Migration of prospindle before the first asymmetric division in germinating spore of Marchantia polymorpha

Abstract

The development of the plant body starts with spore germination in bryophytes. In many cases, the first division of the spore occurs after germination and cell elongation of the spore. In Marchantia polymorpha, asymmetric division occurs upon spore germination to generate two daughter cells: the larger one retains the ability to divide and develops into the thallus via sporeling or protonema, while the smaller one maintains tip growth and differentiates into the first rhizoid, providing a scaffold for initial development. Although spore germination of M. polymorpha was described in the 19th century, the intracellular processes of the first asymmetric division of the spore have not been well characterized. In this study, we used live-cell imaging analyses to elucidate microtubule dynamics during the first asymmetric division concomitantly with germination. In particular, we demonstrated that the preprophase band was not formed in the spore and that the bipolar prospindle, which is a microtubule structure surrounding the nucleus during prophase, migrated from the center to the periphery in the spore, suggesting that it was the earliest visible sign of cell polarity. We also showed that the occurrence of asymmetric division depended on actin filaments. Our findings regarding the first division of the spore in M. polymorpha will lead to a better model for cell-autonomous asymmetric division in plants.

Introduction

Asymmetric cell division is essential for the development of multicellular organisms. It generates distinct cell types, resulting in the differentiation of various tissues and organs and the maintenance of self-renewing stem cell pools. Compared with that in animals, asymmetric cell division is of greater importance for plants and multicellular algae, in which growth and morphogenesis are based on the de novo formation of cell types, tissues, and organs (de Smet and Beeckman 2011; Pillitteri et al. 2016). As plant somatic cells do not move from their original positions because of their cell walls, appropriate control of when, where, and in which direction asymmetric divisions occur is necessary for correct morphogenesis (Smolarkiewicz and Dhonukshe 2013). In particular, the first asymmetric division is crucial in the unicellular developmental stage, such as the zygote and spore, in the sense of the determination of the developmental axis going forward (Bisgrove and Kropf 2007; Zhang and Laux 2011).

In plants and multicellular algae, it has ubiquitously been observed that development starts with asymmetric division of the unicellular spore in the gametophyte generation or zygote in the sporophyte generation. In the fucoid brown algae Fucus spp. and Silvetia spp., which are photosynthetic multicellular eukaryotes in a phylogenetically distant group from terrestrial plants, the first division concomitant with the germination of the zygote is asymmetric (Bisgrove and Kropf 2007). The first asymmetric division generates two daughter cells: the tip-growing smaller cell, which continues to divide and grow to differentiate into the multicellular rhizoids that give rise to the scaffold for anchoring the alga to a rocky substratum, and the large cell, which mainly develops into the photosynthetic and reproductive stipe and fronds. Classical physiological studies on the process of zygote germination demonstrated that the entrance of the sperm and environmental cues, such as light, gravity, and mechanical stimuli (touch), coordinately determine the orientation of the asymmetric division (Kropf 1997). Although it has been suggested that the regulation of filamentous actin and microtubule configuration is also important for the establishment and maintenance of the cell polarity required for asymmetric division of the zygote, information about molecular mechanisms for cytoskeletal regulation in these algae is still limited (Hable 2014; Hable and Hart 2010). In angiosperms, including Arabidopsis thaliana and Oryza sativa, the first division of the zygote is also asymmetric, generating an asymmetric two-celled embryo consisting of an apical cell and a basal cell (Khanday and Sundaresan 2021). Analysis of embryogenesis-defective mutants has revealed some transcription factors involved in the asymmetric division of the zygote (Khanday and Sundaresan 2021; Ueda et al. 2011; Zhang and Laux 2011). However, as the zygote is surrounded by maternal cells in the ovule, it is difficult to observe, and consequently, physiological studies have been delayed. Thus, organizational components downstream of the transcription factors that function in zygote polarization have not been identified. Recently, the development of microscopic culture systems for the Arabidopsis zygote has demonstrated the importance of organelle positioning, actin filaments, and microtubules in the polarization of the zygote; however, the molecular mechanisms of cytoskeletal regulation remain unclear (Kimata and Ueda 2020; Kimata et al. 2016; Matsumoto et al. 2021).

