著者は、トーマス ・ シリングが親切贈与 Tg (sox10:eGFP) 魚とメアリー halloran さん親切贈与 Tg(foxd3:GFP)魚をありがちましょう。

The protocol described here was performed in accordance with the guidelines for the humane treatment of laboratory animals established by the University of Michigan Committee on the Use and Care of Animals (UCUCA).
1. Embryo Collection for Time-lapse Imaging
<ol>
	<li>Between 3 and 9 pm, set up male and female adult Tg(sox10:EGFP) or Tg(foxd3:GFP) transgenic zebrafish in a divided breeding tank for pairwise mating. 
	NOTE: The Tg(sox10:EGFP) and Tg(foxd3:GFP) fish, kind gifts from Thomas Schilling and Mary Halloran, respectively, were crossed into the Casper (roy -/-, nacre -/-) background to decrease auto-fluorescence and interference with pigmentation.
	<ol>
		<li>Assemble the breeding tank (slotted inner tank + solid outer tank) and fill it with reverse osmosis (RO) system water.</li>
		<li>Transfer up to 3 females and 3 males into opposing sides of the tank separated by a divider; up to 6 fish can be bred pairwise in a single breeding tank.</li>
		<li>The next morning, remove the divider shortly after the lights turn on in the vivarium. 
		NOTE: In the present study, the lights are on a 14-hour light (on at 9 am) and 10-hour dark (off at 11 pm) cycle.</li>
		<li>Allow undisturbed mating for 20 min or until sufficient numbers of embryos are produced; the embryos will be at the bottom of the breeding tank. When eggs are observed, lift the slotted inner tank out along with the fish and quickly place them (fish and inner tank) into a clean solid outer tank filled with RO water.</li>
	</ol>
	</li>
	<li>Prepare standard 1X embryo medium by adding the following per 1 L of RO water: 0.287 g (4.9 mM) NaCl, 0.127 g (0.17 mM) KCl, 0.048 g (0.33 mM) CaCl2.2H2O, 0.04 g (0.33 mM) MgSO4 (anhydrous). Stir solution until the salts are completely dissolved.
	<ol>
		<li>Add 100 &#181;L of 0.1% methylene blue as a fungicide. Add 0.25 g of sodium bicarbonate to adjust the pH to 7.75. Store medium at room temperature.</li>
	</ol>
	</li>
	<li>Collect eggs from the outer tank using an egg-collecting screen, and transfer the eggs to Petri dishes (~50 embryos per dish) containing 30 mL of 1X embryo medium. Incubate the collected eggs at 28.5 &#176;C.</li>
	<li>Embryo preparation
	<ol>
		<li>Tg(sox10:EGFP) embryos.
		<ol>
			<li>For Tg(sox10:EGFP) embryos, at 12 h post-fertilization (hpf), remove the dead eggs and assess the developmental stage of the embryos. Screen for GFP-positive embryos using a dissecting fluorescent stereomicroscope with a standard 460-490 nm band-pass excitation filter. Using forceps, remove the chorions from 3-4 embryos that express GFP, and transfer these embryos to a separate Petri dish containing 30 mL of fresh 1X embryo medium.</li>
		</ol>
		</li>
		<li>Tg(foxd3:GFP) embryos.
		<ol>
			<li>For Tg(foxd3:GFP) embryos, at 22-24 hpf, remove the dead eggs and assess the developmental stage of the embryos. Screen for positive embryos using a dissecting fluorescent stereomicroscope with a standard 460-490 nm band-pass excitation filter. Using forceps, remove the chorions from 3-4 embryos that express GFP.</li>
			<li>Prepare 1000X Phenylthiourea (PTU) stock solution by dissolving 0.75 g (3%) of PTU in 25 mL of Dimethylsulfoxide (DMSO). Aliquot and store the stock solution at 4 &#176;C. Add 30 &#181;L of 1000X PTU (3%) stock solution to 30 mL of 1X embryo medium to generate 0.003% PTU solution.</li>
			<li>Place the screened and dechorionated GFP-positive embryos in 0.003% PTU solution to inhibit pigmentation. Do not initiate treatment of the embryos with PTU prior to 20 hpf, as early treatment (at &#60;20 hpf) can have adverse effects on neural crest and neuroepithelial development.28</li>
		</ol>
		</li>
		<li>Prior to conducting the time-lapse experiment, monitor the embryos through live imaging using a standard stereomicroscope at 12-24 h intervals in age-matched comparisons as a control for temperature drift.</li>
	</ol>
	</li>
</ol>
2. Mounting of Embryo for Time-lapse Imaging
<ol>
	<li>Preparation of time-lapse embryo media
	<ol>
		<li>Prepare standard 0.4% tricaine stock solution by adding 0.004 g of tricaine to 100 mL of RO water. Aliquot (~1 mL) the stock solution into fresh 1.5-mL centrifuge tubes and store at -20 &#176;C.</li>
		<li>Prepare time-lapse embryo medium (50 mL of 1X embryo medium, 0.016% tricaine) by adding 2 mL of 0.4% tricaine solution to 48 mL of 1X embryo medium. If using embryos &#62;22 hpf, then also add 50 &#181;L of 3% PTU stock solution (final concentration.003% PTU) to the embryo medium.</li>
	</ol>
	</li>
	<li>Preparation of 2% low-melt agarose
	<ol>
		<li>Add 0.4 g of low-melt agarose powder to 20 mL of 1X embryo medium. Heat for 1-2 min or until the solution is clear and all particles are dissolved. 
		NOTE: Increasing the percentage of the agarose gel decreases the porosity of the matrix, which may physically impede the growth and development of the embryo. Therefore, the agarose solution can be lowered to 1.5% to minimize these effects. However, when the excitation beam is held stationary during laser-scanning microscopy, greater heating can occur, increasing rapidly in a logarithmic relationship with time. In this protocol, heating due to fluorophore absorption is highly localized to the focal region. Thus, in the region of interest, the temperature could increase to a value high enough (&#8805; 30 &#176;C) to melt agarose solutions made at percentages lower than 1.5, thereby directly exposing the embryo to the laser or enabling conditions in which the embryo floats out of the focal plane. For this reason, the use of agarose solutions lower than 1.5% is not recommended.</li>
		<li>Aliquot (~1 mL) the agarose solution into fresh 1.5-mL centrifuge tubes for storage. Store excess agarose solution in liquid form on a heat block (60-70 &#176;C) for 2-3 weeks.</li>
	</ol>
	</li>
	<li>Mounting the Embryo
	<ol>
		<li>To set up the open bath chamber, place a small amount of high vacuum grease on the base of the open bath chamber. Place a circular glass coverslip onto the base and screw the top of the open bath chamber onto the base until tight ( Figure 1A, B).</li>
		<li>Pipet (~500-700 &#181;L) of 2% agarose solution (60-70 &#176;C) into the open bath chamber until the base is ~3/4 filled ( Figure 1C). Note that once the agarose solution is in the open bath chamber on the lab bench, the temperature of the solution decreases approximately 1 &#176;C/s.</li>
		<li>Wait 30 s to allow the agarose to cool slightly (~30-40 &#176;C), without completely polymerizing, and subsequently transfer a single embryo to the center of the base ( Figure 1D, E).</li>
		<li>Under a fluorescent stereomicroscope, position the embryo using a 1-10 &#181;L micropipette tip, making sure that the embryo is placed near the bottom of the agarose, as the embryo may float away when covered with embryo media if it is placed too near the surface of the agarose. 
