Name: multi-photon time lapse imaging to visualize development in real-time: visualization of migrating neural crest cells in zebrafish embryos
Uploaded: 2017-08-09T15:00:00
Description: 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

The authors thank Thomas Schilling for kindly gifting the Tg(sox10:eGFP) fish and Mary Halloran for kindly gifting the Tg(foxd3:GFP) fish.

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:G...

<ol>
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L., Coulombre, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origins+of+avian+ocular+and+periocular+tissues.">Origins of avian ocular and periocular tissues.</a> Exp Eye Res. 29, 27-43 (1979).</li><li>Bohnsack, B. L., 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=Thyroid+hormone+and+retinoic+acid+interact+to+regulate+zebrafish+craniofacial+neural+crest+development.">Thyroid hormone and retinoic acid interact to regulate zebrafish craniofacial neural crest development.</a> Dev Biol. 373, 300-309 (2013).</li><li>Chawla, B., Schley, E., Williams, A. L., Bohnsack, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+and+pitx2+regulate+early+neural+crest+survival+and+migration+in+craniofacial+and+ocular+development.">Retinoic acid and pitx2 regulate early neural crest survival and migration in craniofacial and ocular development.</a> Birth Defects Res B Dev Reprod Toxicol. , (2016).</li><li>Trainor, P. A., Tam, P. P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cranial+paraxial+mesoderm+and+neural+crest+cells+of+the+mouse+embryo-codistribution+in+the+craniofacial+mesenchyme+but+distinct+segregation+in+branchial+arches.">Cranial paraxial mesoderm and neural crest cells of the mouse embryo-codistribution in the craniofacial mesenchyme but distinct segregation in branchial arches.</a> Development. 121 (8), 2569-2582 (1995).</li><li>Hong, C. S., Saint-Jeannet, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sox+proteins+and+neural+crest+development.">Sox proteins and neural crest development.</a> Sem Cell Dev Biol. 16, 694-703 (2005).</li><li>Steventon, B., Carona-Fontaine, C., Mayor, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+network+during+neural+crest+induction:+from+cell+specification+to+cell+survival.">Genetic network during neural crest induction: from cell specification to cell survival.</a> Sem Cell Dev Biol. 16, 647-654 (2005).</li><li>Dressler, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dental+and+craniofacial+anomalies+associated+wth+Axenfeld-Rieger+syndrome+with+PITX2+mutation.">Dental and craniofacial anomalies associated wth Axenfeld-Rieger syndrome with PITX2 mutation.</a> Case Report Med. , 621984 (2010).</li><li>Ozeki, H., Shirai, S., Ikeda, K., Ogura, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anomalies+associated+with+Axenfeld-Rieger+syndrome.">Anomalies associated with Axenfeld-Rieger syndrome.</a> Graefes Arch Clin Exp Ophthalmol. 237 (9), 730-734 (1999).</li><li>Strungaru, M. H., Dinu, I., Walter, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotype-phenotype+correlations+in+Axenfeld-Rieger+malformation+and+glaucoma+patients+with+FOXC1+and+PITX2+mutations.">Genotype-phenotype correlations in Axenfeld-Rieger malformation and glaucoma patients with FOXC1 and PITX2 mutations.</a> Invest Ophthalmol Vis Sci. 48, 228-237 (2007).</li><li>Tumer, Z., Bach-Holm, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axenfeld-Rieger+syndrome+and+spectrum+of+Pitx2+and+Foxc1+mutations.">Axenfeld-Rieger syndrome and spectrum of Pitx2 and Foxc1 mutations.</a> Eur J Hum Genet. 17, 1527-1539 (2009).</li><li>Schoner, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrocephalus,+agenesis+of+the+corpus+callosum,+and+cleft+lip/palate+represent+frequent+associations+in+fetuses+with+Peters+plus+syndrome+and+B3GALTL+mutations.+Fetal+PPS+phenotypes,+expanded+by+Dandy+Walker+cyst+and+encephalocele.">Hydrocephalus, agenesis of the corpus callosum, and cleft lip/palate represent frequent associations in fetuses with Peters plus syndrome and B3GALTL mutations. Fetal PPS phenotypes, expanded by Dandy Walker cyst and encephalocele.</a> Prenat Diagn. 33 (1), 75-80 (2013).</li><li>Aliferis, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+nonsense+B3GALTL+mutation+confirms+Peters+plus+syndrome+in+a+patient+with+multiple+malformations+and+Peters+anomaly.">A novel nonsense B3GALTL mutation confirms Peters plus syndrome in a patient with multiple malformations and Peters anomaly.</a> Ophthalmic Genet. 31 (4), 205-208 (2010).</li><li>Lesnik Oberstein, ., S, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peters+Plus+syndrome+is+caused+by+mutations+in+B3GALTL,+a+putative+glycosyltransferase.">Peters Plus syndrome is caused by mutations in B3GALTL, a putative glycosyltransferase.</a> Am J Hum Genet. 79 (3), 562-566 (2006).</li><li>Brittijn, S. 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Multi-photon time-lapse imaging enables the in vivo tracking of transient and migratory cell populations. This powerful technique can be used to study embryonic processes in real time, and in the present study, the results of this method enhanced the current knowledge of neural crest cell migration and development. Previous time-lapse imaging studies typically utilize confocal laser scanning microscopy.29,30,31,</s...

