We thank Satoshi Kishigami, Sayaka Wakayama, Hiroaki Nagatomo, Satoshi Kamimura, and Kana Kishida for providing critical comments and technical support. This work was partially funded by the Ministry of Education, Culture, Sports, Science and Technology program for promoting the reform of national universities to M.O.; the Japan Society for the Promotion of Science (16H02593), Asada Science Foundation, and the Takeda Science Foundation to T.W. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the University of Yamanashi. The protocol was approved by the Committee on the Ethics of Animal Experiments of the University of Yamanashi (Permit Number: A24-50). All surgeries were performed under tribromoethanol anesthesia, and all efforts were made to minimize suffering. An illustrated overview of the procedures and time-table are shown in Figure 1 and Table 1, respectively.
1. Preparation of Messenger RNA (In Vitro Transcription of eGFP-H2B mRNA)
<ol>
	<li>Linearize 5 &#181;g of template DNA pTOPO-eGFP-H2B3,7 with 30 U of Not I in 50 &#181;L at 37 &#176;C overnight.</li>
	<li>Collect 1.5 &#181;L of digested DNA for confirmation whether completely digested by electrophoresis.
	<ol>
		<li>Apply 1.5 &#181;L and 3 - 5 &#181;L of undigested DNA with loading dye to the well of 2% agarose gel, respectively, and subject to electrophoresis. 
		NOTE: If completely digested, a single band (approx. 5 kb) is observed. Undigested DNA is used as a&#160;control, which appears at a different position.</li>
	</ol>
	</li>
	<li>Add 151.5 &#181;L of ddH2O and 200 &#181;L of phenol/chloroform/isoamyl alcohol (25:24:1) to the remaining digested DNA.
	<ol>
		<li>Mix vigorously by vortex.</li>
		<li>Centrifuge at the maximum speed for 15 min.</li>
	</ol>
	</li>
	<li>Recover the supernatant and then add 200 &#181;L of chloroform.
	<ol>
		<li>Mix vigorously by vortex.</li>
		<li>Centrifuge at maximum speed for 15 min.</li>
	</ol>
	</li>
	<li>Recover the supernatant and then add 20 &#181;L of 3M sodium acetate and 1.5 &#181;L of ethachinmate.
	<ol>
		<li>Mix vigorously by vortex.</li>
		<li>Add 550 &#181;L of 100% ethanol and then mix vigorously by vortex.</li>
		<li>Centrifuge at maximum speed for 15 min.</li>
		<li>Discard the supernatant and then wash the pellet with 70% ethanol.</li>
		<li>Dry the pellet by evaporation for 5 - 10 min.</li>
	</ol>
	</li>
	<li>Dissolve precipitated plasmid DNA in 8 &#181;L of nuclease-free water.</li>
	<li>Assess plasmid concentration (expected concentration is about 300 - 500 ng/&#181;L) with nanodrop by following manufacturer&#39;s instruction.</li>
	<li>Perform in vitro transcription for mRNA encoding eGFP-H2B with 1 &#181;g of purified DNA as a template in 20 &#181;L of total reaction volume by following the manufacturer&#39;s instructions.
	<ol>
		<li>After in vitro transcription, add 36 &#956;L of water then recover 1.5 &#181;L from 56&#160;&#181;L of synthesized mRNA for analysis by electrophoresis (as without poly A).</li>
	</ol>
	</li>
	<li>Perform poly A tailing for synthesized mRNA in a 100 &#181;L reaction volume by following the manufacturer&#39;s instructions.</li>
	<li>Add 60 &#181;L of lithium chloride precipitation solution to the poly A tailed mRNA of eGFP-H2B and then mix vigorously.
	<ol>
		<li>Cool at -30 &#176;C for 30 min.</li>
		<li>Centrifuge at maximum speed for 15 min.</li>
		<li>Discard the supernatant and then wash the pellet with 70% ethanol.</li>
		<li>Dry the pellet by evaporation for 5 - 10 min.</li>
	</ol>
	</li>
	<li>Dissolve the pellet with 20 &#181;L of nuclease-free water by heating at 55 &#176;C for 30 min.</li>
	<li>Assess the concentration of the precipitated mRNA with nanodrop. Dilute to 500 ng/&#181;L with nuclease-free water and store at -80 &#176;C until use.</li>
	<li>Confirm preferred mRNA production; subject 1 &#181;L of mRNA with/without (obtained in 1.8.1 and 1.12 steps, respectively) poly A tail mixed with 1.5 &#181;L of 0.1 mg/mL ethidium bromide and 3 &#181;L of sample loading dye to the electrophoresis analysis. Applied volume was 5.5 &#181;L. 
	NOTE: If Poly A tailing was successful, the shifted band compared to the mRNA without poly A tailing should be observed (Figure 2A).</li>
</ol>
2. Preparation of Vasectomized Male Mice
<ol>
	<li>Weigh the ICR male mice at 8 - 12 months using a weight scale.</li>
	<li>Intraperitoneally inject male mice with 0.7 - 0.9 mL 1.2% tribromoethanol anesthesia. 
	NOTE: The amount of anesthesia depends on the size of the mouse. For example, a mouse weighing 40 g is injected with 0.8 mL of tribromoethanol anesthesia.</li>
	<li>Wet the berry with 70% ethanol.</li>
	<li>Make a small cut (3 - 5 mm) on the berry and then make an incision on the muscle wall with the help of scissors. 
	NOTE: Scissors and forceps should be cleaned with 70% ethanol before starting the surgery.</li>
	<li>Grip a fat pad with forceps and gently pull it out. Then, the testis and vas deferens appear. 
	NOTE: Vas deferens is a U-shaped tube and with a red blood vessel.</li>
	<li>Tie off the vas deferens at two points with surgical sutures and then cut out the middle part between the tied points.</li>
	<li>Gently push back the fat pad and testis into the berry.
	<ol>
		<li>Repeat the surgery with the remaining testis.</li>
	</ol>
	</li>
	<li>Sew the muscle wall with two stitches with surgical suture.</li>
</ol>
3. IVF
<ol>
	<li>Inject 8 - 10-week old female B6D2F1 (BDF1) mice intraperitoneally with 7.5 IU equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) at 46 - 50 h interval.</li>
	<li>Prepare drops of 300 &#181;L human tubal fluid (HTF)8 media for capacitation and insemination 1 day before IVF in a 30-mm dish, cover these drops with the mineral oil on the laboratory bench and then preincubate in an incubator overnight.</li>