		NOTE: In the presented videos (Videos 1-3), the embryos are oriented laterally, but depending on the area of interest, the embryos can be oriented ventrally or dorsally.</li>
		<li>Monitor and reposition the embryo until the agarose has set. Once the agarose has set, use a transfer pipet to fill the assembled open chamber entirely with time-lapse embryo media ( Figure 1F).</li>
		<li>Place the open bath chamber in the quick exchange platform, which fits onto the stage adapter ( Figure 1G). Place the entire setup (embryo, open bath chamber, quick exchange platform and stage adaptor) onto the stage of the microscope immediately above the condenser ( Figure 1H).</li>
	</ol>
	</li>
</ol>
3. Microscope Set-up for Time-lapse Imaging
<ol>
	<li>Determining laser settings
	<ol>
		<li>Determining laser wavelength to use. Use a wavelength that is twice the excitation wavelength of the fluorophore of interest (e.g. wavelength between 880 and 940 nm for GFP). 
		NOTE: In the present study, the wavelength setting for GFP was between 880 and 940 nm. The higher the wavelength, the lower the output power of the laser.</li>
		<li>Determine laser transmission. A high percent of laser transmission will kill the embryo, use the lowest level of transmission (recommended). For 24 to 48 h time-lapse studies, as presented herein, keep transmission below 5%.</li>
		<li>Determine microscope detection systems and ensure that the correct filters for the fluorophore are in place. 
		NOTE: For multi-photon microscopes, there are multiple detection systems with various sensitivities for the emitted fluorescence. In general, an internal detection system has less sensitivity than an external detection system. For transgenic lines with high levels of GFP expression, the internal detection system is adequate. For transgenic lines with low levels of GFP expression or with other fluorophores (e.g., red fluorescent protein), an external detection system may be required to maintain the percent of laser transmission at a reasonable level. Regardless the detection system used, the correct filters for the fluorophore must be in place.</li>
	</ol>
	</li>
	<li>Adjusting software settings on the multi-photon microscope
	<ol>
		<li>Using the 5X objective, locate the embryo. Manually raise the stage to the highest position, and use the fine focus to position the embryo in the middle of the microscope range.</li>
		<li>Manually lower the stage, and change the 5X objective to the 25X water immersion objective (numerical aperture NA, 0.95). Carefully raise the stage to bring the embryo back into focus.</li>
		<li>In the software, click on the &#34;xyzt&#34; mode for obtaining multiple images at time intervals (t) in the x-y plane over a depth of &#34;z&#34;. Use the epifluorescence or brightfield view to find the depth of focus in the area of interest, which will demarcate the Z-stack.
		<ol>
			<li>In the software, click on &#34;begin&#34; button; for these experiments, the lateral edge of the eye was the beginning of the Z-stack. Click on &#34;end&#34; button; the midline of the embryo was the end of the Z-stack. 
			NOTE: The step size was 0.3-0.6 &#181;m and there was a total of ~200 steps for a z-stack size of 60 to 120 &#181;m).</li>
		</ol>
		</li>
		<li>Click on the menu for adjusting acquisition time and imaging frequency. For the present system, ~200 steps requires approximately 5 min for each z-stack acquisition. For adequate recovery of the fluorophore and survival of the embryo, allow for a ratio of at least 1:3 between z-stack acquisition (laser power on) and recovery time (laser power off). 
		NOTE: For example, z-stacks are acquired every 20 min with 5 min of z-stack acquisition and 15 min of recovery. For this protocol, larger z-stacks can be obtained, but would appropriately increase the time between z-stack acquisitions, resulting in fewer images over the time-lapse course.
		<ol>
			<li>With appropriate time for embryo recovery, set the time between z-stacks in the designated window. Set the total length of time for the experiment in the appropriate window.</li>
		</ol>
		</li>
		<li>Final software, laser, and embryo adjustments.
		<ol>
			<li>Turn on the live image setting to make final adjustments to the laser settings. Adjust laser transmission (see 3.1.2), gain and offset slider bars within the software to optimize the fluorescent image. Also, adjust the orientation of the embryo, as needed, depending on the length of the experiment, anticipated growth of the embryo, etc. Make sure that the area of interest remains within the frame through the duration of the experiment.</li>
			<li>Turn off the epifluorescent light source as it is no longer needed during time-lapse acquisition. Cover the stage with the laser safety box ( Figure 1I). When using the internal detection system, the laser safety box is adequate for protection against background light. Press &#34;start&#34;. 
			NOTE: However, with more sensitive external detection systems, the laser safety box does not block enough background light, and additional covers are required to prevent the disruption of image acquisition.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. During Time-lapse Acquisition
<ol>
	<li>Refill the open bath chamber with time-lapse embryo media every 8-12 h (at least 2 times per day) during time-lapse acquisition through the sliding doors on the laser safety box ( Figure 1I).</li>
	<li>Before opening the doors of the laser safety box, ensure that the microscope is not actively acquiring an image. 
	NOTE: The use of heaters and circulating media systems is not necessary for time-lapse imaging experiments lasting 24 to 48 h. Indeed, the temperatures of both stage and in-line heaters are difficult to control, and during image acquisition the embryo exhibits an adequate development rate at a temperature range from 25-28 &#176;C. Moreover, circulating media systems tend to overflow and potentially damage the equipment. Thus, all embryos are routinely staged post-acquisition.</li>
</ol>
5. Post-acquisition processing
<ol>
	<li>In the software, click on the &#34;file&#34; menu and choose &#34;save&#34;.</li>
	<li>Open the file in image processing software (see the Table of Materials). Highlight the correct image series. In the software, choose the &#34;Process&#34; menu. Click on 3D Deconvolution and &#34;Apply&#34; to deconvolve each z-stack. 
	NOTE: The file is large; therefore, this step may take many hours.</li>
	<li>In the software, under the &#34;Process&#34; menu, click on &#34;maximum projection.&#34; Click on &#34;Apply&#34; to initiate maximum projection to generate 1 image per z-stack. Export each maximum projected file (1 image per z-stack) as a tiff.</li>
	<li>Import individual tiff files into video processing software. Select all tiff files and drag them into the video editor. Adjust length of each image within the video to 0.1s. Export video as mov or mp4 file.</li>
</ol>

