Technology Live-Cell Imaging and Optical Manipulation of Arabidopsis Early Embryogenesis Graphical Abstract Authors Keita Gooh, Minako Ueda, Kana Aruga, ..., Hideyuki Arata, Tetsuya Higashiyama, Daisuke Kurihara Correspondence [email protected] In Brief Gooh et al. establish a live-embryo imaging system for Arabidopsis and generate a complete lineage tree from double fertilization in early embryogenesis. They provide a platform for real-time analysis of cell division dynamics and cell fate specification using optical manipulation and micro- engineering techniques such as laser irradiation of specific cells. Highlights d A live-embryo imaging system for Arabidopsis is established d A complete lineage tree from double fertilization to 64-cell embryo uses this system d Endosperm development is not required for cell patterning during early embryogenesis d Damage to an embryo initial cell induces rapid cell fate conversion in the suspensor Gooh et al., 2015, Developmental Cell 34, 242–251 July 27, 2015 ª2015 Elsevier Inc. http://dx.doi.org/10.1016/j.devcel.2015.06.008 Developmental Cell Technology Live-Cell Imaging and Optical Manipulation of Arabidopsis Early Embryogenesis Keita Gooh,1 Minako Ueda,1,2 Kana Aruga,1 Jongho Park,1,3,4 Hideyuki Arata,1,3 Tetsuya Higashiyama,1,2,3 and Daisuke Kurihara1,3,* 1Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8602, Japan 2Institute of Transformative Bio-Molecules (ITbM), Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8602, Japan 3Higashiyama Live-Holonics Project, ERATO, JST, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8602, Japan 4Present address: Precision and Intelligence Laboratory, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8503, Japan *Correspondence: [email protected] http://dx.doi.org/10.1016/j.devcel.2015.06.008 SUMMARY to the embryo (Kawashima and Goldberg, 2010; Lau et al., 2012; Wendrich and Weijers, 2013). In addition to cell fate specification Intercellular communications are essential for cell of the apical and basal cells, the fate of a variety of other cell proliferation and differentiation during plant embryo- types is determined during embryogenesis, such as epidermis genesis. However, analysis of intercellular commu- formation when the radial axis is established at the 16-cell em- nications in living material in real time is difficult bryo stage. However, the regulation of pattern formation during owing to the restricted accessibility of the embryo embryogenesis remains unknown. within the flower. We established a live-embryo Flowering plants produce two sperm cells; one fuses with the egg cell to form the zygote and the other fuses with the other imaging system to visualize cell division and cell Arabidopsis thaliana female gamete, the central cell, to form the endosperm. This fate specification in from process is termed double fertilization (Mansfield et al., 1991). zygote division in real time. We generated a cell- The central cell also contributes to early embryogenesis. For division lineage tree for early embryogenesis in example, a central-cell-derived small cysteine-rich peptide, Arabidopsis. Lineage analysis showed that both ESF1, is required for normal pattern formation of embryos (Costa the direction and time course of cell division be- et al., 2014). The endosperm provides nutrition to the embryo (Li tween sister cells differed along the apical-basal and Berger, 2012) and is considered to be an important contrib- or radial axes. Using the Arabidopsis kpl mutant, utor to embryogenesis in developing seeds. In addition, the sus- in which single-fertilization events are frequent, pensor is believed to supply nutrients and growth factors to the we showed that endosperm development is not proembryo and thus play an important role in embryogenesis by required for pattern formation during early embryo- connecting the proembryo to the surrounding tissue (Kawashima and Goldberg, 2010). In spite of these distinct morphological fea- genesis. Optical manipulation demonstrated that tures, little is known about the contribution of the endosperm and damage to the embryo initial cell induces cell fate suspensor to the timing of cell division timing and pattern forma- conversion of the suspensor cell to compensate tion of the embryo. for the disrupted embryo initial cell even after cell Along the apical-basal axis, several factors are expressed in fate is specified. specific regions of the embryo to regulate embryo development (Wendrich and Weijers, 2013). Among the first genes to become activated in the apical cell, DORNRO¨SCHEN (DRN) (also known INTRODUCTION as ENHANCER OF SHOOT REGENERATION1; ESR1) encodes an AP2-type transcription factor and is expressed in the apical In plants, the first division