In pteridophytes and bryophytes, unicellular spores are the starting point for the development of haploid plant bodies. It is generally accepted that asymmetric division occurs simultaneously with spore germination in most pteridophytes and some bryophytes; however, information about the process of the first asymmetric division of the spore is limited. In the fern Onoclea sensibilis, the nuclei of chemically fixed spores were visualized using N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl) pyridinium dibromide (DAPI) staining, and the results suggested that the nucleus, which was near the center of the dormant spore, moved to the germination site in the imbibed and light-irradiated spore (Miller and Greany 1974). In the liverwort Marchantia polymorpha, classical studies with light microscopy have shown that spores divide asymmetrically upon germination under sufficient light conditions, producing large and small daughter cells with distinct cell fates (Inoue 1960; O’Hanlon 1926). The large cell maintains division activity as the stem cell, which divides further to develop into the main body of the plant, while the small cell elongates and differentiates into the first rhizoid, which seems to serve as a scaffold for initial development (Inoue 1960; Shimamura 2016). However, little is known about the subcellular processes (including cytoskeletal dynamics or organelle localization) that occur during the asymmetric division of the spore.

We believe that the spores of M. polymorpha provide a good model for the study of stem cell maintenance by asymmetric division, especially for cell-autonomous stem cell renewing process, because division is not affected by any other cells; additional reasons include the ease of observing these spores under a microscope and recent technological breakthroughs that have enabled straightforward applications of molecular biology (Bowman et al. 2017; Ishizaki et al. 2008, 2015; Sugano et al. 2014, 2018). In this study, we investigated microtubule dynamics during the first asymmetric division of spores using live-imaging analysis. In particular, we elucidated that the prospindle relocated from the center to the periphery, demonstrating the earliest recognizable cell polarity in the spore. Furthermore, the effects of cytoskeleton-disrupting reagents suggested that both microtubules and actin filaments are required for the asymmetric division of the spore.

Materials and methods

Plant materials and growth conditions

Wild-type spores were obtained by crossing female and male accessions of M. polymorpha: Takaragaike-2 (Tak-2) and Tak-1, respectively (Ishizaki et al. 2008). Transgenic spores expressing Citrine-MpTUB2 were obtained by crossing female pro35S:Citrine-MpTUB2 line #6 (Otani et al. 2018) and male pro35S:Citrine-MpTUB2 #6-BC1, which is a progeny of female pro35S:Citrine-MpTUB2 line #6 and Tak-1. Harvested mature sporangia were dried with a few pieces of silica gel (approximately 2 mm in diameter) in a tube and stored at −80°C. Immediately before use, spores of surface-sterilized sporangia were suspended in a liquid medium (half-strength Gamborg’s B5 salts [Gamborg’s B5 Medium Salt Mixture, Wako] and 0.5% [w/v] 2-morpholinoethanesulfonic acid [MES], pH 5.5) (Gamborg et al. 1968). The spore suspension was incubated at 22°C in the dark or under white light (120 µmol m⁻² s⁻¹) for 24 h and then incubated for another 24 h under white light (120 µmol m⁻² s⁻¹) (Supplementary Figure S1A, B). In the case of treatments with cytoskeleton-disrupting reagents, the spore suspension was spread on cellophane on agar medium (1% agar, half-strength Gamborg’s B5 salts, 0.5% [w/v] MES, pH 5.5), containing 100 µM oryzalin (Oryz), 100 µM latrunculin B (LatB), or 1/500 volume of DMSO for the mock treatment, respectively, and was incubated according to the regimes described in Supplementary Figure S1C, D.

Cell staining

To visualize the plasma membrane, the spores were treated with 8 µM N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)-phenyl)hexatrienyl)pyridinium dibromide (FM4-64) (Life Technologies) in a liquid medium containing half-strength Gamborg’s B5 salts and 0.5% (w/v) MES (pH 5.5) for 5 min at 22–25°C, immediately before observation.