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L., Gallina, D., Kahana, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenothiourea+sensitizes+zebrafish+cranial+neural+crest+and+extraocular+muscle+developmnt+to+changes+in+retinoic+acid+and+insulin-like+growth+factor+signaling.">Phenothiourea sensitizes zebrafish cranial neural crest and extraocular muscle developmnt to changes in retinoic acid and insulin-like growth factor signaling.</a> PLoS ONE. 6, e22991 (2011).</li><li>Gfrerer, L., Dougherty, M., Laio, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+craniofacial+development+in+the+sox10:kaede+transgenic+zebrafish+line+using+time-lapse+confocal+microscopy.">Visualization of craniofacial development in the sox10:kaede transgenic zebrafish line using time-lapse confocal microscopy.</a> J Vis Exp. (79), e50525 (2013).</li><li>Lopez, A. L. R., Garcai, M. D., Dickinson, M. E., Larina, I. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+confocal+microscopy+of+the+developing+mouse+embryonic+yolk+sac+vasculature.">Live confocal microscopy of the developing mouse embryonic yolk sac vasculature.</a> Methods Mol Biol. 1214, 163-172 (2015).</li><li>McGurk, P. D., Lovely, C. B., Eberhart, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+craniofacial+morphogenesis+in+zebrafish+using+4D+confocal+microscopy.">Analyzing craniofacial morphogenesis in zebrafish using 4D confocal microscopy.</a> J Vis Exp. (83), e51190 (2014).</li><li>Nowotschin, S., Ferrer-Vaquer, A., Hadjantonakis, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+mouse+development+with+confocal+time-lapse+microscopy.">Imaging mouse development with confocal time-lapse microscopy.</a> Methods Enzymol. 476, 351-377 (2010).</li></ol>