	<li>Sacrifice matured male ICR mice by cervical dislocation. Dissect cauda epididymis with a 27-G needle and collect spermatozoon. Culture them for capacitation in 300 &#181;L of HTF media for 1 - 2 h at 38 &#176;C.</li>
	<li>16 h after hCG injection, sacrifice super ovulated female mice by cervical dislocation. Transfer the oviduct ampulla into mineral oil next to HTF media and then draw out cumulus cells and oocytes complex from this oviduct ampulla by a 30-G needle into the preincubated insemination-HTF medium. 
	NOTE: Some female mice do not often show ovulation despite super ovulation treatment. Enlarged oviduct ampulla is a sign of ovulation.</li>
	<li>After capacitation, transfer 2 - 3 &#181;L of the capacitated spermatozoon into insemination-HTF media in which the ovulated oocytes were collected.</li>
	<li>1 h after insemination, collect the fertilized zygotes and treat them with hyaluronidase at 100 &#181;g/mL for 10 min in CZB9 media. 
	NOTE: When insemination is performed properly, 1 h after insemination, the fertilized zygotes with the 2nd polar body are observed. If less or low-quality sperm is used, only little zygotes with the 2nd polar body are observed. Also, if many sperms are used for insemination, polyspermy occurs. Proper insemination leads to zygotes with male and female pronuclei. By using these criteria, it is possible to find whether insemination is done well or not.
	<ol>
		<li>After washing in CZB media, subject the zygotes with a second polar body to microinjection with eGFP-H2B mRNA.</li>
	</ol>
	</li>
</ol>
4. Preparation of Chamber and Microinjection Pipettes
<ol>
	<li>Dilute RNA encoding eGFP-H2B using nuclease-free water to obtain a concentration of 250 ng/&#181;L.</li>
	<li>Prepare 2 - 3 drops of PVP-HTF and eGFP-H2B drops mRNA, and 5 - 6 drops of Hepes buffered CZB (H-CZB) drops on the 60-mm dish. Cover these drops with mineral oil. 
	NOTE: These preparations can be performed on the laboratory bench. There is no requirement for using laminar airflow cabinet.</li>
	<li>Pull borosilicate glass with a micropipette puller (e.g., P = 500, heat = 825, pull = 30, vel = 120, and time = 200)</li>
	<li>After breaking the tip of the pipette, bend the pulled glass microinjection pipette close to the tip (&#8722;300 &#181;m back) at around 30&#176; using a microforge.</li>
</ol>
5. Microinjection
<ol>
	<li>Turn off the plate heater on the stage of micromanipulator to prevent the killing of the zygotes with a piezo during mRNA injection.</li>
	<li>Wash and coat the inside of injection needles in HEPES-buffered-HTF containing 10% PVP (PVP-HTF).</li>
	<li>Collect the fertilized zygotes with a 2nd polar body (Figure 2B) and transfer 20 - 25 zygotes onto the chamber of the microscope stage attached with a micromanipulator.</li>
	<li>Fill the diluted eGFP-H2B mRNA into borosilicate glass capillaries from the tips.</li>
	<li>Hold the zygote with a holding pipette and then break the zona pellucida and the cytosolic membrane with a piezo drive.</li>
	<li>Inject about 10 pL of mRNA into the cytoplasm of the zygotes. Finish mRNA-injection within 10 min to prevent damage caused by longer culturing the zygotes outside the incubator. 
	NOTE: The injection volume should be as large as the 2nd polar body. If the amount of mRNA injected is too large, it is possible to cause the free eGFP-H2B fraction, which may affect the mobile fraction.</li>
	<li>10 min after microinjection, wash in at least 3 drops and then culture the injected zygotes in CZB at 38 &#176;C until zFRAP analysis.</li>
</ol>
6. zFRAP Analysis
<ol>
	<li>1 h prior to zFRAP analysis, turn on the laser and hot plate attached to the stage of the confocal microscope. Set the temperature at 38 &#176;C.</li>
	<li>Put 6 - 10 &#181;L of HEPES-buffered CZB media covered with mineral oil on the 60-mm glass bottom dish and warm on the heater until zygote collection.&#160;</li>
	<li>8 - 12 h after insemination, collect 5-10 eGFP-H2B-expressing zygotes and transfer into 6 - 10 &#181;L of pre-warmed HEPES-buffered CZB medium drop. 
	NOTE: To avoid longer culturing outside incubator, 5 - 10 eGFP-H2B-expressing zygotes were used at each analysis. 5 to 10 zygotes can be analyzed within 1 h.</li>
	<li>Set the bleaching and imaging conditions according to the manufacturer&#39;s instructions. The case a confocal laser scanning microscope (Table of Materials) is shown below:
	<ol>
		<li>Use 60X&#160;oil lens (60X 60X/1.35 oil). 
		NOTE: Lenses with higher magnification (higher numerical aperture) show higher sensitivity.</li>
		<li>Set scanning speed as 4.0 &#181;s/Pixel and size as &#34;512&#34; on &#34;Acquisition Setting&#34; (Supplemental Figure 1).</li>
		<li>Increase Digital zoom to &#34;5&#34; (Supplemental Figure 1). 
		NOTE: A higher zoom is required to recognize whether focus drift occurs or not.</li>
		<li>Open dye list (Supplemental Figure 1) and then chose &#34;EGFP&#34;. Set observation time as 1.6 s (interval is set as &#34;free run&#34; setting) (Supplemental Figure 1) 
		NOTE: More observation points may cause more detailed data, but it may also cause phototoxicity. In the zFRAP analysis, a 1.6 s observation time seems to be enough to detect the parental asymmetry of chromatin looseness.</li>
		<li>Set the number of pictures as 12 in total (Supplemental Figure 1).</li>
		<li>Start &#34;Stimulus setting&#34; panel and then click &#34;Main&#34; on UseScanner. Set scanning speed as 10.0 &#181;s/Pixel. Click &#34;Activation in Series&#34; and then set PreActivation as &#34;3 frames&#34; and Activation Time as &#34;5 sec&#34; (Supplemental Figure 1).</li>
		<li>Click &#34;Focus x 2&#34; and set the focus and then &#34;Stop&#34;.</li>
		<li>Set bleaching and imaging laser power (477 nm) at 110 and 15 &#181;W, respectively, by adjusting &#34;laser gage power&#34; (Supplemental Figure 1). 
