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

Författarna vill tacka Thomas Schilling för vänligt gifting Tg (sox10:eGFP) fisk och Mary Halloran för vänligt gifting Tg(foxd3:GFP) fisken.

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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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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Flera photon time-lapse imaging gör i vivo spårning av övergående och flyttande cellpopulationer. Denna kraftfulla teknik kan användas för att studera embryonala processer i realtid, och i den aktuella studien, förbättrade resultaten av denna metod den aktuella kunskapen om neural crest cellmigration och utveckling. Tidigare time-lapse imaging studier utnyttjar vanligtvis confocal microscopy för laserscanning. 29 , 30 , <sup class="xr...

Medfödda ögonsjukdomar kan orsaka barndom blindhet och beror ofta på avvikelser i den kraniala neural crest. Neural crest celler är övergående stamceller som kan uppstå från neuralrörsdefekter och bildar många vävnader i hela kroppen. 1 , 2 , 3 , 4 , 5 Neural crest celler, härrör från prosencephalon och mesencephalon, ge upphov till ben och brosk i mellanansiktet och frontala regioner, och iris, hornhinnan, trabekelverket och sklera i främre segmentet av ögat. 4 , 6 , 7 , 8 Neural crest celler från rhombencephalon form faryngeal arches, käken och hjärt utflöde tarmkanalen. 1 , 3 , 4 , 9 , 10 studier har belyst de bidrag av den neural crest till okulär och periokulära utveckling, betonar vikten av dessa celler i ryggradsdjur öga utveckling. Faktiskt, störningar av neural crest cellmigration och differentiering leda till kraniofaciala och okulär anomalier som observerats i Axenfeld-Rieger syndrom och Peters Plus syndrom. 11 , 12 , 13 , 14 , 15 , 16 , 17 således en omfattande förståelse av migration, proliferation och differentiering av dessa neural crest celler kommer att ge inblick i komplexiteten bakom medfödda ögonsjukdomar.
Zebrafiskar är en kraftfull modellorganism för att studera okulär utveckling, som strukturerar av zebrafisk ögat liknar motsvarigheterna hos däggdjur, och många gener är evolutionärt bevarade mellan zebrafiskar och däggdjur. 18 , 19 , 20 dessutom Sebrafisken embryon är transparent och ovipar, underlättar visualisering av ögat utveckling i realtid.
Expanderande på tidigare publicerade arbete,6,7,20 flyttande mönster av neural crest celler beskrevs med flera photon fluorescens time-lapse imaging på transgena zebrafiskar linjer märkt med grönt fluorescerande protein (GFP) under SRY (sex-bestämma region Y) transkriptionell kontroll-fält 10 (sox10) eller Forkhead låda D3 (foxd3) genen regulatoriska regioner. 21 , 22 , 23 , 24. flera photon fluorescens time-lapse imaging är en kraftfull teknik som kombinerar de avancerade optiska teknikerna av laserscanning mikroskopi med lång våglängd flera photon fluorescens magnetisering att fånga högupplösta, tredimensionella bilder av prov märkta med fluorophores. 25 , 26 , 27 användning av flera photon lasern har tydliga fördelar jämfört med standard konfokalmikroskopi, inklusive ökad vävnad penetration och minskad fluorophore blekning.
Två distinkta populationer av neural crest celler varierar i tidpunkten för migration och flyttande vägar med den här metoden och var diskriminerade, nämligen foxd3-positiv neural crest celler i periokulära mesenchyme och utveckla öga och sox10-positiv neural crest celler i de kraniofaciala mesenchyme. Med den här metoden introduceras en strategi att visualisera migration av okulär och kraniofaciala neural crest migration i zebrafiskar, vilket gör det lätt att observera reglerade neural crest migrering i realtid under utveckling.
Detta protokoll ger information för att generera tidsförlopp videor under tidig öga utveckling i Tg (sox10:EGFP) och Tg (foxd3:GFP) transgena zebrafiskar, som ett exempel. Detta protokoll kan tillämpas ytterligare för högupplösta, tredimensionella, realtid visualisering av den tidiga utvecklingen av någon okulär och kraniofaciala struktur som härrör från neural crest celler i zebrafiskar. Denna metod kan dessutom ytterligare tillämpas för visualisering av utvecklingen av andra vävnader och organ i zebrafiskar och andra djurmodeller.

