Name: in vitro ovule cultivation for live-cell imaging of zygote polarization and embryo patterning in arabidopsis thaliana
Uploaded: 2017-09-11T14:00:00
Description: Watch this Scientific Journal Video about In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana at JoVE.com

Microscopy in this work was conducted at the Institute of Transformative Bio-Molecules (WPI-ITbM) of Nagoya University and supported by the Japan Advanced Plant Science Network. This work was supported by grants from the Japan Science and Technology Agency (ERATO project to T.H. and M.U.) and from the Japan Society for the Promotion of Science: a Grant-in-Aid for Scientific Research on Innovative Areas (Nos. JP24113514, JP26113710, JP15H05962, and JP15H05955 for M.U., and Nos. JP16H06465, JP16H06464 and JP16K21727 for T.H), a Grant-in-Aid for Young Scientists (B, Nos. JP24770045 and JP26840093 for M.U.), and a Grant-in-Aid for challenging Exploratory Research (No. JP16K14753 for M.U.).

1. Preparation of the In Vitro Ovule Culture Medium
<ol>
	<li>Make the liquid medium forthe in vitro ovule culture (&#34;N5T medium&#34;) containing 1x Nitsch basal salt mixture, 5% (w/v) trehalose dihydrate, 0.05% (w/v) 2-(N-morpholino)ethanesulfonic acid (MES)-KOH (pH 5.8), and 1x Gamborg&#39;s vitamin solution.</li>
	<li>Adjust the pH to 5.8 with KOH.</li>
	<li>Sterilize the medium by autoclaving it (121 &#176;C, 20 min) or filtering it through a 0.22-&#181;m filter. 
	NOTE: The sterilized med...

<ol>
	<li>Natesh, S., Rau, M. A., Johri, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Embryology of Angiosperms. , 377-443 (1984).</li><li>Kranz, E., Lorz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+fertilisation+of+maize+by+single+egg+and+sperm+cell+protoplast+fusion+mediated+by+high+calcium+and+high+pH.">In vitro fertilisation of maize by single egg and sperm cell protoplast fusion mediated by high calcium and high pH.</a> Zygote. 2 (2), 125-128 (1994).</li><li>Uchiumi, T., Uemura, I., Okamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+an+in+vitro+fertilization+system+in+rice+(Oryza+sativa+L.).">Establishment of an in vitro fertilization system in rice (Oryza sativa L.).</a> Planta. 226 (3), 581-589 (2007).</li><li>Sun, M. X., Moscatelli, A., Yang, H. Y., Cresti, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+double+fertilization+in+Nicotiana+tabacum+(L.):+the+role+of+cell+volume+in+cell+fusion.">In vitro double fertilization in Nicotiana tabacum (L.): the role of cell volume in cell fusion.</a> Sex Plant Rep. 13 (4), 225-229 (2001).</li><li>Tian, H. Q., Yuan, T., Russell, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+double+fertilization+and+the+cell+cycle+in+male+and+female+gametes+of+tobacco.">Relationship between double fertilization and the cell cycle in male and female gametes of tobacco.</a> Sex Plant Rep. 17 (5), 243-252 (2004).</li><li>Costa, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+cell-derived+peptides+regulate+early+embryo+patterning+in+flowering+plants.">Central cell-derived peptides regulate early embryo patterning in flowering plants.</a> Science. 344 (6180), 168-172 (2014).</li><li>Sauer, M., Friml, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+culture+of+Arabidopsis+embryos+within+their+ovules.">In vitro culture of Arabidopsis embryos within their ovules.</a> Plant J. 40 (5), 835-843 (2004).</li><li>Gooh, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+imaging+and+optical+manipulation+of+Arabidopsis+early+embryogenesis.">Live-cell imaging and optical manipulation of Arabidopsis early embryogenesis.</a> Dev Cell. 34 (2), 242-251 (2015).</li><li>Park, J., Kurihara, D., Higashiyama, T., Arata, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+microcage+arrays+to+fix+plant+ovules+for+long-term+live+imaging+and+observation.">Fabrication of microcage arrays to fix plant ovules for long-term live imaging and observation.</a> Sens Act B-Chem. 191, 178-185 (2014).</li><li>Kimata, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoskeleton+dynamics+control+the+first+asymmetric+cell+division+in+Arabidopsis+zygote.">Cytoskeleton dynamics control the first asymmetric cell division in Arabidopsis zygote.</a> Proc Natl Acad Sci U S A. 113 (49), 14157-14162 (2016).</li><li>Nambo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+of+Synthetic+Chemistry+and+Live-Cell+Imaging+Identified+a+Rapid+Cell+Division+Inhibitor+in+Tobacco+and+Arabidopsis+thaliana.">Combination of Synthetic Chemistry and Live-Cell Imaging Identified a Rapid Cell Division Inhibitor in Tobacco and Arabidopsis thaliana.</a> Plant Cell Physiol. 57 (11), 2255-2268 (2016).</li><li>Mizuta, Y., Kurihara, D., Higashiyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+with+longer+wavelength+excitation+in+intact+Arabidopsis+tissues.">Two-photon imaging with longer wavelength excitation in intact Arabidopsis tissues.</a> Protoplasma. 252 (5), 1231-1240 (2015).</li></ol>