		NOTE: Very strong bleaching may cause a larger bleached area, which causes difficulties for the quantitative analysis. For measurement of the laser power, use a power meter. It is important to confirm the laser power to avoid the effect of the time-related deterioration of the laser power.</li>
		<li>Click &#34;Clip Rect&#34; (Supplemental Figure 1) in Stimulus Setting panel. Set the region of interest (ROI; red; ROI #1B) as 40 x 40 pixels (7.6 &#181;m2). Prepare a reference region (REF; green; ROI #2), and a background (BG; blue; ROI #3) using &#34;copy ROI&#34; and &#34;paste ROI&#34; (right-click button on the mouse). 
		NOTE: Pixel size can be set through &#34;ROI manager&#34; which can be called by clicking the right button of the mouse when the cursor is on the ROI. Larger ROIs are thought to be better since they can standardize the dispersion of the zFRAP score. However, too large of an ROI cannot be used for this analysis because it makes it difficult to avoid the nucleolus precursor body (NPB). eGFP-H2B in this compartment was thought to be free from chromatin and showed remarkable higher mobility3. ROI and REF areas should be separated as much as possible. If these two regions are close, bleaching may also affect the region of REF.</li>
		<li>Click &#34;Live&#34; on the tool bar and then start &#34;Live Plot&#34; (Supplemental Figure 1). 
		NOTE: Live plot indicates fluorescence signal intensity within each ROI and is used for adjusting laser power using HV. Note that if ROI is not selected, no intensity would be detected on Live plot panel.</li>
		<li>Determining the ROI position 
		NOTE: ROI can be moved by dragging into the desired position in pronuclei. (1) Be sure to avoid the nucleolus, and (2) fit the entire ROI into the nucleoplasm. Do not contain the region outside the area of the nuclear membrane (cytoplasm) in ROI. The localization of eGFP-H2B is highly restricted into the nucleoplasm so that it is easy to find whether ROI contains cytoplasm or not by the fluorescence of eGFP-H2B. Male pronuclei tend to be larger than those of females, which are often localized near the 2nd polar body. While using these criteria, judge the male or female pronuclei.</li>
		<li>Click &#34;Focus x 2&#34; (Supplemental Figure 1) and then adjust the HV power gage of the channel for EGFP into fluorescence intensity to around 2000 (fluorescence intensity can be seen in the &#34;live plot&#34; window).</li>
	</ol>
	</li>
	<li>Click &#34;Time&#34; and then &#34;XY&#34; (Supplemental Figure 1) to start the zFRAP analysis. 
	NOTE: If &#34;Time&#34; button is suitably selected, &#34;t&#34; appears on the &#34;XY&#34; button.
	<ol>
		<li>Click &#34;Series Done&#34; (Supplemental Figure 1), which appears after FRAP imaging near the &#34;XY button&#34; and then obtain FRAP-analyzed data.</li>
		<li>Save the file &#34;2D View-Image XXXXX&#34; as a preferred named file (oib. file format) by &#34;save as&#34; (Ctrl + Shift + S can be also used).</li>
		<li>Select ROI, REF, and BG and then click (Ctrl+A can be also used) &#34;Series Analysis&#34; in &#34;2D View Image&#34; window.</li>
		<li>Obtain row FRAP data and then click &#34;save&#34; button in the live plot window. 
		NOTE: Row FRAP quantitative data is saved as a csv file.</li>
	</ol>
	</li>
	<li>For no zFRAP control, put some of the zygotes injected with mRNA on the drop, which is near to the rim of the glass bottom dishes to avoid the effect from fluorescence observation.</li>
</ol>
7. Data Calculation
<ol>
	<li>While using the obtained quantitative data, make the graph of the &#34;recovery curve&#34; and &#34;mobile fraction&#34; by Excel as shown in the references3,7 with some modifications (see below and supplemental excel file).</li>
	<li>Calculate the relative intensity by subtracting the value of BG from those of ROI and REF in 12 pictures for each time point.</li>
	<li>Divide the value of ROI by that of the REF. As a result, 12 standardized relative intensity scores at each time point can be obtained.</li>
	<li>Calculate the average of the relative score for ROI in 3 images taken before photo bleaching.</li>
	<li>Calculate the recovery rate. Divide the 12 obtained relative scores for ROI in the images taken at each time point (obtained at 7.1.2.) by the average of the relative score before photo bleaching (obtained at 7.1.3.). Draw recovery curve with using these scores (Figure 2E).</li>
	<li>Calculate the bleaching rate. 
	NOTE: Bleaching rate (%) = 100 - recovery rate of 4th image.</li>
	<li>Calculate the mobile fraction (MF) by using the formula as follows10,11,12 
	MF (%) = 12th recovery rate (at the end point) - 4th recovery rate/ bleaching rate &#215; 100.</li>
</ol>
8. Embryo Transfer
<ol>
	<li>Mate matured ICR females at 8 - 12 months with vasectomized male ICR mice the night before the embryo transfer by letting them be in the same cage overnight.</li>
	<li>Collect the embryos that were zFRAP-analyzed and determined to develop to the two-cell stage.</li>
	<li>Intraperitoneally inject female mice with 0.7 - 0.9 mL 1.2% tribromoethanol anesthesia or with 0.35 - 0.45 mL of 0.75/4/5mg/kg medetomidine/midazolam/butorphanol mixed agents prior to embryo transfer. 
	NOTE: The volume of anesthesia is determined by the body weight of mice. Tribromoethanol: 0.2 mL/10 g of body weight; medetomidine/midazolam/butorphanol mixed agents: 0.1 mL/ 10 g of body weight.
	<ol>
		<li>Transfer these two-cell stage embryos into the oviducts of pseudopregnant female ICR mice with a vaginal plug at 0.5 days post coitum (dpc).</li>
		<li>After the embryo transfer, put the female mice on the heater set at 37 &#176;C until they wake up.</li>
	</ol>
	</li>
	<li>At 18.5 dpc, sacrifice the surrogate mother by cervical dislocation and recover the pups derived from zFRAP-analyzed zygotes by cesarean section. Some of the recovered pups were delivered to the foster mother and their body weights were assessed each week.</li>
</ol>

