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 medium can be stored at 4 &#176;C for 2 - 3 months.</li>
</ol>
2. Preparation of the PDMS Micropillar Array Device
<ol>
	<li>Cut the PDMS micropillar device by a knife, razor blade or scissors to fit into the glass part (14 mm &#966;) of a 35 mm glass-bottom dish. 
	NOTE: The full procedure of the PDMS device construction is described in previous papers8,9, and thus the details are omitted here. Glass-bottom dishes with smaller volumes, such as 4-well or 96-well plates, can be used for short-term imaging without the device.</li>
	<li>Sterilize the upper side of the PDMS device under UV light for 15 min.</li>
	<li>Turn the PDMS device over by using square tip tweezers, and keep it under UV light for 15 min to sterilize the bottom.</li>
	<li>Transfer the sterilized PDMS device into a 35 mm culture dish and add the N5T medium until the device is completely soaked in the medium (approximately 5 - 7 mL).</li>
	<li>Put the dish into a vacuum chamber, and reduce the pressure to degas. Keep vacuuming for 3 h to overnight until the air in the device is replaced by medium. 
	NOTE: The device can be detached by air bubbles under a strong vacuum.</li>
	<li>Take the device from the dish by holding it with square tip tweezers and put it on a paper towel to remove the extra medium on the side and bottom. 
	NOTE:The medium on the micropillar array (upper surface) should not be removed because this will be used for the ovule cultivation.</li>
	<li>Transfer the device onto a 76 mm x 26 mm slide glass, ensuring to keep the micropillar part as the upper side, and cover the device with 35 mm culture dish lid to prevent the medium from drying out during the following ovule extraction.</li>
</ol>
3. Silique Dissection and Ovule Extraction
<ol>
	<li>Sterilize a needle (0.40 mm G) and fine tweezers by wiping with 70% ethanol. The needle is easily handled by attaching it to a syringe or wood stick.</li>
	<li>Check the inflorescence of the plant, and select the proper siliques for the experiment. The approximately 5 mm siliques contain zygotes, and the 8 - 10 mm siliques include young globular embryos.</li>
	<li>Excise the siliques by using tweezers, and place them on double-sided tape on a 76 x 26 mm slide glass. One silique contains about 40 - 60 ovules, and 3 - 4 siliques (i.e., 120 - 240 ovules) are sufficient for one device.</li>
	<li>Open the silique (ovary wall) to see the ovules inside using the sterilized needle and tweezers under a stereomicroscope. Cut only the ovary wall so that the ovules are not damaged.</li>
	<li>Transfer the opened silique into the N5T medium on the PDMS device (prepared in step 2.7), and release the ovules into the medium with the needle or tweezers.</li>
	<li>Put a small cover glass (18 mm x 18 mm) onto the PDMS device to push the ovules into the spaces in the micropillar array.</li>
	<li>Take the cover glass off by pulling it horizontally using tweezers or fingers to remove extra medium.</li>
	<li>Turn the PDMS device upside down, place it into a 35 mm glass-bottom dish, and slightly press the device by pushing using the square tip tweezers to stick it to the glass.</li>
	<li>Pour N5T medium gently into the glass-bottom dish by decanting the medium bottle until the PDMS device is completely soaked in the medium, and seal the dish with paraffin film. 