Microscopy

To observe the fluorescent images of chlorophyll and the bright-field images in the same cell, we used a fluorescence microscope (BX51; Olympus) equipped with a charge coupled device (CCD) camera system (DP70; Olympus). To capture confocal images, spores were mounted with liquid 1/2 Gamborg’s B-5 medium (pH 5.5) on 35-mm diameter Petri dishes with 14-mm diameter coverslip windows at the bottom (Matsunami Glass Ind. Ltd.) and covered with a cover glass (Matsunami Glass Ind. Ltd.) to prevent drying. The specimens were observed using an inverted microscope (IX70, Olympus) equipped with a UPlanSApo 60×/1.2 water immersion objective lens and a spinning disk confocal laser scanning unit (CSU10; Yokogawa Electric Co., Ltd.) with a cooled CCD camera (CoolSNAP HQ2; Photometrics). For Citrine imaging, 488 nm excitation lasers (HPU-50101-PFS2; Furukawa, Tokyo, Japan), with strength 10 or 12.5 mW and 524–546 nm emission filters (FF01-535/22-25; Semrock, Rochester, NY, USA), were used. For FM4-64 imaging, 561 nm excitation lasers (85-YCA-025-040; CVI Melles Griot, Albuquerque, NM, USA) and 604–644 nm emission filters (FF01-624/40-25, Semrock) were used. To detect autofluorescence of chloroplasts, 488 nm excitation lasers (HPU-50101-PFS2; Furukawa, Tokyo, Japan) and 604–644 nm emission filters (FF01-624/40-25, Semrock) were used. Using METAMORPH software (Universal Imaging A), a series of optical sections were acquired at 0.5-µm intervals, and time-lapse images were captured at a single focal length in 2-min intervals for 120–180 min. Maximum intensity projection images were constructed from serial optical sections using the FIJI software (Schindelin et al. 2012).

Results

Spores are divided asymmetrically upon germination

The mature spores of M. polymorpha had a rounded, hemispherical shape, and no chlorophyll signal was detected (Supplementary Figure S2D, Figures 1A, D). After incubation on the medium in the dark for 24 h, water-absorbed spores became slightly enlarged into a rounder shape, in which chlorophyll signals were recognized (Figures 1B, E). Further 24-h incubation under continuous white light induced enlargement and germination in many spores, which possessed mature enlarged chloroplasts (Figures 1C, F). These results are consistent with observations by Nakazato et al. (1999). Visualization of the plasma membrane with FM4-64 showed that teardrop-shaped or tadpole-shaped germinated spores underwent the first division and that even round-shaped spores, which did not appear to have germinated, often completed the first division (Figures 1G, H). Therefore, the first cell division of spores occurs before the visible shape changes directed by tip growth.

Figure 1. Spore germination in Marchantia polymorpha. A–F. Spores before and after germination. Mature spores (A, D), spores after the 24-h incubation in the dark (B, E), and those after 24-h incubation under light following the 24-h dark incubation (C, F). The fluorescent images of chloroplasts shown in panels D, E, and F are of the same area as the bright-field images shown in panels A, B, and C, respectively. Bar=10 µm. G, H. First division plane, observed after the 48-h incubation under light (Supplementary Figure S1A). The fluorescent image was a maximum intensity projection of 30 serial images with 0.5-µm interval. Arrowheads, the first cell division; red, chloroplasts; cyan, FM4-64. Bar=10 µm.

