Arabidopsis leptotene to be 5 h, zygotene -6 h, pachytene -10 h and a diplotene duration of approximately 1 h. We estimate G2 duration to be approximately 7 h based on the timing of the initial appearance of EdU signal in early leptotene meiocytes. In addition we have found that DNA replication in meiocytes is not done synchronously with the associated tapetal layer of cells. The EdU labeling suggests that S-phase replication of meiocyte DNA precedes the duplication of tapetal cell DNA.ConclusionsThe increased number of meiotic staging criteria that can be assessed in our confocal analysis, as compared to chromosome spreading or squashing, makes the identification of even the early and late portions of the prophase I substages attainable. This enhanced staging coupled with the ability to easily generate large data sets at hourly time points makes it possible to more exactly determine substage duration and to detect modest temporal abnormalities involving meiocyte entrance into and/or exit from leptotene, zygotene and pachytene. Confocal analysis also makes it possible to study the relationships between different cell types within the flower bud as meiosis proceeds.Electronic supplementary materialThe online version of this article (doi:10.1186/1746-4811-10-33) contains supplementary material, which is available to authorized users.

The Arabidopsis Cdk1/Cdk2 homolog CDKA ;1 controls chromosome axis assembly during plant meiosis

“…The meiotic stage was determined by four morphological criteria: The squared cell shape of meiocytes, the centered position of the nucleolus, the chromosome axis being labeled by a previously generated functional ASY3 reporter (PRO ASY3 :ASY3:RFP), and the finding that tapetum cells were still single-nucleated (Wang et al, 2004;Yang et al, 2006;Stronghill et al, 2014;Prusicki et al, 2019). In wild-type male meiocytes at late G2, numerous foci and short stretches of ASY1 signal were present (n = 18 out of 20 male meiocytes analyzed).…”

“…In wild-type male meiocytes at late G2, numerous foci and short stretches of ASY1 signal were present (n = 18 out of 20 male meiocytes analyzed). The meiotic stage was determined by four morphological criteria: The squared cell shape of meiocytes, the centered position of the nucleolus, the chromosome axis being labeled by a previously generated functional ASY3 reporter (PRO ASY3 :ASY3:RFP), and the finding that tapetum cells were still single-nucleated (Wang et al, 2004;Yang et al, 2006;Stronghill et al, 2014;Prusicki et al, 2019). In contrast, only a diffuse ASY1: GFP signal without any foci could be detected in the nuclei of meiocytes of CDKA;1 T161D plants (25 out of 25) at a moment when ASY3 forms foci and short stretches (Fig 2A).…”

SWITCH 1/DYAD is a novel WINGS APART-LIKE antagonist that maintains sister chromatid cohesion in meiosis

“…S4). In addition, the SWI1-GFP signal was present in leptotene and became even stronger as cells progressed through leptotene as staged by the migration of the nucleolus to one side of the nucleus [33][34][35] and the appearance of an ASY3 signal on condensing chromosomes ( Fig. 2a, Supplementary Fig.…”

“…1a,b, 5c,d, Supplementary Video S4). Note that due to the failure of chromosome axis formation and of the aberrant migration of nucleolus in swi1 mutants, the meiotic stages in swi1 mutants were determined by the morphology of meiocytes in combination with the number of nuclei in tapetal cells 29,34 . The restoration of cohesion in the swi1 wapl1 wapl2 and the resemblance to the wapl1 wapl2 mutant phenotype was further confirmed by chromosome spread analysis ( Fig.…”

Live cell imaging of meiosis in Arabidopsis thaliana - a landmark system

“…The poly-nuclearization of tapetum cells was 348 clearly visible in our KINGBIRD line (Supplement movie 2, from minute 980 to minute 349 1207), possibly representing a sixth cell parameter next to the five meiotic 350 parameters presented above ( Figure 3A). Notably, tapetum cell differentiation was 351 previously suggested as a criterion to judge stages of meiosis (Stronghill et al, 2014; before A4/zygotene and conversely, when all tapetum cells are poly-nucleated, 354 meiosis has progressed into A7/diplotene. However, endomitosis only poorly 355 correlated with any of the meiotic stages between A4 and A7 ( Figure 3B) and hence, 356…”