この仕事は健康の国民の協会 (K08EY022912-01) とビジョン研究コア (P30 EY007003) の国立眼研究所からの助成金によって財政上支えられます。

多光子タイムラプス イメージングが一過性と渡り鳥の細胞集団の生体内で追跡を可能にします。リアルタイムで萌芽期のプロセスを研究するこの強力な技術を使用ことができます、現在の研究では、このメソッドの結果は神経堤細胞の移動と開発の現在の知識を強化します。以前のタイムラプス イメージング研究は、通常、共焦点レーザ走査型顕微鏡を利用します。29</s...

先天性眼疾患小児失明を引き起こすことができるおよび頭蓋神経堤の異常によることが多い。神経堤細胞は神経管から発生し、全身の多くの組織を形成する非定常の幹細胞です。1,2,3,4,5前と、中脳由来神経堤細胞は、骨と軟骨の顔面と前頭葉、虹彩、角膜、小柱網と目の眼の強膜に上昇を与えます。4,6,7,8神経堤細胞、咽頭アーチ菱フォーム、あご及び心臓流出路から。1,3,4,9,10研究を強調しますしている眼に神経の頂上の貢献や眼周囲の開発、脊椎動物の目の開発のこれらのセルの重要性を強調します。確かに、神経堤細胞の移動と分化の混乱は、Axenfeld リーガー症候群および Peters プラス症候群における顎顔面と眼球の異常に します。11,12,13,14,15,16,17したがって、これら神経堤細胞の分化、増殖、移行の包括的な理解は、先天性眼疾患の基になる複雑さに洞察を提供します。
ゼブラフィッシュはゼブラフィッシュ眼の構造は、哺乳類に類似し、多くの遺伝子は、ゼブラフィッシュと哺乳類の間保存された眼の開発を研究するため強力なモデル生物です。18,19,20また、zebrafish の胚が透明で、卵生、目におけるリアルタイムの可視化を促進します。
以前に発行された仕事で拡大して、6、7,20神経堤細胞の移動パターン多光子蛍光タイムラプス イメージング (性決定領域 Y) sry 遺伝子の転写制御の下で緑色蛍光タンパク質 (GFP) の付いたトランスジェニックゼブラフィッシュ線を使用して記述された-ボックス 10 (sox10) またはフォーク ヘッド ボックス D3 (foxd3) 遺伝子の規定する領域。21,22,23,24. 多光子蛍光タイムラプス イメージングはレーザ走査型顕微鏡標本 fluorophores が付いているタグの立体化、高解像度の画像をキャプチャする長い波長多光子蛍光励起の高度な光学技術を組み合わせた強力な手法です。25,26,27多光子レーザーの使用増加組織浸透と減少蛍光漂白などを含む標準の共焦点顕微鏡上の明確な利点があります。
このメソッドを使用すると、移行と渡り鳥の経路のタイミングでさまざまな神経堤細胞の 2 つの異なる集団が眼周囲間充織と発展途上の目で判別、すなわち foxd3 陽性の神経堤細胞と顔面の間充織で sox10 陽性神経堤細胞。この方法では、開発中にリアルタイムで規制の神経堤の移行を観察しやすくゼブラフィッシュ眼と顔面神経の頂上の移行の移行を可視化へのアプローチは導入されました。
このプロトコルは、Tg (sox10:EGFP) の例として、Tg (foxd3:GFP) 遺伝子導入ゼブラフィッシュ初期の目の開発の間に時間経過のビデオを生成するための情報を提供します。このプロトコルは、ゼブラフィッシュ神経堤細胞由来眼と顔面の構造体の早い開発の高解像度、リアルタイム 3次元可視化のためさらに適用できます。また、このメソッドは、他の組織や臓器ゼブラフィッシュと他の動物モデルの開発の可視化のためさらに適用できます。