Congenital eye diseases can cause childhood blindness and are often due to abnormalities of the cranial neural crest. Neural crest cells are transient stem cells that arise from the neural tube and form numerous tissues throughout the body.1,2,3,4,5 Neural crest cells, derived from the prosencephalon and mesencephalon, give rise to the bone and cartilage of the midface and frontal regions, and the iris, cornea, trabecular meshwork, and sclera in the anterior segment of the eye.4,6,7,8 Neural crest cells from the rhombencephalon form the pharyngeal arches, jaw, and cardiac outflow tract.1,3,4,9,10 Studies have highlighted the contributions of the neural crest to ocular and periocular development, emphasizing the importance of these cells in vertebrate eye development. Indeed, disruption of neural crest cell migration and differentiation lead to craniofacial and ocular anomalies as observed in Axenfeld-Rieger Syndrome and Peters Plus Syndrome.11,12,13,14,15,16,17 Thus, a comprehensive understanding of the migration, proliferation and differentiation of these neural crest cells will provide insight into the complexities underlying congenital eye diseases.
The zebrafish is a powerful model organism for studying ocular development, as the structures of the zebrafish eye are similar to their mammalian counterparts, and many genes are evolutionarily conserved between zebrafish and mammals.18,19,20 In addition, zebrafish embryos are transparent and oviparous, facilitating the visualization of eye development in real-time.
Expanding on previously published work,6,7,20 the migratory pattern of neural crest cells was described using multi-photon fluorescence time-lapse imaging on transgenic zebrafish lines labeled with green fluorescent protein (GFP) under the transcriptional control of the SRY (sex-determining region Y)-box 10 (sox10) or Forkhead Box D3 (foxd3) gene regulatory regions.21,22,23,24. Multi-photon fluorescence time-lapse imaging is a powerful technique that combines the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation to capture high-resolution, three-dimensional images of specimens tagged with fluorophores.25,26,27 The use of the multi-photon laser has distinct advantages over standard confocal microscopy, including increased tissue penetration and decreased fluorophore bleaching.
Using this method, two distinct populations of neural crest cells varying in timing of migration and migratory pathways were discriminated, namely foxd3-positive neural crest cells in the periocular mesenchyme and developing eye and sox10-positive neural crest cells in the craniofacial mesenchyme. With this method, an approach to visualize the migration of ocular and craniofacial neural crest migration in zebrafish is introduced, making it easy to observe regulated neural crest migration in real time during development.
This protocol provides information for generating time-lapse videos during early eye development in Tg(sox10:EGFP) and Tg(foxd3:GFP) transgenic zebrafish, as an example. This protocol can be further applied for the high-resolution, three-dimensional, real-time visualization of the early development of any ocular and craniofacial structure derived from neural crest cells in zebrafish. Moreover, this method can further be applied for the visualization of the development of other tissues and organs in zebrafish and other animal models.