Microtubule arrangements dynamically change before the first asymmetric division

To reveal intracellular processes during the first asymmetric division, we investigated the dynamics of microtubule structures in germinating spores using transgenic spores expressing fluorescently labeled tubulin and Citrine-MpTUB2 under the control of the CaMV 35S promoter. A small number of fine microtubules were observed on the surface of the water-absorbed spores after incubation in the dark for 24 h (Figure 2A). In a population of spores that had been incubated for 24 h under white light after incubation in the dark for 24 h, various types of microtubule arrangements were observed (Figure 2B). Imbibed unicellular spores before the first cell division were classified into five groups according to their microtubule configuration: 1) cortical meshwork, 2) inner radial array with cortical meshwork, 3) prospindle, 4) metaphase spindle, and 5) phragmoplast (Figure 2B). The most abundant microtubule configuration among the unicellular spores was the cortical meshwork recognized in groups 1 and 2 (Figure 2C). As the microtubule arrays in groups 3–5 (prospindle, metaphase spindle, and phragmoplast) were characteristic of the mitotic phase of the cell cycle, it was obvious that mitosis proceeded in light-irradiated spores. We occasionally observed a sixth type, bicellular sporelings (group 6), which had undergone the first cell division (Figure 2B). The ratio of the populations in group 1 decreased depending on the duration of the light irradiation, while the ratio of the number of spores and sporelings in and after the mitotic phase (groups 3–5 and group 6, respectively) increased (Figure 2C).

Figure 2. Microtubule arrangements in germinating spores. A. A water-absorbed spore after the dark incubation. Top, transmitting image; middle, peripheral image; bottom, medial image. Green, Citrine-MpTUB2; red, chloroplast autofluorescence. Bar=10 µm. B. Classification of six microtubule arrangements in spores, in which germination is induced by 24-h incubation under light after water absorption in the dark. Microtubule arrangements belonging to groups 1–5 were observed in pre-dividing unicellular spores and those belonging to group 6 were observed in post-dividing bicellular spores. Top, transmitting images; middle, confocal images of Citrine-MpTUB2 in the peripheral region of the spore (peri); bottom, confocal images of Citrine-MpTUB2 in the medial region of the spore (med). Bar=10 µm. C. The ratio of variations in microtubule arrangements in the spores after different incubation times under light. The numbers on the right indicate the groups classified in A. D. Size distribution of unicellular spores belonging to group 1, group 2, and groups 3–5 after 20–24 h of light incubation. n=213.

Although the size of spores was identical before or after imbibition in the dark, the spores enlarged into various sizes after 24 h of light irradiation (Supplementary Figures S2A, B, C). Classification by size indicated that the smaller spores showed the cortical microtubule meshwork, while the larger ones exhibited mitotic microtubule arrays (Figure 2D, top and bottom). The spores belonging to group 2, which had radial microtubule organization in addition to the cortical meshwork, were predominant in the middle size (Figure 2D). These results suggest that light irradiation induces a series of dynamic changes in the microtubule organization in the imbibed spores; the cortical microtubule meshwork is organized first, an inner radial array appears, and then mitotic structures are composed.

The “prospindle” (or “prophase spindle”), which appears in prophase before the break down of the nuclear envelope, is a microtubule array surrounding the nucleus, defined by two oppositely positioned bright poles in M. polymorpha (Buschmann et al. 2016; Yamada and Goshima 2017). We recognized the astral structure of microtubule fibers, or the “polar organizer”, arranged at the pole of the prospindle (Figure 3A). Furthermore, we noticed that the prospindle was localized in the center of some spores, but unilaterally in others (Figures 2A, 3B, C). In the latter case, one of the poles seemed to be near the periphery (Figure 3C, arrowhead). We calculated the ratios of the spores with center-stayed prospindles to those with periphery-localized prospindles using the data set in Figure 2C (Supplementary Figure S3A). The ratio of the periphery-localized prospindles was 23.5% after 20-h of incubation and the ratio increased to 52.5% after 24-h incubation, showing an increase in the peripheral localization of prospindles in an incubation time-dependent manner (Supplementary Figure S3B). These results indicate the migration of the prospindle from the center to the periphery.

Figure 3. Prospindle localization in the spore. A. Top view of the prospindle. B. Side view of the prospindle localized in the center of the spore. C. Side view of the prospindle localized in the peripheral region of the spore. Green, Citrine-MpTUB2; magenta, chlorophyll auto fluorescence. Fluorescence images: maximum intensity projection images of six serial images with 0.5-µm interval. Right panels, transmitting images; arrowhead, a prospindle pole close to the periphery. Bars=10 µm.