<table border="0" cellpadding="0" cellspacing="0" width="919">
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			<td height="42" style="height:42px;width:251px;">Breeding Tanks with Dividers</td>
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			<td height="42" style="height:42px;width:251px;">Potassium Chloride</td>
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			<td style="width:104px;">1049380500</td>
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			<td style="width:391px;">Poly bottle; 500 g. Powder, GR ACS. CAS No. 144-55-8, EC Number 205-633-8</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:251px;">N-Phenylthiourea</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:104px;">P7629-25G</td>
			<td style="width:391px;">&#62;98%. CAS Number 103-85-5, EC Number 203-151-2</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Dimethylsulfoxide</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:104px;">D8418-500ML</td>
			<td style="width:391px;">Molecular Biology grade. CAS Number 67-68-5, EC Number 200-664-3</td>
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			<td height="21" style="height:21px;width:251px;">Tricaine Methanesulfonate</td>
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			<td style="width:391px;">Tricaine-S</td>
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			<td style="width:391px;">GeneMate Sieve GQA Low Melt Agarose, 25 g</td>
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			<td height="21" style="height:21px;width:251px;">Open Bath Chamber</td>
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			<td style="width:391px;">High Profile</td>
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			<td height="21" style="height:21px;width:251px;">Glass Coverslips</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:104px;">12-545-102</td>
			<td style="width:391px;">Circle cover glass. 25 mm diameter</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:251px;">High Vacuum Grease</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:104px;">14-635-5C</td>
			<td style="width:391px;">2.0-lb. tube. DOW CORNING CORPORATION 
			1658832</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Quick Exchange Platform</td>
			<td style="width:175px;">Warner Instruments</td>
			<td style="width:104px;">QE-1</td>
			<td style="width:391px;">35 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Stage Adapter</td>
			<td style="width:175px;">Warner Instruments</td>
			<td style="width:104px;">SA-20LZ-AL</td>
			<td style="width:391px;">16.5 x 10 cm</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:251px;">TC SP5 MP multi-photon microscope</td>
			<td style="width:175px;">Leica Microsystems CMS GmbH</td>
			<td style="width:104px;"></td>
			<td style="width:391px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Mai Tai DeepSee Ti-Sapphire Laser</td>
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			<td style="width:104px;"></td>
			<td style="width:391px;"></td>
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			<td height="40" style="height:40px;width:251px;">Laser Safety Box</td>
			<td style="width:175px;">Leica Microsystems CMS GmbH</td>
			<td style="width:104px;"></td>
			<td style="width:391px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Leica Application Suite X (LAS X)&#160; Software</td>
			<td style="width:175px;">Leica Microsystems CMS GmbH</td>
			<td style="width:104px;"></td>
			<td style="width:391px;"></td>
		</tr>
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			<td height="42" style="height:42px;width:251px;">Photoshop CS 6 Version 13.0 x64 Software</td>
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			<td style="width:104px;"></td>
			<td style="width:391px;"></td>
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			<td height="21" style="height:21px;width:251px;">iMovie Version 10.1.4 Software</td>
			<td style="width:175px;">Apple</td>
			<td style="width:104px;"></td>
			<td style="width:391px;"></td>
		</tr>
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</table>

multi-photon time lapse imaging to visualize development in real-time: visualization of migrating neural crest cells in zebrafish embryos