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The authors have nothing to disclose.

As revealed in this study, the zFRAP analysis does not cause critical damage to full-term development, suggesting this method is a very useful tool to reveal the association between molecular events and the embryonic developmental potential. During reprogramming, into the two-cell like state of ES cells, by which the differential potential of those cells becomes as high as that of two-cell stage embryos, chromatin looseness changes into that comparable with two-cell stage embryos5. Accordingly, it...

After fertilization, the chromatin structure is altered dynamically, and zygotic chromatin structure is then eventually established1,2. During this period, in paternal pronuclei, the dominant chromatin protein is changed from protamine into histone. The resulting chromatin is extremely different from that of sperms and female oocytes in several points (e.g., histone variant composition, histone modification). Thus, formed zygotic chromatin is thought to be important for subsequent embryonic development. However, despite efforts to reveal the details of zygotic chromatin structure over long periods, methods to evaluate the quality of zygotes or to predict their full-term development at the one-cell stage by analyzing their chromatin structure have never been established.
In the previous study, it is discovered that zygotes have an extremely loosened chromatin structure3. Currently, chromatin looseness or openness is believed to be an important factor for cellular differentiation potential in embryonic stem (ES) cells4. ES cells do not exhibit homogeneity in nature, but are rather heterogeneous; in ES cell colonies, some transiently acquire a higher differentiation potential comparable to blastomeres of two-cell stage embryos. During this transition into the two-cell like state, chromatin looseness in ES cells changes into what is comparable with two-cell stage embryos5. Thus, chromatin looseness seems to be important for cellular differentiation potential and it is possible that extensively open chromatin in zygotes is useful for the evaluation of zygotic developmental potential.
Live imaging is a powerful tool that allows for the analysis of molecular events during ontogenesis since this method allows for subsequent development and even full-term development6. As one of the live imaging methods, FRAP analysis has been used to examine chromatin looseness in preimplantation embryos and ES cells3,4,5. If zygotic chromatin looseness can be analyzed without a detrimental effect on full-term development by FRAP analysis, it may be a valuable tool for the evaluation of the quality of embryos at the one-cell stage. However, the effects on full-term development by this experimental method have not been examined. Recently, an experimental system using FRAP to evaluate zygotic chromatin looseness was developed. Because this was a new observation system for zygotes, it was termed as zygotic FRAP (zFRAP). zFRAP did not critically affect full-term development and has been reported elsewhere7. In this report, the protocol of this experimental method is described.