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

Medfödd öga och kraniofaciala anomalier återspeglar störningar i den neural crest, en övergående befolkning av flyttande stamceller som ger upphov till många typer av celler i hela kroppen. Förstå biologin av den neural crest har begränsats, vilket återspeglar bristande genetiskt eftertraktansvärda modeller som kan studeras i vivo och i realtid. Zebrafisk är en särskilt viktig utvecklingsmässiga modell för att studera flyttande cellpopulationer, såsom den neural crest. För att undersöka neural crest migration in i tredje ögat, implementerades en kombination av avancerade optiska tekniker för laserscanning mikroskopi med lång våglängd flera photon fluorescens excitation för att fånga högupplösta, tredimensionella, realtid videor av utveckla ögat i transgena zebrafiskar embryon, nämligen Tg (sox10:EGFP) och Tg (foxd3:GFP), som sox10 och foxd3 har visats i ett flertal djurmodeller att reglera tidiga neural crest differentiering och sannolikt representerar markörer för neural crest celler. Flera photon time-lapse imaging användes för att urskilja den beteende och flyttande mönster två neural crest cellpopulationer bidrar till tidig öga utveckling. Detta protokoll ger information för att generera tidsförlopp videor under zebrafiskar neural crest migreringen, som ett exempel, och ytterligare kan användas för att visualisera den tidiga utvecklingen av många strukturer i zebrafiskar och andra modellorganismer.

Flera photon fluorescens time-lapse imaging genereras en serie videoklipp som avslöjade flyttmönster av kraniala neural crest celler som ger upphov till de kraniofaciala strukturer och främre segmentet av ögat i Tg (sox10:EGFP) och Tg (foxd3:GFP) zebrafiskar linjer. Som ett exempel migrera sox10 -positiva neural crest celler mellan 12 och 30 hpf från kanten av neuralrörsdefekter i regionen kraniofaciala (Video 1, figu...

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

Flera Photon Time Lapse Imaging att visualisera utvecklingen i realtid: visualisering av migrera Neural Crest celler hos zebrafiskar embryon

En kombination av avancerade optiska tekniker för laserscanning mikroskopi med lång våglängd flera photon fluorescens excitation implementerades för att fånga högupplösta, tredimensionella, realtid avbildning av neural crest migration i Tg (sox10:EGFP) och Tg (foxd3:GFP) zebrafiskar embryon.

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

Research

JoVE Journal

Developmental Biology

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

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

analysera<em&gt; In Vivo</em&gt; Cell Migration använder Cell Transplantationer och Time-lapse avbildning i Zebrafish embryon

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

Analys av zebrafisk njure utveckling med Time-lapse avbildning med ett dissektionsmikroskop Utrustad för optisk Sektione

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

Kinetic Mätning och Real Time Visualisering av somatiska Omprogrammering

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

Induktion av hypoxi i levande groda och zebrafiskembryon

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

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

Utveckling av ett In Vitro -test att kvantifiera hematopoetiska stamceller och stamceller celler (HSPCs) utveckla zebrafiskar embryon

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

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

Realtid Live-cell flödescytometri att undersöka kalcium tillströmning, Pore bildandet och fagocytos av P2X7-receptorer i vuxen neurala stamceller

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

En skiktad monteringsmetod för utökad tidsfördröjning konfokalmikroskopi av hela Zebrafish embryon

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

Real tid bioluminescence avbildning av notch signalering dynamik under Murine neurogenes

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

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

Isolering, kultur och adipogenic Induktion av neurala Crest Original Fett-Härledda stamceller från periaorektisk fettvävnad

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