先天性の目と頭蓋顔面の異常、神経の頂上、全身に様々 な細胞タイプに上昇を与える渡り鳥の幹細胞の一時的な人口の混乱を反映します。神経の頂上の生物学を理解することは、制限、リアルタイムで生体内で調査することができます遺伝的扱いモデルの不足を反映してされています。ゼブラフィッシュは、渡り鳥細胞群、神経堤などを勉強するため特に重要な発達モデルです。Sox10とfoxd3初期神経分化と神経堤細胞のマーカーを表す可能性があります多数の動物モデルで示されている、発展途上の目に神経の頂上の移行を検討するには、長波長多光子蛍光励起顕微鏡レーザーの高度な光学技術の組み合わせはトランスジェニックゼブラフィッシュ胚で、Tg (sox10:EGFP) と Tg (foxd3:GFP)、すなわち開発の目の高解像度、立体化、リアルタイムのビデオをキャプチャする実装されました。多光子タイムラプス イメージングは、動作や目の早期開発に貢献する 2 つの神経堤細胞集団の移住性パターンを識別する使用されました。このプロトコルは、例としてゼブラフィッシュ神経堤移行中に時間経過のビデオを生成するための情報を提供し、ゼブラフィッシュと他のモデル有機体の多くの構造の初期の開発を視覚化するさらに適用することができます。

多光子蛍光タイムラプス イメージングは、ゼブラフィッシュ行 craniofacial 構造と Tg (sox10:EGFP) と Tg (foxd3:GFP) の眼の前眼部に上昇を与える頭部神経堤細胞の移行パターンを明らかにしたビデオのシリーズを生成されます。例としてsox1012 と 30 の hpf の間肯定的な神経堤細胞は神経管の端から顎顔面領域 (ビデオ 1 図 2</strong...

Watch this Scientific Journal Video about Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos at JoVE.com

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

リアルタイムで開発を可視化する多光子タイムラプス イメージング: ゼブラフィッシュ胚の神経堤細胞の可視化

長い波長多光子蛍光励起顕微鏡レーザーの高度な光学技術の組み合わせは、Tg (sox10:EGFP) および Tg (foxd3:GFP) ゼブラフィッシュ胚の神経堤移行の高解像度、リアルタイム三次元画像をキャプチャする実装されました。

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

multi-photon-time-lapse-imaging-to-visualize-development-real-time

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

7.5K ビュー.  University of Michigan. 長い波長多光子蛍光励起顕微鏡レーザーの高度な光学技術の組み合わせは、Tg (sox10:EGFP) および Tg (foxd3:GFP) ゼブラフィッシュ胚の神経堤移行の高解像度、リアルタイム三次元画像をキャプチャする実装されました。

ビデオ: Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

分析<em&gt;インビボ</emゼブラフィッシュ胚における細胞移植とタイムラプスイメージングを使用して&gt;細胞移動

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

発生生物学

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.

解剖顕微鏡を使用したタイムラプスイメージングとゼブラフィッシュ腎臓の開発の分析は、光学切片のための装備します

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

Kinetic Measurement and Real Time Visualization of Somatic Reprogramming

Somatic reprogramming has enabled the conversion of adult cells to induced pluripotent stem cells (iPSC) from diverse genetic backgrounds and disease phenotypes. Recent advances have identified more efficient and safe methods for introduction of reprogramming factors. However, there are few tools to monitor and track the progression of reprogramming. Current methods for monitoring reprogramming rely on the qualitative inspection of morphology or staining with stem cell-specific dyes and antibodies. Tools to dissect the progression of iPSC generation can help better understand the process under different conditions from diverse cell sources. 
This study presents key approaches for kinetic measurement of reprogramming progression using flow cytometry as well as real-time monitoring via imaging. To measure the kinetics of reprogramming, flow analysis was performed at discrete time points using antibodies against positive and negative pluripotent stem cell markers. The combination of real-time visualization and flow analysis enables the quantitative study of reprogramming at different stages and provides a more accurate comparison of different systems and methods. Real-time, image-based analysis was used for the continuous monitoring of fibroblasts as they are reprogrammed in a feeder-free medium system. The kinetics of colony formation was measured based on confluence in the phase contrast or fluorescence channels after staining with live alkaline phosphatase dye or antibodies against SSEA4 or TRA-1-60. The results indicated that measurement of confluence provides semi-quantitative metrics to monitor the progression of reprogramming.

The protocol presented in this study describes methods for the real-time monitoring of reprogramming progression via the kinetic measurement of positive and negative pluripotent stem cell markers using flow cytometry analysis. The protocol also includes the imaging-based assessment of morphology, and marker or reporter expression during iPSC generation.