<table border="0" cellpadding="0" cellspacing="0" width="919">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Breeding Tanks with Dividers</td>
			<td style="width:175px;">Aquaneering</td>
			<td style="width:104px;">ZHCT100</td>
			<td style="width:391px;">Crossing Tank Set (1.0-liter) Clear Polycarbonate with Lid and Insert</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">M205 FA Combi-Scope</td>
			<td style="width:175px;">Leica Microsystems CMS GmbH</td>
			<td style="width:104px;"></td>
			<td style="width:391px;">Stereofluorescence Microscope - FusionOptics and TripleBeam</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Sodium Chloride</td>
			<td style="width:175px;">Millipore (EMD)</td>
			<td style="width:104px;">7760-5KG</td>
			<td style="width:391px;">Double PE sack. CAS No. 7647-14-5, EC Number 231-598-3</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Potassium Chloride</td>
			<td style="width:175px;">Millipore (EMD)</td>
			<td style="width:104px;">1049380500</td>
			<td style="width:391px;">Potassium chloride 99.999 Suprapur. CAS No. 7447-40-7, EC Number 231-211-8.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Calcium Chloride Dihydrate</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:104px;">C79-500</td>
			<td style="width:391px;">Poly bottle; 500 g. CAS No. 10035-04-8</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Magnesium Sulfate (Anhydrous)</td>
			<td style="width:175px;">Millipore (EMD)</td>
			<td style="width:104px;">MX0075-1</td>
			<td style="width:391px;">Poly bottle; 500 g. CAS No. 7487-88-9, EC Number 231-298-2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Methylene Blue</td>
			<td style="width:175px;">Millipore (EMD)</td>
			<td style="width:104px;">284-12</td>
			<td style="width:391px;">Glass bottle; 25 g. Powder, Certified Biological Stain</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Sodium Bicarbonate</td>
			<td style="width:175px;">Millipore (EMD)</td>
			<td style="width:104px;">SX0320-1</td>
			<td style="width:391px;">Poly bottle; 500 g. Powder, GR ACS. CAS No. 144-55-8, EC Number 205-633-8</td>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Tricaine Methanesulfonate</td>
			<td style="width:175px;">Western Chemical Inc.</td>
			<td style="width:104px;">MS222</td>
			<td style="width:391px;">Tricaine-S</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Low-Melt Agarose</td>
			<td style="width:175px;">ISC Bioexpress</td>
			<td style="width:104px;">E-3112-25</td>
			<td style="width:391px;">GeneMate Sieve GQA Low Melt Agarose, 25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Open Bath Chamber</td>
			<td style="width:175px;">Warner Instruments</td>
			<td style="width:104px;">RC-40HP</td>
			<td style="width:391px;">High Profile</td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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>
			<td style="width:175px;">SpectraPhysics</td>
			<td style="width:104px;"></td>
			<td style="width:391px;"></td>
		</tr>
		<tr height="40">
			<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">
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multi-photon time lapse imaging to visualize development in real-time: visualization of migrating neural crest cells in zebrafish embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.

Multi-photon fluorescence time-lapse imaging generated a series of videos that revealed the migration patterns of cranial neural crest cells that give rise to the craniofacial structures and anterior segment of the eye in the Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish lines. As an example, sox10-positive neural crest cells between 12 and 30 hpf migrate from the edge of the neural tube into the craniofacial region (Video 1, F...

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

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.