	NOTE: The PDMS device can be recycled, but devices that are too old are easily detached from the glass bottom. Additionally, the device may float if it is not completely degassed in step 2.5 or the medium is poured too quickly.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55975/55975fig2.jpg" /> 
Figure 2: Schematic Procedure for Sample Preparation. 
This schematic flow corresponds to steps 3.5 to 4.1. <a href="//cloudfront.jove.com/files/ftp_upload/55975/55975fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Time-lapse Imaging
<ol>
	<li>Place the glass-bottom dish that was prepared in step 3.9 on an inverted microscope, and select suitable ovules to focus on. 
	NOTE: Because the ovules vary in stage, position, and direction, suitable ovules should be found by checking their marker fluorescence.</li>
	<li>Start the live-imaging according tothe manufacturer&#39;s instructions of the microscope system. 
	NOTE: The microscope equipment and parameters are diverse, and thus the users should choose the proper settings that fit to the experiment purpose. As an example, the settings used in this manuscript are listed in Table 1.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Equipment/setting</td>
			<td>Figure 3 (A) and Supplemental Videos 1 and 4</td>
			<td>Figure 3 (B) and Supplemental Video 2</td>
			<td>Supplemental Video 3</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>laser-scanning inverted microscope (A1R MP)</td>
			<td>spinning-disk confocal inverted microscope (CSU-W1)</td>
			<td>box-type inverted confocal microscope system with a stable incubation chamber (CV1000)</td>
		</tr>
		<tr>
			<td>Laser</td>
			<td>Ti:sapphire femtosecond pulse laser</td>
			<td>488-nm and 561-nm LD lasers</td>
			<td>488-nm and 561-nm LD laser</td>
		</tr>
		<tr>
			<td>Objective lens&#160;</td>
			<td>40X water-immersion objective lens (NA = 1.15) with immersion medium</td>
			<td>60X silicone oil immersion objective lens (NA = 1.30), mounted on a Piezo focus drive</td>
			<td>40X objective lens (NA = 0.95)</td>
		</tr>
		<tr>
			<td>Detector</td>
			<td>external non-descanned GaAsP PMT detector</td>
			<td>EMCCD camera</td>
			<td>EMCCD camera</td>
		</tr>
		<tr>
			<td>Dichroic mirror</td>
			<td>DM495 and DM560</td>
			<td>DM488/561</td>
			<td>DM400-410/488/561</td>
		</tr>
		<tr>
			<td>Filter</td>
			<td>band-pass filters; 534/30 nm and 578/105 nm</td>
			<td>band-pass filters; 520/35 nm, and 593/46 nm</td>
			<td>band-pass filters; 520/50 nm and 617/73 nm</td>
		</tr>
		<tr>
			<td>Slice along z-axis</td>
			<td>31 z-stacks with 1-&#181;m intervals</td>
			<td>17 z-stacks with 3-&#181;m intervals</td>
			<td>7 z-stacks with 5-&#181;m intervals</td>
		</tr>
		<tr>
			<td>Time interval</td>
			<td>20 min</td>
			<td>5 min</td>
			<td>10 min</td>
		</tr>
	</tbody>
</table>
Table 1: The Microscope Systems and Settings Used in This Manuscript. 
The microscopes and parameters are diverse, and thus each user should choose the suitable system for the experiment.
<ol start="3">
	<li>Check the acquired image sequence, and convert it to the general movie format, such as .avi or .mov for presentation. 
	NOTE: If the microscope software cannot output the images as a movie format, it is possible to save the images as .tif and then open them in ImageJ, an open source program inspired by NIH Image, to change the file type. ImageJ is provided at: https://imagej.nih.gov/ij/. ImageJ can also be used to make a Z-stuck movie, such as the maximum-intensity projection, as described at: https://imagej.net/Z-functions.</li>
</ol>