体細胞の再プログラミングの反応速度測定とリアルタイムの可視化

Somatic reprogramming has enabled the conversion of adult cells to induced pluripotent stem cells (iPSC) from diverse genetic backgrounds and disease ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and zebrafish embryos. Our system comprises a chamber featuring a simple setup that is nevertheless robust to induce and maintain a specific oxygen concentration and temperature in any experimental solution of choice. The presented system is very cost-effective but highly functional, it allows induction and sustainment of hypoxia for direct experiments in vivo and for various time periods up to 48 h.
To monitor and study the effects of hypoxia, we have employed two methods - measurement of levels of hypoxia-inducible factor 1alpha (HIF-1&#945;) in whole embryos or specific tissues and determination of retinal stem cell proliferation by 5-ethynyl-2&#8242;-deoxyuridine (EdU) incorporation into the DNA. HIF-1&#945; levels can serve as a general hypoxia marker in the whole embryo or tissue of choice, here embryonic retina. EdU incorporation into the proliferating cells of embryonic retina is a specific output of hypoxia induction. Thus, we have shown that hypoxic embryonic retinal progenitors decrease proliferation within 1 h of incubation under 5% oxygen of both frog and zebrafish embryos.
Once mastered, our setup can be employed for use with small aquatic model organisms, for direct in vivo experiments, any given time period and under normal, hypoxic or hyperoxic oxygen concentration or under any other given gas mixture.

We introduce a novel hypoxic chamber system for use with aquatic organisms such as frog and zebrafish embryos. Our system is simple, robust, cost-effective and allows the induction and sustainment of hypoxia in vivo and for up to 48 h. We present 2 reproducible methods to monitor the effectiveness of hypoxia.

生きた蛙およびゼブラフィッシュの胚における低酸素の誘導

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells (HSPCs) in Developing Zebrafish Embryos

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell lineages that comprise mature blood. Isolation and identification of these HSPCs is difficult because they are defined ex post facto; they can only be defined after their differentiation into specific cell lineages. Over the past few decades, the zebrafish (Danio rerio) has become a model organism to study hematopoiesis. Zebrafish embryos develop ex utero, and by 48 h post-fertilization (hpf) have generated definitive HSPCs. Assays to assess HSPC differentiation and proliferation capabilities have been developed, utilizing transplantation and subsequent reconstitution of the hematopoietic system in addition to visualizing specialized transgenic lines with confocal microscopy. However, these assays are cost prohibitive, technically difficult, and time consuming for many laboratories. Development of an in vitro model to assess HSPCs would be cost effective, quicker, and present fewer difficulties compared to previously described methods, allowing laboratories to quickly assess mutagenesis and drug screens that affect HSPC biology. This novel in vitro assay to assess HSPCs is performed by plating dissociated whole zebrafish embryos and adding exogenous factors that promote only HSPC differentiation and proliferation. Embryos are dissociated into single cells and plated with HSPC-supportive colony stimulating factors that cause them to generate colony forming units (CFUs) that arise from a single progenitor cell. These assays should allow more careful examination of the molecular pathways responsible for HSPC proliferation, differentiation, and regulation, which will allow researchers to understand the underpinnings of vertebrate hematopoiesis and its dysregulation during disease.

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos

Here, we present a simple method to quantitate hematopoietic stem and progenitor cells (HSPCs) in embryonic zebrafish. HSPCs from dissociated zebrafish are plated in methylcellulose with supportive factors, differentiating into mature blood. This allows the detection of blood defects and allows drug screening to be easily conducted.

ゼブラフィッシュ初期胚細胞 (HSPCs) を造血幹・前駆細胞の量的にIn Vitroアッセイの開発

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

ゼブラフィッシュ初期胚および腫瘍の細胞の電気的活動の可視化

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues or cells. A difficulty with imaging whole embryo development is that zebrafish embryos grow substantially in length. When mounted as regularly done in 0.3-1% low melt agarose, the agarose imposes growth restriction, leading to distortions in the soft embryo body. Yet, to perform confocal time-lapse microscopy, the embryo must be immobilized. This article describes a layered mounting method for zebrafish embryos that restrict the motility of the embryos while allowing for the unrestricted growth. The mounting is performed in layers of agarose at different concentrations. To demonstrate the usability of this method, whole embryo vascular, neuronal and muscle development was imaged in transgenic fish for 55 consecutive hours. This mounting method can be used for easy, low-cost imaging of whole zebrafish embryos using inverted microscopes without requirements of molds or special equipment.

This article describes a method to mount fragile zebrafish embryos for extended time-lapse confocal microscopy. This low-cost method is easy to perform using regular glass-bottom microscopy dishes for imaging on any inverted microscope. The mounting is performed in layers of agarose at different concentrations.

ゼブラフィッシュ胚全体の延長タイムラプス共焦点顕微鏡法の層状取り付け法

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues ...