This manuscript introduces a simple in vitro ovule cultivation protocol that is efficient for use in the live-cell imaging of developing zygotes and embryos.
The design of the PDMS device may need optimization according to the embryo stage. The first developed device was a microcage array to adjust the orientation and to fix the position of the ovules9, and then a micropillar device was constructed to trap ovules more efficiently8. Furth...

The basic body plan of an organism develops from a unicellular zygote. In most flowering plants, zygotic division generates an apical and a basal cell, which develop into the shoot and root, respectively1. Therefore, it is important to understand how the plant body is formed during embryogenesis, but there has not been an effective tool to directly observe the dynamics of living zygotes and embryos because they develop deep in the flower. In several monocot species, such as maize and rice, an in vitro fertilization method has been established2,3. In this method, isolated sperm and egg cells are fused electrically or chemically, and the generated cell can develop into a fertile plant. However, in dicot plants, there is no in vitro fertilization method that can produce proper embryos, presumably because of the non-synchronized cell cycle state of male and female gametes4,5. In addition, the embryo-surrounding tissue (endosperm) plays important roles in embryo development6.
In a model dicot species, A. thaliana, an in vitro cultivation method was developed by focusing on the whole ovule, which contains both the embryo and endosperm7. This system was successfully used to analyze the effects of various chemical reagents on embryogenesis, but it is not suitable for time-lapse imaging because it has a low survival rate. Therefore, a novel in vitro ovule cultivation system was developed in order to start as early as the zygote stage and produce fertile plants at a high ratio8. After various trials, it was found that Nitsch medium and trehalose significantly improved the survival rate of ovules8. In addition, because the ovule expands as it grows and thus frequently moves away from the observation field of the microscope, a PDMS device was developed to fix the ovule in the medium9. The PDMS device enabled the long-term imaging for 3 - 4 days, which is sufficient to trace the development from a zygote to a heart-stage embryo. Using this method, it becomes possible to visualize the dynamics of zygote polarization and embryo patterning, not only under normal conditions, but also in the presence of chemical inhibitors or in various mutant backgrounds8,10,11.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55975/55975fig1.jpg" /> 
Figure 1: Schematic Diagram of the Specific Fluorescent Markers used to Visualize Zygotes and Embryos Through the Ovule. 
The Arabidopsis zygote develops into an embryo in the ovule, which is generated deep inside the flower. In this in vitro cultivation system, the zygote and embryo are observed through the ovule, and thus it is important to use specific fluorescent markers that are not expressed in other ovule tissues. <a href="//cloudflare.jove.com/files/ftp_upload/55975/55975fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table width="606">
	<tbody>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">Nitsch basal salt mixture</td>
			<td style="width:104px;">Duchefa</td>
			<td style="width:119px;">N0223</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">trehalose dihydrate</td>
			<td style="width:104px;">Wako Pure Chemical</td>
			<td style="width:119px;">206-18455</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">MES</td>
			<td style="width:104px;">Dojindo</td>
			<td style="width:119px;">345-01625</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">Gamborg&#8217;s vitamin solution</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">G1019</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">35-mm glass-bottom dish</td>
			<td style="width:104px;">Matsunami Glass</td>
			<td style="width:119px;">D111300</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">35 mm culture dish</td>
			<td style="width:104px;">Corning</td>
			<td style="width:119px;">430588</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">PDMS</td>
			<td style="width:104px;">Dow Corning Co.</td>
			<td style="width:119px;">Sylgard184</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">76 &#215; 26 mm slide glass</td>
			<td style="width:104px;">Matsunami Glass</td>
			<td style="width:119px;">S1225</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">18 &#215; 18 mm slide glass</td>
			<td style="width:104px;">Matsunami Glass</td>
			<td style="width:119px;">C018181</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">needle (gauge 0.40mm)</td>
			<td style="width:104px;">Terumo</td>
			<td style="width:119px;">NN-2719S</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">Immersion medium Immersol W 2010</td>
			<td style="width:104px;">Zeiss</td>
			<td style="width:119px;">444969-0000-000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">A1R MP</td>
			<td style="width:104px;">Nikon</td>
			<td style="width:119px;">A1RsiMP(1080) Ti-E-TIRF</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="width:216px;height:84px;">CSU-W1</td>
			<td style="width:104px;">Yokogawa Electric</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">It is a customized equipment, and thus Catalog Number is not avairable.</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">CV1000</td>
			<td style="width:104px;">Yokogawa Electric</td>
			<td style="width:119px;">CV1000-SP84</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