of the fertilized egg cell (zygote) is crit- cell derivatives after the first zygotic division (Cole et al., 2009; ical to establish the apical-basal axis and to produce two Kirch et al., 2003). Given that embryo pattern formation fails in daughter cells with different developmental fates (Mansfield the double mutant of DRN and its closest homolog, DRN-LIKE and Briarty, 1991). The zygote divides asymmetrically to form a (DRNL), resulting in development of an embryo without cotyle- small cytoplasm-rich apical cell and a large vacuolated basal dons, DRN is thought to be a key regulator of embryo formation cell (1-cell embryo stage). This asymmetric zygotic division is (Cole et al., 2009; Kirch et al., 2003). common in plants. The apical cell, which acts as the embryo WUSCHEL RELATED HOMEOBOX (WOX) transcription fac- initial cell, proliferates in a well-organized pattern to generate tors also show region-specific expression and are essential the proembryo, which is the precursor of almost all cells of the for the formation and maintenance of the apical-basal axis plant body (Ueda and Laux, 2012). The basal cell, which acts (Breuninger et al., 2008; Haecker et al., 2004; Ueda et al., as the suspensor initial cell, repeatedly divides horizontally to 2011). After zygote division, WOX2 expression is restricted to generate the filamentous suspensor, which connects the proem- the apical cell derivatives and regulates embryo development. bryo to the surrounding maternal tissues and transports nutrients In contrast, WOX8 expression is restricted to the basal cell 242 Developmental Cell 34, 242–251, July 27, 2015 ª2015 Elsevier Inc. derivatives and regulates suspensor development (Breuninger RESULTS AND DISCUSSION et al., 2008; Ueda et al., 2011). In addition, WOX8 regulates em- bryo development via non-cell-autonomous activation of WOX2 Development of Live-Cell Imaging System for (Breuninger et al., 2008). However, this mechanism remains un- Arabidopsis Embryogenesis clear, and it is unknown how the apical and basal cell fates are Angiosperm embryogenesis in vivo occurs within multiple layers related. of maternal tissues in the flower; therefore, it is difficult to observe and analyze the real-time dynamics in living material DESIGN (Figure 1A). To enable live-cell analysis of early embryogenesis, we developed an in vitro ovule culture system using Arabidopsis The spatiotemporal development of the apical and basal cells, thaliana. First, we examined the effect of medium composition and how their distinct fates are specified, are also poorly under- on ovule growth and embryo development. Nitsch medium sup- stood. Early embryogenesis in vivo occurs within the ovule in the plemented with 5% trehalose resulted in the highest percentage flower. Sauer and Friml (2004) developed an in vitro culture of ovule survival (Figure 1B). Compared with the previously method for fertilized ovules in Arabidopsis, but direct observa- developed in vitro culture medium (Sauer and Friml, 2004), the tion of early stages of embryogenesis was difficult due to the frequency of normal embryo development was significantly low survival rate of young ovules containing a zygote. An effi- higher after incubation for 24, 48, and 72 hr in Nitsch medium cient in vitro culture system for development of the zygote into supplemented with 5% trehalose (Figure 1C). Furthermore, using a mature seed is yet to be established in Arabidopsis. Currently, the latter medium, the embryos were able to develop into adult our knowledge of early embryo development in plants is mostly plants (Figures 1D and 1E). Thus, trehalose was effective for derived from snapshot images of fixed samples. Recently, con- ovule growth and embryo development in A. thaliana. In several struction of a four-dimensional map from a large number of fixed angiosperms, including monocots and dicots, exogenous treha- embryo samples has contributed to an improved understanding lose application confers high desiccation tolerance to somatic of cell division patterns during early embryogenesis in Arabidop- embryos of barley (Ryan et al., 1999) and a greater adaptability sis (Yoshida et al., 2014). However, studies that use fixed sam- to environmental conditions during acclimatization in jojoba ples lack real-time temporal information such as the length of (Llorente et al., 2007), and promotes long-term in vitro vegetative the cell cycle in specific cell types; therefore, how cell fates culture in Torenia fournieri (Yamaguchi et al., 2011). Trehalose are spatiotemporally specified remains largely unknown both accumulation is associated with exposure to abiotic and biotic at the cellular and molecular levels. Moreover, direct interven- stress in many plants (Fernandez