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

The overall goal of multi-photon time lapse imaging is to capture a high-resolution three dimensional real time images of migratory cell populations. This method can help answer key questions in developmental biology, such as mapping migration pathways of different cell populations. The main advantage of this technique over traditional confocal microscopy is that multi-photon lasers have deeper tissue penetration and decreased phototoxicity.This increases the usability in thicker tissues and allows for longer periods of image acquisition. To set up for embryo collection between 3:00 and 9:00 p.m. assemble breeding tanks and fill them with reverse osmosis, or RO water.Transfer up to three females and three males into opposing sides of the divided tank. The next morning, remove the divider shortly after the lights turn on in the vivarium. Allow undisturbed mating for 20 minutes 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 into a clean, solid outer tank filled with RO water. Use an egg collecting screen to collect eggs from the outer tank and transfer the eggs to Petri dishes containing 30 milliliters of 1X embryo medium.Incubate the collected eggs at 28.5 degrees Celsius. For transgenic embryos, at 24 hours post-fertilization or HPF, remove the dead eggs and assess the developmental stage of the embryos. Using forceps, remove the chorions from three to four embryos that express GFP.Under a dissecting fluorescence stereo microscope with a standard 460 to 490 nanometer band pass excitation filter, screen for GFP positive embryos. Transfer these embryos to a separate Petri dish containing 30 milliliters of fresh 1X embryo medium. After 20 HPF, place the screened and dechorionated GFP positive embryos in 0.003%PTU solution to inhibit pigmentation.Avoid initiating treatment earlier as it can have adverse effects on neural crest and neuroepithelial development. Add 0.4 grams of LowMelt Agarose powder to 20mL of 1x embryo medium. Heat the mixture for one to two minutes or until the solution is clear and all particles are dissolved.Aliquot approximately 1mL of the Agarose solution into fresh 1.5mL centrifuge tubes for storage. 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 cover slip onto the base and screw the top of the open bath chamber onto the base until it is tight.Pipette approximately 500-700 microliters of 2%Agarose solution into the open bath chamber until the base is approximately 3/4s filled. Wait 30 seconds to allow the Agarose to cool slightly without completely polymerizing and subsequently transfer a single embryo to the center of the base. Under a fluorescent stereo microscope, use a 10 microliter micro pipette tip to orient the embryo and position it near the bottom of the Agarose so that it doesn't float away when the embryo medium is added.A critical step is to make sure that the embryo is positioned correctly in the Agarose. In these experiments, we orient the embryo laterally but the embryo can also be positioned to focus on dorsal and ventral areas. Monitor and reposition the embryo until the Agarose has set.Then use a transfer pipette to entirely fill the assembled open chamber with time-lapse embryo medium. After placing the entire set up onto the stage of the microscope, according to the text protocol, use the five times objective to locate the embryo. Then manually raise the stage to the highest position and use the fine focus to position the embryo in the middle of the microscope range.Manually lower the stage and change the five time objective to the 25 time water immersion objective. Carefully raise the stage to bring the embryo back into focus. In the software, click on the XYTZ mode for obtaining multiple images at time or T intervals in the XY plane over a depth of Z.Use the epifluorescence or bright field view to find the depth of focus in the area of interest which will demarcate the z-stack.For these experiments, the lateral edge of the eye is the beginning of the z-stack. When focused here, in the software click the begin button. After focusing down to the mid-line of the embryo, which is the end of the z-stack, click the end button.A critical step is to make sure that the Z step encompasses the area of interest. In our case, we want to capture the width of the eye from the lateral to the medial edges. Click on the menu for adjusting the acquisition time and the imaging frequency.For adequate recovery of the fluor4 and survival of the embryo, allow for a ratio of at least one to three between z-stack acquisition when laser power is on and recovery time when laser power is off. With appropriate time for embryo recovery, set the time between z-stacks in the designated window. Then set the total length of time for the experiment in the appropriate window.Now, turn on the live image setting to make final adjustments to the laser settings. Adjust laser transmission, gain and offset slider bars within the software to optimize the fluorescence image. Also, adjust the orientation of the embryo as needed depending on the length of the experiment, anticipated growth of the embryo, et cetera.Make sure that the area of interest remains within the frame through the duration of the experiment. During time-lapse acquisition, turn off the epifluorescence light source, cover the stage with the laser safety box which is adequate for protection against background light, then press start. Access the open bath chamber through the sliding doors on the laser safety box to refill it with time lapse embryo medium every 8-12 hours.Following imaging acquisition, open the files in the image processing software, highlight the correct image series. In the software, choose the process menu, click on 3D deconvolution and apply to deconvolve each z-stack. Import individual tif files into video processing software, then select all tif files and drag them into the video editor.Adjust the length of each image within the video to 0.1 seconds. Finally, export the video as a mov or mp4 file. Seen here by multi-photon fluorescence imaging are the migration patterns of cranial neural crest cells that give rise to the cranial facial structures and anterior segment of the eye in transgenic sox10:EGFP and foxd3:GFP zebrafish lines.Sox 10 positive neural crest cells between 12 and 30 hpf migrate from the edge of the neural tube into the cranial facial region. The cells from the prosencephalon and mesencephalon migrate dorsally and ventrally to the developing eye to populate the periocular mesencine. As indicated here, the sox10 positive cells form the frontal nasal process.Neural crest cells from the rhombencephalon migrate ventrally and give rise to the pharyngeal arches. Time lapse imaging of foxd3 positive neural crest cells between 30 and 60 hpf show that these cells migrated between the optic cup and surface ectoderm and though the ocular fissure marked by the arrow and coalesced around the lens marked by the asterisk forming the iris stroma. Once mastered, this technique from start to finish can be done in four days if it is performed properly.When attempting this procedure, it is important to remember to choose embryos with a high amount of fluorescence. Setting up the software initially can be challenging but once the parameters are determined, additional changes are typically minimal. After watching this video, you should have a good understanding of how to use multi-photon time-lapse microscopy to image migrating cells in the embryo.

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

Research

JoVE Journal

Developmental Biology

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.