Polarized migration of the prospindle

To examine migration, we performed live-cell confocal imaging of the prospindle, both of poles of which were separated from the periphery, in the unicellular spore. As shown in Figure 4, the prospindle was globose in shape during the first 60 min of observation (Figure 4, Supplementary Movie S4). The prospindle then elongated into a fusiform shape and gradually migrated from the center to the periphery in the direction of one of the poles (Figure 4, Supplementary Movie S4). Finally, one of the poles arrived near the periphery and stopped migration (Figure 4, arrowhead). Six of the 10 prospindles showed similar polarized migration within our 180-min observations. The other prospindles stayed at the center and did not progress to mitosis during observation. It is possible that the cellular activity of these 4 spores was damaged by laser irradiation, had insufficient aeration in the liquid medium on the microscope, or there was insufficient light for germination. Consequently, these results support the possibility that the prospindle migrates from the center to the periphery, either according to cell polarity or to establish it, before the first asymmetric division.

Figure 4. Prospindle migration from the center to the periphery. A series of 10-min interval images of the prospindle at the center of the spore. Numbers indicate the time (min) after the beginning of the observation. Arrowheads, prospindle pole moving close to periphery. Bar=10 µm.

The polarized prospindle proceeds to mitosis

Further observation of the polarized prospindle revealed that the prospindle at the periphery was reassembled into the mitotic spindle. Microtubules constituting the prospindle seemed to be excluded from the nucleus, consistent with the fact that the nuclear envelope was intact during prophase (t=−08 to −04) (Figure 5). We observed that several microtubule fibers were stretching into the previous intra-nuclear area, as shown in Figure 5 (t=00), suggesting the occurrence of nuclear envelope breakdown in prometaphase. Four minutes after the breakdown of the nuclear envelope, a typical barrel-shaped mitotic spindle appeared (t=04) (Figure 5, Supplementary Movie S5). Approximately 15 min after spindle formation, some microtubules seemed to accumulate near the spindle poles (t=16–20), suggesting a chromosome separation process. Furthermore, we recognized the organization of the phragmoplast (t=24) (Figure 5, Supplementary Movie S5). Finally, the ring-shaped phragmoplast expanded centrifugally (t=28–56) (Figure 5, Supplementary Movie S5). These results suggest that the duration from nuclear envelope breakdown to chromosome separation in the first mitotic division of the spore is less than 30 min, which could be the reason only a few spores showing the mitotic spindle were observed among the population of light-irradiated spores (Figure 2).

Figure 5. Polarized prospindle proceeds to the mitotic rearrangements. A transmitting image and a series of fluorescent images of the polarized prospindle at 4-min intervals focused on the medial plane of the spore. Numbers indicate the time (min) before and after the appearance of the mitotic spindle. Green, Citrine-MpTUB2; magenta, chlorophyll auto-fluorescence. Bar=10 µm.

Localization of chloroplasts is coordinated during the asymmetric division

Live-cell imaging of the spores in this study also revealed that the localization of chloroplasts was coordinated during the first asymmetric division. Chloroplasts were localized randomly in the spores after imbibition in the dark (Figures 1E, 2B) and in the spores exhibiting the cortical meshwork of microtubules after the subsequent light irradiation. However, they accumulated centripetally to the poles of the prospindle (Figure 3A). Even during polarized prospindle migration, each chloroplast always appeared as being attracted to either one of the poles. It was occasionally observed that some chloroplasts that had originally accumulated at one pole did not stay at that location and instead became attracted to the other pole. All chloroplasts were released upon the organization of the phragmoplast (Figure 5, Supplementary Movie S5). After the first division, not only the larger daughter cell but also the smaller cell contained chloroplasts (Figures 1G, H). This suggests that the accumulation of chloroplasts to the poles of the prospindle ensures chloroplast allocation to both asymmetric daughter cells.