in vitro ovule cultivation for live-cell imaging of zygote polarization and embryo patterning in arabidopsis thaliana

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

By using this ovule cultivation system, this method can trace the living dynamics of zygote polarization and embryo patterning. This is an achievement because previously there was no technique to visualize the real-time behavior of the zygote and embryo, which are hidden deep in the mother tissue. Figure 3A and Supplemental Video 1 show that the microtubules (MTs) in the young zygote accumulate as a transverse ring in the subapical region<sup...

Watch this Scientific Journal Video about In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana at JoVE.com

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

This manuscript describes an in vitro ovule cultivation method that enables live-cell imaging of Arabidopsis zygotes and embryos. This method is utilized to visualize the intracellular dynamics during zygote polarization and the cell fate specification in developing embryos.

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

The overall goal of this procedure is to enable live-cell imaging of Arabidopsis zygotes and embryos for visualizing the dynamics of their embryogenesis. This method can help answer key questions in the plant development field about how zygote assymetry and embryo morphology are generated. The main advantage of this technique is that we can trace the living dynamics of zygote polarization and embryo patterning.Visual demonstration of this method is critical as preparation with micropillar device and the ovules are difficult to explain by text only. To prepare PDMS micropillar array device, first cut the device so that it fits into a 35 millimeter glass-bottom dish and sterilize the top and bottom sides of the device with one 15-minute UV light exposure per side. When all of the sides have been sterilized, transfer the PDMS device into a new 35 millimeter culture dish and add five to seven milliliters of fresh N5T medium to the dish, until the device is completely submerged.Then place the dish into a vacuum chamber and reduce the pressure leaving the device in the vacuum until all of the air in the device had been replaced with medium. When the device has been degassed, use square tip tweezers to dab the device on a piece of paper towel to remove any excess medium from the sides and bottom. Then place the device onto a 76 by 26 millimeter slide glass and cover it with a 35 millimeter culture dish lid to keep the device hydrated.Before beginning the dissection, sterilize a 0.40 millimeter gauge needle and fine tweezers with 70 percent ethanol. Next, check the inflorescence of the plant to allow selection of the appropriate siliques for the experiment. The approximately five millimeter siliques contain zygotes and the eight to 10 millimeter siliques include young globular embryos.Using tweezers, transfer three to four siliques of interest onto a piece of double-sided tape on a 76 by 26 millimeter slide glass. Then transfer the slide under a stereo microscope and use the needle and tweezers to open the ovary walls. Transfer the open siliques onto the micropillar array of the PDMS device and release the ovules into the medium.Then place and 18 by 18 millimeter cover glass onto the device to push the ovules into the spaces of the micropillar array. When the ovules are in place, use tweezers or fingers to pull the cover glass horizontally off of the device to remove any excess medium, and place the device upside down into a new 35 millimeter glass-bottom dish. Use the tip of the square tweezers to slightly press the device into the bottom of the dish and pour fresh N5T medium into the dish until the device is completely submerged.Then, seal the dish with parafilm. For time lapse imaging of the ovules, transfer the dish onto the stage of an inverted microscope and use the marker fluorescence to select the ovules for imaging. When the optimal ovules have been located, begin live-imaging the samples according to the manufacturer's recommendations.This ovule cultivation system allows tracing of the living dynamics of zygote polarization and embryo patterning. For example, here at the accumulation of microtubules as a transverse ring, followed by their formation into a spindle, to be observed. The embryonic cells then quarternately divide to form a radially symmetrical globular shape.Treatment of the cultured ovules with an actin polymerization inhibitor, however, inhibits polar nuclear migration, resulting in a more symmetric division of the zygote and suggesting a role for actin filaments in zygote polarization. Once mastered, this technique can be completed in two hours if it is performed properly. Following this procedure laser disruption can be performed to answer additional questions about the role of cell to cell communication in embryo patterning.After watching this video, you should have a good understanding of how to visualize tight events during embryogenesis.