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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:260px;">Confocal laser scaning microscope</td>
			<td style="width:208px;">Olympus</td>
			<td style="width:119px;">FV1200</td>
			<td style="width:435px;">FV1000 can be also used for FRAP analysis (reference #7)</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">Thermo plate</td>
			<td style="width:208px;">Tokai Hit</td>
			<td style="width:119px;">TP-110RH26</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">Inverted microscope</td>
			<td style="width:208px;">Olympus</td>
			<td style="width:119px;">IX71</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Micro manupilator</td>
			<td style="width:208px;">Narishige</td>
			<td style="width:119px;">MMO-202ND</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">Pieze Micro Micromanipulator</td>
			<td style="width:208px;">Prime tech</td>
			<td style="width:119px;">PMAS-CT150</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">35 mm culture dish</td>
			<td style="width:208px;">Falcom</td>
			<td style="width:119px;">351008</td>
			<td>35 x 100 mm style; for IVF</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">60 mm culture dish</td>
			<td style="width:208px;">Falcom</td>
			<td style="width:119px;">351007</td>
			<td>60 x 15 mm style; for embryo culture</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">50 mm culture dish</td>
			<td style="width:208px;">Falcom</td>
			<td style="width:119px;">351006</td>
			<td>50 x 9 mm style; for manipulation on the stage</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">50 mm glass bottom dish</td>
			<td style="width:208px;">Matsunami</td>
			<td style="width:119px;">D910400</td>
			<td>50 mm dish, 27 mm &#966; hole size; for FRAP analysis</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">Mineral oil</td>
			<td style="width:208px;">Organic spceiality chemicals</td>
			<td style="width:119px;">625071</td>
			<td>For FRAP analysis on the glass bottom dishes</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">Mineral oil</td>
			<td style="width:208px;">Sigma</td>
			<td style="width:119px;">M8410-1L</td>
			<td>For IVF and embryo culture</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">glass capillary</td>
			<td style="width:208px;">Drummond</td>
			<td style="width:119px;">1-000-1000</td>
			<td>For handling mouse zygotes</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">Borosilicate glass</td>
			<td style="width:208px;">Prime tech</td>
			<td style="width:119px;">B100-75-10-PT</td>
			<td>For microinjection of mRNA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:260px;">Micropipett puller</td>
			<td style="width:208px;">Sutter instrument</td>
			<td style="width:119px;">P-97/IVF</td>
			<td style="width:435px;">For preparation of the injection or holding neadle (out side diameter: 80 &#181;m, inner diameter: 10 &#181;m)&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">Microforge</td>
			<td style="width:208px;">&#160;Narishige</td>
			<td>MF-900</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:260px;">mMESSAGE MACHINE SP6 Transcription kit</td>
			<td style="width:208px;">Thermo 
			Fisher scientific</td>
			<td style="width:119px;">AM1340</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:260px;">Poly (A) tailing kit</td>
			<td style="width:208px;">Thermo 
			Fisher scientific</td>
			<td style="width:119px;">AM1350</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">PVP solution 10% (PVP-HTF)</td>
			<td style="width:208px;">IrvineSceientific</td>
			<td style="width:119px;">99311</td>
			<td>HEPES buffered HTF containing 10% PVP</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">Sodium HEPES</td>
			<td style="width:208px;">Sigma</td>
			<td style="width:119px;">H3784</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">pTOPO eGFP-H2B</td>
			<td style="width:208px;"></td>
			<td style="width:119px;"></td>
			<td>Template plasmid for eGFP-H2B mRNA (reference #6)</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:260px;">NorthernMax Gly Sample loading Dye&#160;</td>
			<td style="width:208px;">Thermo 
			Fisher scientific</td>
			<td style="width:119px;">AM8551</td>
			<td>For electrophoresis of in vitro transcribed mRNA</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">Phenol chloroform isamyl alcohol</td>
			<td style="width:208px;">nacalai tesque</td>
			<td style="width:119px;">25970-14</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">Chloroform</td>
			<td style="width:208px;">nacalai tesque</td>
			<td style="width:119px;">&#160;08401-65</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">Not I</td>
			<td style="width:208px;">TOYOBO</td>
			<td style="width:119px;">NOT-111X</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">Ethachinmate</td>
			<td style="width:208px;">WAKO</td>
			<td style="width:119px;">318-01793</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">3664 Otical power meter</td>
			<td style="width:208px;">Hioki</td>
			<td style="width:119px;">3664</td>
			<td>&#160;Power meter for laser power</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:260px;">Stereomicmicroscope</td>
			<td style="width:208px;">Olympus</td>
			<td style="width:119px;">SZX16</td>
			<td></td>
		</tr>
	</tbody>
</table>