Disruption of the cytoskeletons inhibited asymmetric division

Finally, we examined the effects of cytoskeleton disruption on asymmetric division. On the mock medium, the spores germinated into a tear-drop shape after 2 day of incubation under continuous white light, and after 7 day, grew into a multicellular cluster with some rhizoids (Figure 6A). In the presence of the cytoskeleton-disrupting reagents Oryz (100 µM) or LatB (100 µM), the spores did not germinate after 2 day of incubation. After incubation for 7 day, the spores treated with Oryz were still unicellular, but had become large and spherical in shape (Figure 6A), suggesting that microtubule disruption inhibits cell division and tip growth, while cell expansion occurs. On the other hand, the sporelings treated with LatB grew into small multicellular clusters with no rhizoid after a 7-d incubation (Figure 6A). Furthermore, a short treatment with LatB before the first cell division resulted in the tendency of cell plate formation at a more symmetric position (Figure 6B). The ratio of the spore with expanding cell plate or complete cell wall was 27% (n=172) in the mock-treated spores in Figure 6B, and the average ratio of cell size between the daughter cells (small-cell area/large-cell area) was calculated to be 0.25±0.0094 (n=28) from bicellular spores. On the other hand, cell plate formation was hardly observed in LatB-treated spores, in which the ratio of the spores with expanding cell plates was 1.2% (n=284). Although no bicellular spores were observed, the average ratio of predicted cell size between the daughter cells (predicted small-cell area/predicted large-cell area) was calculated to be 0.77 (n=3). The positions of the occasionally observed prospindles were neither center-localized nor polarize-positioned in LatB-treated spores, as shown in Figure 6C. These results suggest that disruption of the actin filaments inhibits “asymmetric” division and tip growth, resulting in cell clusters with no differentiated rhizoids.

Figure 6. Disruption of the cytoskeleton inhibits asymmetric division. A. Spores and sporelings incubated under continuous white light in the absence or presence of cytoskeleton disrupting reagents oryzalin (Oryz, 100 µM) and latrunculin B (LatB, 100 µM) for 2 and 7 day. B. The nascent cell plate visualized using FM4-64, in the sporelings incubated in the dark for 24 h followed by incubation in continuous white light for 24 h (incubation in the absence or presence of LatB for the final 8 h). C. The microtubule arrangements in a spore incubated under the dark for 24 h followed by incubation in continuous white light for 22 h (incubation in the absence or presence of LatB for the final 6 h). Left panel, the peripheral region (peri); right panel, the medial region (med). Green, Citrine-MpTUB2; magenta, chlorophyll auto fluorescence. Bars=10 µm.

Discussion

Asymmetric division upon spore germination in M. polymorpha

“Germination” is a well-known phenomenon and is the first step of the developmental process in plants. It is generally recognized as a change in morphology accompanied by protrusion when dormant plant reproductive products, such as spores, pollen, or seeds, become active and bud. In M. polymorpha, spore germination is characterized by a change in form from round to tadpole shape (Inoue 1960) (Figure 1B, C). In a sporeling, which is formed after spore germination under sufficient light, it is easy to distinguish the two types of cells: one with a high density of chloroplasts, and the other with a low density (Inoue 1960; Kohchi et al. 2021; Shimamura 2016). Even in the teardrop-shaped spore at an earlier stage, the two cells are distinguishable under a differential interference microscope. Based on these observations, it has been accepted that asymmetric cell division occurs during germination under sufficient light, generating two different daughter cells: the small rhizoid cell, and the large germ cell (Kohchi et al. 2021; Shimamura 2016). However, the subcellular processes involved in asymmetric cell division during spore germination are largely unknown. In this study, we elucidated that not only the teardrop-shaped spores but also the globose-shaped spores underwent the first division (Figure 1G, H). Live-cell imaging analysis also confirmed that the first cell division progressed in the spores that appeared as being more rounded (Figures 3, 4). These results suggest that the first asymmetric cell division can proceed earlier than the initiation of tip growth in the spores of M. polymorpha.