<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>

The authors have nothing to disclose.

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; ...

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10.8K Views.  Nagoya University. 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.

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

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

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the &#8220;hanging drop&#8221; method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3+ cells and gave rise to the canonical &#947;&#948; T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3-/- mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.

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

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

In Vitro Differentiation of Mature Myofibers for Live Imaging

Skeletal muscles are composed of myofibers, the biggest cells in the mammalian body and one of the few syncytia. How the complex and evolutionarily conserved structures that compose it are assembled remains under investigation. Their size and physiological features often constrain manipulation and imaging applications. The culture of immortalized cell lines is widely used, but it can only replicate the early steps of differentiation.
Here, we describe a protocol that enables easy genetic manipulation of myofibers originating from primary mouse myoblasts. After one week of differentiation, the myofibers display contractility, aligned sarcomeres and triads, as well as peripheral nuclei. The entire differentiation process can be followed by live imaging or immunofluorescence. This system combines the advantages of the existing ex vivo and in vitro protocols. The possibility of easy and efficient transfection as well as the ease of access to all differentiation stages broadens the potential applications. Myofibers can subsequently be used not only to address relevant developmental and cell biology questions, but also to reproduce muscle disease phenotypes for clinical applications.

In Vitro Differentiation of Mature Myofibers for Live Imaging

Muscle cells are among the most complex eukaryotic cells. We present a protocol for the in vitro differentiation of highly mature myofibers that allows for genetic manipulation and clear imaging during all developmental stages.

Skeletal muscles are composed of myofibers, the biggest cells in the mammalian body and one of the few syncytia. How the complex and evolutionarily ...

Live Confocal Imaging of Developing Arabidopsis Flowers

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. Recent advances in confocal microscopy, combined with the development of numerous fluorophores, have overcome these issues and opened the possibility to study the expression of several genes simultaneously, with a good cellular resolution, in live samples. Live confocal imaging provides plant biologists with a powerful tool to study development, and has been extensively used to study root growth and the formation of lateral organs on the flanks of the shoot apical meristem. However, it has not been widely applied to the study of flower development, in part due to challenges that are specific to imaging flowers, such as the sepals that grow over the flower meristem, and filter out the fluorescence from underlying tissues. Here, we present a detailed protocol to perform live confocal imaging on live, developing Arabidopsis flower buds, using either an upright or an inverted microscope.

Live confocal imaging provides biologists with a powerful tool to study development. Here, we present a detailed protocol for the live confocal imaging of developing Arabidopsis flowers.

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. ...

A Method for Characterizing Embryogenesis in Arabidopsis

Given the highly predictable nature of their development, Arabidopsis embryos have been used as a model for studies of morphogenesis in plants. However, early stage plant embryos are small and contain few cells, making them difficult to observe and analyze. A method is described here for characterizing pattern formation in plant embryos under a microscope using the model organism Arabidopsis. Following the clearance of fresh ovules using Hoyer's solution, the cell number in and morphology of embryos could be observed, and their developmental stage could be determined by differential interference contrast microscopy using a 100X oil immersion lens. In addition, the expression of specific marker proteins tagged with Green Fluorescent Protein (GFP) was monitored to annotate cell identity specification during embryo patterning by confocal laser scanning microscopy. Thus, this method can be used to observe pattern formation in wild-type plant embryos at the cellular and molecular levels, and to characterize the role of specific genes in embryo patterning by comparing pattern formation in embryos from wild-type plants and embryo-lethal mutants. Therefore, the method can be used to characterize embryogenesis in Arabidopsis.

A Method for Characterizing Embryogenesis in Arabidopsis

This protocol outlines a method for observing embryogenesis in Arabidopsis via ovule clearance followed by the inspection of embryo pattern formation under a microscope.

Given the highly predictable nature of their development, Arabidopsis embryos have been used as a model for studies of morphogenesis in plants. However, ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

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

Human blood flukes, Schistosoma spp., have a complex life cycle that involves asexual and sexual developmental phases within a snail intermediate and mammalian final host, respectively. The ability to isolate and sustain the different life cycle stages under in vitro culture conditions has greatly facilitated investigations of the cellular, biochemical and molecular mechanisms regulating parasite growth, development and host interactions. Transmission of schistosomiasis requires asexual reproduction and development of multiple larval stages within the snail host; from the infective miracidium, through primary and secondary sporocysts, to the final cercarial stage that is infective to humans. In this paper we present a step-by-step protocol for mass hatching and isolation of Schistosoma mansoni miracidia from eggs obtained from livers of infected mice, and their subsequent introduction into in vitro culture. It is anticipated that the detailed protocol will encourage new researchers to engage in and broaden this important field of schistosome research.

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 ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...