zygotic fluorescence recovery after photo-bleaching analysis for chromatin looseness that allows full-term development

Live imaging is a powerful tool that allows for the analysis of molecular events during ontogenesis. Recently, chromatin looseness or openness has been shown to be involved in the cellular differentiation potential of pluripotent embryonic stem cells. It was previously reported that compared with embryonic stem cells, zygotes harbor an extremely loosened chromatin structure, suggesting its association with their totipotency. However, until now, it has not been addressed whether this extremely loosened/open chromatin structure is important for embryonic developmental potential. In the present study, to examine this hypothesis, an experimental system in which zygotes that were analyzed by fluorescence recovery after photo-bleaching can develop to term without any significant damage was developed. Importantly, this experimental system needs only a thermos-plate heater in addition to a confocal laser scanning microscope. The findings of this study suggest that fluorescence recovery after photo-bleaching analysis (FRAP) analysis can be used to investigate whether the molecular events in zygotic chromatin are important for full-term development.

zFRAP analysis with eGFP-H2B
Properly produced mRNA encoding eGFP-H2B, which is seen as a shifted band caused by poly A tailing (Figure 2A), was injected into the cytoplasm of zygotes with a 2nd polar body at 1 - 3 h after insemination (Figure 2B). Eight to 12 h after insemination, zygotes with 2 pronuclei showing the expression of eGFP-H2B were collected and subjected to ...

Watch this Scientific Journal Video about Zygotic Fluorescence Recovery After Photo-bleaching Analysis for Chromatin Looseness That Allows Full-term Development at JoVE.com

Zygotic Fluorescence Recovery After Photo-bleaching Analysis for Chromatin Looseness That Allows Full-term Development

Chromatin looseness appears to be involved in the developmental potential of blastomeres. However, it is not known whether chromatin looseness can be used as a reliable index for the developmental potential for embryos. Here, an experimental system in which chromatin looseness-evaluated zygotes can develop to full term has been described.

Live imaging is a powerful tool that allows for the analysis of molecular events during ontogenesis. Recently, chromatin looseness or openness has been ...

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7.8K Views.  University of Yamanashi. Chromatin looseness appears to be involved in the developmental potential of blastomeres. However, it is not known whether chromatin looseness can be used as a reliable index for the developmental potential for embryos. Here, an experimental system in which chromatin looseness-evaluated zygotes can develop to full term has been described.