Microtubule dynamics during asymmetric division of spores

Our live-cell imaging analyses demonstrated the microtubule dynamics during the first asymmetric division of spores as follows: 1) light irradiation induces organization of the peripheral random meshwork in the water-absorbed spore, 2) radial microtubules appear, 3) the prospindle with two poles is organized around the nucleus, 4) metaphase spindle formation occurs, and 5) the phragmoplast expands to complete division. These microtubule dynamics were similar to those observed during somatic cell division in the thallus of M. polymorpha, except for preprophase bands (PPBs) (Buschmann et al. 2016; Lee and Liu 2019; Yamada and Goshima 2017). PPBs were never observed during the first cell division of the spore in this study. However, in the process of somatic cell division in the thallus, after the appearance of polar organizers on the nuclear envelope in the early mitotic phase, cortical PPBs are assembled before the characteristic bipolar prospindle formation (Buschmann et al. 2016). Our results are consistent with the fact that PPBs are absent from protonema cells at an early stage of development, including sporelings, in the moss Physcomitrium patens (Doonan et al. 1987). On the other hand, the unique changes in microtubule organization have been elucidated in the meiotic division of spore-mother cells of M. polymorpha (Brown et al. 2010). During sporogenesis, microtubule organizing centers were formed on the chloroplasts, and the prospindle was formed in a tetrad structure with four poles. Prior to meiosis I, the chloroplast was enlarged and then divided into four chloroplasts, which located themselves at the periphery of the tetrad position. This chloroplast-dependent microtubule organization seems to allow each spore to possess at least one plastid. During the asymmetric division of the spore, polar organizers seemed to be located on the nuclear envelope, similar to the case in thallus cells (Figures 2, 3). However, chloroplasts accumulated at the prospindle poles before chromosome segregation in the spore (Figure 3), suggesting a possible association between the mitotic microtubule apparatus and chloroplasts.

The most remarkable finding in this study of microtubule dynamics is that the position of the prospindle changes from the center to the periphery in the globose-shaped spores (Figures 3, 4). Time-lapse imaging revealed that the prospindle migrated to the direction of one of the poles before the nuclear envelope broke down (Figure 4). Our findings suggest that polarized prospindle localization causes the following division to be unequal, producing large and small daughter cells. Prospindle migration is the earliest intracellular asymmetry reported in spores.

Because the spores are generally formed in a tetrahedral arrangement through the meiotic division from the spore mother cells, it has been suggested that the spores have a proximal-distal polar axis. In most Marchantiales species, mature spores have distinct triradiate marks on the coat at the proximal site of the spore because of the former tetrahedral positioning (Inoue 1960). These spores germinate at certain positions, which can be predicted based on the coat structure. On the other hand, in M. polymorpha, the mature spores have no distinct triradiate marks on the coat, and thus, the proximal-distal polar axis cannot be predicted from the coat structure (Inoue 1960) (Supplementary Figure S2D, Figure 1B). Furthermore, during germination, the spore coat became fragmented and adhered to the surface of the spores after water absorption; therefore, the germination site was unrecognizable on the coat structure (Inoue 1960) (Supplementary Figure S2E). The detailed observations of microtubule organization in this study revealed that no obvious structure predicting the future division plane, including PPBs, was formed during the first division of the spore, similar to the case of the suspension cells (Buschmann et al. 2016). Thus, it is still unclear whether the germination site is predetermined in the spores of M. polymorpha, i.e., whether the prospindle migrates according to cell polarity or certain marks. However, we determined the mechanism underlying spore asymmetry. The first division was not asymmetric when the actin cytoskeleton was disrupted, and the spores occasionally continued to divide and form a cluster of several cells in the presence of actin-filament disrupting reagents (Figure 6), suggesting that actin filaments are required for the division to be asymmetric. Further investigation to explore the roles of actin filaments in the asymmetric division of spores should be the next step toward the elucidation of the molecular mechanism of spore germination.

Abbreviations

CCD

charge coupled device

DMSO

dimethyl sulfoxide

FM4-64

N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl) pyridinium dibromide

LatB

latrunculin B

MES

2-morpholinoethanesulfonic acid

Oryz

oryzalin

PPB

preprophase band

TUB

β-tubulin

Conflict of interest statement

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.