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

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 ...

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.

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.

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 ...

Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a method whereby live-cell flow cytometry is extended upon to analyze the multiple functions of P2X7 receptor activation in real-time. Using a time module installed on a flow cytometer, live-cell functionality can be assessed and plotted over a given time period to explore the kinetics of calcium influx, transmembrane pore formation, and phagocytosis. This simple method is advantageous as all three canonical functions of the P2X7 receptor can be assessed using one machine, and the gathered data plotted over time provides information on the entire live-cell population rather than single-cell recordings often obtained using technically challenging patch-clamp methods. Calcium influx experiments use a calcium indicator dye, while P2X7 pore formation assays rely on ethidium bromide being allowed to pass through the transmembrane pore formed upon high agonist concentrations. Yellow-green (YG) latex beads are utilized to measure phagocytosis. Specific agonists and antagonists are applied to investigate the effects of P2X7 receptor activity. Individually, these methods can be modified to provide quantitative data on any number of calcium channels and purinergic and scavenger receptors. Taken together, they highlight how the use of real-time live-cell flow cytometry is a rapid, cost-effective, reproducible, and quantifiable method to investigate P2X7 receptor function.

Providing single-cell sensitivity, real-time flow cytometry is uniquely suited to quantify multimodal receptor functions of live cultures. Using adult neural progenitor cells, the P2X7 receptor function was assessed via calcium influx detected by calcium indicator dye, transmembrane pore formation by ethidium bromide uptake, and phagocytosis using fluorescent latex beads.

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a ...

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.

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

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.

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.

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

Real-Time Imaging of CCL5-Induced Migration of Periosteal Skeletal Stem Cells in Mice

Periosteal skeletal stem cells (P-SSCs) are essential for lifelong bone maintenance and repair, making them an ideal focus for the development of therapies to enhance fracture healing.&#160; Periosteal cells rapidly migrate to an injury to supply new chondrocytes and osteoblasts for fracture healing. Traditionally, the efficacy of a cytokine to induce cell migration has only been conducted in vitro by performing a transwell or scratch assay. With advancements in intravital microscopy using multiphoton excitation, it was recently discovered that 1) P-SSCs express the migratory gene CCR5 and 2) treatment with the CCR5 ligand known as CCL5 improves fracture healing and the migration of P-SSCs in response to CCL5. These results have been captured in real-time. Described here is a protocol to visualize P-SSC migration from the calvarial suture skeletal stem cell (SSC) niche towards an injury after treatment with CCL5. The protocol details the construction of a mouse restraint and imaging mount, surgical preparation of the mouse calvaria, induction of a calvaria defect, and acquisition of time-lapse imaging.

This protocol describes the detection of CCL5-mediated periosteal skeletal stem cell migration in real-time using live animal intravital microscopy.

Periosteal skeletal stem cells (P-SSCs) are essential for lifelong bone maintenance and repair, making them an ideal focus for the development of ...

An Optimized O9-1/Hydrogel System for Studying Mechanical Signals in Neural Crest Cells

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to various organs and tissues. Tissue stiffness produces mechanical force, a physical cue that plays a critical role in NCC differentiation; however, the mechanism remains unclear. The method described here provides detailed information for the optimized generation of polyacrylamide hydrogels of varying stiffness, the accurate measurement of such stiffness, and the evaluation of the impact of mechanical signals in O9-1 cells, a NCC line that mimics in vivo NCCs.
Hydrogel stiffness was measured using atomic force microscopy (AFM) and indicated different stiffness levels accordingly. O9-1 NCCs cultured on hydrogels of varying stiffness showed different cell morphology and gene expression of stress fibers, which indicated varying biological effects caused by mechanical signal changes. Moreover, this established that varying the hydrogel stiffness resulted in an efficient in vitro system to manipulate mechanical signaling by altering gel stiffness and analyzing the molecular and genetic regulation in NCCs. O9-1 NCCs can differentiate into a wide range of cell types under the influence of the corresponding differentiation media, and it is convenient to manipulate chemical signals in vitro. Therefore, this in vitro system is a powerful tool to study the role of mechanical signaling in NCCs and its interaction with chemical signals, which will help researchers better understand the molecular and genetic mechanisms of neural crest development and diseases.

Detailed step-by-step protocols are described here for studying mechanical signals in vitro using multipotent O9-1 neural crest cells and polyacrylamide hydrogels of varying stiffness.

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to ...