Video: Zygotic Fluorescence Recovery After Photo-bleaching Analysis for Chromatin Looseness That Allows Full-term Development

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological systems. In FDAP experiments, the clearance of proteins within living organisms can be measured. A protein of interest is tagged with a photoconvertible fluorescent protein, expressed in vivo and photoconverted, and the decrease in the photoconverted signal over time is monitored. The data is then fitted with an appropriate clearance model to determine the protein half-life. Importantly, the clearance kinetics of protein populations in different compartments of the organism can be examined separately by applying compartmental masks. This approach has been used to determine the intra- and extracellular half-lives of secreted signaling proteins during zebrafish development. Here, we describe a protocol for FDAP experiments in zebrafish embryos. It should be possible to use FDAP to determine the clearance kinetics of any taggable protein in any optically accessible organism.

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion FDAP

Protein levels in cells and tissues are often tightly regulated by the balance of protein production and clearance. Using Fluorescence Decay After Photoconversion (FDAP), the clearance kinetics of proteins can be experimentally measured in vivo.

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological ...

Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq

The recruitment of chromatin regulators and the assignment of chromatin states to specific genomic loci are pivotal to cell fate decisions and tissue and organ formation during development. Determining the locations and levels of such chromatin features in vivo will provide valuable information about the spatio-temporal regulation of genomic elements, and will support aspirations to mimic embryonic tissue development in vitro. The most commonly used method for genome-wide and high-resolution profiling is chromatin immunoprecipitation followed by next-generation sequencing (ChIP-Seq). This protocol outlines how yolk-rich embryos such as those of the frog Xenopus can be processed for ChIP-Seq experiments, and it offers simple command lines for post-sequencing analysis. Because of the high efficiency with which the protocol extracts nuclei from formaldehyde-fixed tissue, the method allows easy upscaling to obtain enough ChIP material for genome-wide profiling. Our protocol has been used successfully to map various DNA-binding proteins such as transcription factors, signaling mediators, components of the transcription machinery, chromatin modifiers and post-translational histone modifications, and for this to be done at various stages of embryogenesis. Lastly, this protocol should be widely applicable to other model and non-model organisms as more and more genome assemblies become available.

Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq

The question of how chromatin regulators and chromatin states affect the genome in vivo is key to our understanding of how early cell fate decisions are made in the developing embryo. ChIP-Seq&#8212;the most popular approach to investigate chromatin features at a global level&#8212;is outlined here for Xenopus embryos.

The recruitment of chromatin regulators and the assignment of chromatin states to specific genomic loci are pivotal to cell fate decisions and tissue and ...

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

Early embryogenesis starting from a single cell zygote goes through rapid cell division and morphogenesis, and is morphologically characterized by pre-globular, globular, heart, torpedo and cotyledon stages. This progressive development is under the tight regulation of a complex molecular network. Harvesting sufficient early embryos at a similar stage of development is essential for investigating the cellular and molecular regulation of early embryogenesis. This is not straightforward since early embryogenesis undergoes rapid morphogenesis in a short while e.g. 8 days for Medicago truncatula to reach the early cotyledon stage. Here, we address the issue by two approaches. The first one establishes a linkage between embryo development and pod morphology in helping indicate the stage of the zygotic embryo. This is particularly based on the number of pod spirals and development of the spines. An alternative way to complement the in vivo studies is via culturing leaf explants to produce somatic embryos. The medium includes an unusual hormone combination - an auxin (1-naphthaleneacetic acid), a cytokinin (6-benzylaminopurine), abscisic acid and gibberellic acid. The different stages can be discerned growing out of the callus without dissection.

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

The goal is to illustrate that the model legume Medicago truncatula can be readily utilized to investigate the regulation of early plant embryogenesis to complement the non-legume Arabidopsis model. Pod morphology is linked to zygotic embryogenesis stages and a protocol to collect embryos using tissue culture is also provided.

Early embryogenesis starting from a single cell zygote goes through rapid cell division and morphogenesis, and is morphologically characterized by ...

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated discs, and costameres requires the coordinated assembly of multiple components in time and space. Disruption in assembly of these components leads to developmental heart defects. Immunofluorescent staining techniques are used commonly in cultured cardiomyocytes to probe myofibril maturation, but this ex vivo approach is limited by the extent to which myocytes will fully differentiate in culture, lack of normal in vivo mechanical inputs, and absence of endocardial cues. Application of immunofluorescence techniques to the study of developing mouse heart is desirable but more technically challenging, and methods often lack sufficient sensitivity and resolution to visualize sarcomeres in the early stages of heart development. Here, we describe a robust and reproducible method to co-immunostain multiple proteins or to co-visualize a fluorescent protein with immunofluorescent staining in the embryonic mouse heart and use this method to analyze developing myofibrils, intercalated discs, and costameres. This method can be further applied to assess cardiomyocyte structural changes caused by mutations that lead to developmental heart defects.

Mutations that lead to congenital heart defects benefit from in vivo investigation of cardiac structure during development, but high-resolution structural studies in the mouse embryonic heart are technically challenging. Here we present a robust immunofluorescence and image analysis method to assess cardiomyocyte-specific structures in the developing mouse heart.

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded Nucleolus) or unable (NSN, Not Surrounded Nucleolus) to develop to the blastocyst stage after in vitro maturation (IVM) and in vitro fertilization (IVF). It makes use of Hoeschst33342 (or any other DNA intercalating dye) able to bind to the heterochromatin of the nucleolus showing a ring in the SN oocytes or not, like in the NSN oocytes. This represents the easiest and quickest way to sort both antral oocytes that can be eventually used for IVM or IVF procedures. 
Briefly, the protocol consists of the following steps: hormone injection to stimulate follicular growth; isolation of the oocytes at the GV stage from the antral compartment by puncturing the ovary with a sterile needle; preparation of thin glass pipettes for mouth pipetting of the oocytes; sorting of the oocytes with Hoechst33342 prepared at a supravital concentration; IVM, IVF or any other molecular/cellular analysis.
Unfortunately there are still few evidences to sort SN and NSN oocytes using less invasive techniques. If and once they will be identified, they could be potentially applied to human assisted reproductive technologies, although with several aspects that should be modified. To date, this technique has potential implications to dramatically increase IVM and IVF successful procedures in both endangered and species with economic interest.

Here we present a protocol for the characterization of mouse antral oocytes based on nucleolar organization.

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded ...

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development.
The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed.
In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning&#160;or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance.
The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

The method described here allows time-lapse analysis of organ development in zebrafish embryos by using a fluorescence dissecting microscope capable of performing optical sectioning and simple strategies of readjustment to correct focal and planar drift.

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM compared to confocal systems are its low phototoxicity, gentle mounting strategies, fast acquisition with high signal to noise ratio and the possibility of imaging samples from various angles (views) for long periods of time. Imaging from multiple views unleashes the full potential of LSFM, but at the same time it can create terabyte-sized datasets. Processing such datasets is the biggest challenge of using LSFM. In this protocol we outline some solutions to this problem. Until recently, LSFM was mostly performed in laboratories that had the expertise to build and operate their own light sheet microscopes. However, in the last three years several commercial implementations of LSFM became available, which are multipurpose and easy to use for any developmental biologist. This article is primarily directed to those researchers, who are not LSFM technology developers, but want to employ LSFM as a tool to answer specific developmental biology questions. 
Here, we use imaging of zebrafish eye development as an example to introduce the reader to LSFM technology and we demonstrate applications of LSFM across multiple spatial and temporal scales. This article describes a complete experimental protocol starting with the mounting of zebrafish embryos for LSFM. We then outline the options for imaging using the commercially available light sheet microscope. Importantly, we also explain a pipeline for subsequent registration and fusion of multiview datasets using an open source solution implemented as a Fiji plugin. While this protocol focuses on imaging the developing zebrafish eye and processing data from a particular imaging setup, most of the insights and troubleshooting suggestions presented here are of general use and the protocol can be adapted to a variety of light sheet microscopy experiments.

Light sheet fluorescence microscopy is an excellent tool for imaging embryonic development. It allows recording of long time-lapse movies of live embryos in near physiological conditions. We demonstrate its application for imaging zebrafish eye development across wide spatio-temporal scales and present a pipeline for fusion and deconvolution of multiview datasets.

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM ...

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is the formation of fibroblastic foci, which is associated with the disease severity. Mesenchymal cells consisting of the fibroblastic foci are proposed to be derived from several cell sources, including originally resident intrapulmonary fibroblasts and circulating fibrocytes from bone marrow. Recently, mesenchymal cells that underwent epithelial-mesenchymal transition (EMT) have been also supposed to contribute to the pathogenesis of fibrosis. In addition, EMT can be induced by transforming growth factor &#946;, and EMT can be enhanced by pro-inflammatory cytokines like tumor necrosis factor &#945;. The gel contraction assay is an ideal in vitro model for the evaluation of contractility, which is one of the characteristic functions of fibroblasts and contributes to wound repair and fibrosis. Here, the development of a gel contraction assay is demonstrated for evaluating contractile ability of mesenchymal cells that underwent EMT.

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Here, we describe the development and application of a gel contraction assay for evaluating contractile function in mesenchymal cells that underwent epithelial-mesenchymal transition.

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...