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic expression. Notch signaling effector Hes1 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner in neural stem/progenitor cells. Because the period of the oscillatory expression of these genes is very short (2 h), it is difficult to monitor their cyclic expression. To examine such rapid changes in the gene expression or protein dynamics, fast response reporters are required. Because of its fast maturation kinetics and high sensitivity, the bioluminescence reporter luciferase is suitable to monitor rapid gene expression changes in living cells. We used a destabilized luciferase reporter for monitoring the promoter activity and a luciferase-fused reporter for visualization of protein dynamics at single cell resolution. These bioluminescence reporters show rapid turnover and generate very weak signals; therefore, we have developed a highly sensitive bioluminescence imaging system to detect such faint signals. These methods enable us to monitor various gene expression dynamics in living cells and tissues, which are important information to help understand the actual cellular states.

Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

マウス神経新生時のノッチシグナル伝達ダイナミクスのリアルタイム生物発光イメージング

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic ...

Visualizing the Developing Brain in Living Zebrafish using Brainbow and Time-lapse Confocal Imaging

Development of the vertebrate nervous system requires a precise coordination of complex cellular behaviors and interactions. The use of high resolution in vivo imaging techniques can provide a clear window into these processes in the living organism. For example, dividing cells and their progeny can be followed in real time as the nervous system forms. In recent years, technical advances in multicolor techniques have expanded the types of questions that can be investigated. The multicolor Brainbow approach can be used to not only distinguish among like cells, but also to color-code multiple different clones of related cells that each derive from one progenitor cell. This allows for a multiplex lineage analysis of many different clones and their behaviors simultaneously during development. Here we describe a technique for using time-lapse confocal microscopy to visualize large numbers of multicolor Brainbow-labeled cells over real time within the developing zebrafish nervous system. This is particularly useful for following cellular interactions among like cells, which are difficult to label differentially using traditional promoter-driven colors. Our approach can be used for tracking lineage relationships among multiple different clones simultaneously. The large datasets generated using this technique provide rich information that can be compared quantitatively across genetic or pharmacological manipulations. Ultimately the results generated can help to answer systematic questions about how the nervous system develops.

In vivo imaging is a powerful tool that can be used to investigate the cellular mechanisms underlying nervous system development. Here we describe a technique for using time-lapse confocal microscopy to visualize large numbers of multicolor Brainbow-labeled cells in real time within the developing zebrafish nervous system.

Brainbowとタイムラプス共焦点イメージングを用いた生きているゼブラフィッシュの発達脳の可視化

Development of the vertebrate nervous system requires a precise coordination of complex cellular behaviors and interactions. The use of high resolution in ...

Isolation, Culture, and Adipogenic Induction of Neural Crest Original Adipose-Derived Stem Cells from Periaortic Adipose Tissue

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of cardiovascular disease. ADSCs derived from different adipose tissues show distinct features, and those from the PVAT have not been well characterized. In a recent study, we reported that some ADSCs in the periaortic arch adipose tissue (PAAT) descend from the neural crest cells (NCCs), a transient population of migratory cells originating from the ectoderm.
In this paper, we describe a protocol for isolating red fluorescent protein (RFP)-labeled NCCs from the PAAT of Wnt-1 Cre+/-;Rosa26RFP/+ mice and inducing their adipogenic differentiation in vitro. Briefly, the stromal vascular fraction (SVF) is enzymatically dissociated from the PAAT, and the RFP+ neural crest derived ADSCs (NCADSCs) are isolated by fluorescence activated cell sorting (FACS). The NCADSCs differentiate into both brown and white adipocytes, can be cryopreserved, and retain their adipogenic potential for ~3&#8211;5 passages. Our protocol can generate abundant ADSCs from the PVAT for modeling PVAT adipogenesis or lipogenesis in vitro. Thus, these NCADSCs can provide a valuable system for studying the molecular switches involved in PVAT differentiation.

We present a protocol for the isolation, culture, and adipogenic induction of neural crest derived adipose-derived stem cells (NCADSCs) from the periaortic adipose tissue of Wnt-1 Cre+/-;Rosa26RFP/+ mice. The NCADSCs can be an easily accessible source of ADSCs for modeling adipogenesis or lipogenesis in vitro.

神経紋の分離、培養、脂肪形成誘導、周食脂肪組織由来の脂肪由来幹細胞

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of ...

リアルタイムで開発を可視化する多光子タイムラプス イメージング: ゼブラフィッシュ胚の神経堤細胞の可視化

リアルタイムで開発を可視化する多光子タイムラプスイメージング: ゼブラフィッシュ胚の神経堤細胞の可視化