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Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

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A Method for Characterizing Embryogenesis in Arabidopsis

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A Method for Characterizing Embryogenesis in Arabidopsis

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Mass Isolation and In Vitro Cultivation of Intramolluscan Stages of the Human Blood Fluke Schistosoma Mansoni

This article describes a protocol for the large-scale axenic isolation and collection of free-swimming miracidia of the human blood fluke Schistosoma mansoni and their subsequent processing for introduction into in vitro culture.

Human blood flukes, Schistosoma spp., have a complex life cycle that involves asexual and sexual developmental phases within a snail intermediate and ...

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal and postnatal consequences. To ensure the innocuousness of ART applications, studies in animal models are necessary. In addition, as a last step, embryo development studies require evaluation of their capacity to develop full-term healthy offspring. Here, embryo transfer to the uterus is indispensable to perform any ARTs-related experiment.
The rabbit has been used as a model organism to study mammalian reproduction for over a century. In addition to its phylogenetic proximity to the human species and its small size and low maintenance cost, it has important reproductive characteristics such as induced ovulation, a chronology of early embryonic development similar to humans and a short gestation that allow us to study the consequences of ART application easily. Moreover, ARTs (such as intracytoplasmic sperm injection, embryo culture, or cryopreservation) are applied with suitable efficiency in this species.
Using the laparoscopic embryo transfer technique and the cryopreservation protocol presented in this article, we describe 1) how to transfer embryos through an easy, minimally invasive technique and 2) an effective protocol for long-term storage of rabbit embryos to provide time-flexible logistical capacities and the ability to transport the sample. The outcomes obtained after transferring rabbit embryos at different developmental stages indicate that morula is the ideal stage for rabbit embryo recovery and transfer. Thus, an oviductal embryo transfer is required, justifying the surgical procedure. Furthermore, rabbit morulae are successfully vitrified and laparoscopically transferred, proving the effectiveness of the described techniques.

Assisted reproductive techniques (ARTs) are in continuous evaluation to improve outcomes and reduce the associated risks. This manuscript describes a minimally invasive embryo transfer procedure with an efficient cryopreservation protocol that allows the use of rabbits as an ideal animal model of human reproduction.

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal ...

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits are required to control this behavior. Failure to produce the rhythmic pattern of contractions can be indicative of neurological abnormalities. We previously found that defects in protein O-mannosylation, a posttranslational protein modification, affect the axon morphology of sensory neurons and result in abnormal coordinated muscle contractions in embryos. Here, we present a relatively simple method for recording and analyzing the pattern of peristaltic muscle contractions by live imaging of late stage embryos up to the point of hatching, which we used to characterize the muscle contraction phenotype of protein O-mannosyltransferase mutants. Data obtained from these recordings can be used to analyze muscle contraction waves, including frequency, direction of propagation and relative amplitude of muscle contractions at different body segments. We have also examined body posture and taken advantage of a fluorescent marker expressed specifically in muscles to accurately determine the position of the embryo midline. A similar approach can also be utilized to study various other behaviors during development, such as embryo rolling and hatching.

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Here, we present a method to record embryonic muscle contractions in Drosophila embryos in a non-invasive and detail-oriented manner.

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits ...