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

Forfatterne takker Thomas Schilling for venligt gifting Tg (sox10:eGFP) fisk og Mary Halloran for venligt gifting Tg(foxd3:GFP) fisk.

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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Multi photon time-lapse imaging giver i vivo sporing af forbigående og vandrende cellepopulationer. Denne kraftfulde teknik kan bruges til at studere embryonale processer i realtid, og i den nuværende undersøgelse, resultaterne af denne metode forbedret den nuværende viden om neurale crest celle migration og udvikling. Tidligere time-lapse imaging studier udnytter typisk Konfokal laser scanning mikroskopi. 29 , 30 , 31</s...

Medfødt øjensygdomme kan forårsage barndommen blindhed og er ofte på grund af deformiteter i den kranielle neurale crest. Neurale crest celler er forbigående stamceller, der opstår som følge af neuralrøret og danne talrige væv i hele kroppen. 1 , 2 , 3 , 4 , 5 neurale crest celler, der stammer fra prosencephalon og mesencephalon, give anledning til knogle og brusk af midface og frontal regioner, og iris, hornhinden, trabekelværket og sclera i den forreste segment af øjet. 4 , 6 , 7 , 8 neurale crest celler fra rhombencephalon form svælg arches, kæbe og hjerte udstrømning tarmkanalen. 1 , 3 , 4 , 9 , 10 undersøgelser har fremhævet bidrag af neural crest til okulær og periocular udvikling, understreger betydningen af disse celler i hvirveldyr øje udvikling. Faktisk, afbrydelse af neurale crest celler migrering og differentiering føre til kraniofacial og okulær anomalier som observeret i Axenfeld-Rieger-syndrom og Peters Plus syndrom. 11 , 12 , 13 , 14 , 15 , 16 , 17 således, en omfattende forståelse af migration, spredning og differentiering af disse neurale crest celler vil give indsigt i kompleksiteten bag medfødt øjensygdomme.
For zebrafisk er en kraftfuld model organisme for at studere okulær udvikling, som strukturerne af zebrafisk øjet ligner deres pattedyr modparter, og mange gener er evolutionært bevaret mellem zebrafisk og pattedyr. 18 , 19 , 20 Desuden zebrafisk embryoner er gennemsigtig og hunkøns, lette visualisering af øjet udvikling i realtid.
Uddybe tidligere offentliggjorte arbejde,6,7,20 vandrende mønstret af neurale crest celler blev beskrevet ved hjælp af multi photon fluorescens time-lapse imaging på transgene zebrafisk linjer mærket med grøn fluorescerende proteiner (NGL) under transcriptional kontrol af SRY (sex-bestemmende region Y)-box 10 (sox10) eller Forkhead boks D3 (foxd3) genet lovgivningsmæssige områder. 21 , 22 , 23 , 24. multi photon fluorescens time-lapse imaging er en kraftfuld teknik, der kombinerer de avancerede optiske teknikker i laser scanning mikroskopi med lang bølgelængde multi photon fluorescens excitation at fange høj opløsning, tre-dimensionelle billeder af prøver markeret med fluorophores. 25 , 26 , 27 anvendelse af multi foton laser har klare fordele over standard Konfokal mikroskopi, herunder øget væv penetration og nedsat fluorophore blegning.
Brug denne metode, var to forskellige populationer af neurale crest celler varierende i timing af migration og vandrende veje diskriminerede, nemlig foxd3-positive neurale crest celler i periocular udkom og udviklende øje og sox10-positive neurale crest celler i det kraniofaciale udkom. Med denne metode, er der indført en tilgang til at visualisere migration af okulær og craniofacial neurale crest migration i zebrafisk, gør det let at observere regulerede neurale crest migration i realtid under udvikling.
Denne protokol giver oplysninger til at generere time-lapse videoer i den tidlige øje udvikling i Tg (sox10:EGFP) og Tg (foxd3:GFP) transgene zebrafisk, som et eksempel. Denne protokol kan anvendes yderligere for høj opløsning, tre-dimensionelle, real-time visualisering af den tidlige udvikling af enhver okulær og craniofacial struktur, afledt af neurale crest celler i zebrafisk. Denne metode kan desuden yderligere anvendes til visualisering af udviklingen af andre væv og organer i zebrafisk og andre dyremodeller.

<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>
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			<td height="21" style="height:21px;width:251px;">Mai Tai DeepSee Ti-Sapphire Laser</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>
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			<td height="42" style="height:42px;width:251px;">Leica Application Suite X (LAS X)&#160; Software</td>
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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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multi-photon time lapse imaging to visualize development in real-time: visualization of migrating neural crest cells in zebrafish embryos

Medfødt øje og kraniofaciale anomalier afspejler forstyrrelser i de neurale crest, en forbigående befolkning af vandrende stamceller, der giver anledning til mange celletyper i hele kroppen. Forståelse af neural crest biologi har været begrænset, hvilket afspejler en manglende genetisk tractable modeller, der kan blive undersøgt i vivo og real-time. Zebrafisk er et særlig vigtigt udviklingsmæssige model til at studere vandrende cellepopulationer, f.eks i neurale crest. For at undersøge neurale crest migration ind i tredje øjet, blev en kombination af de avancerede optiske teknikker i laser scanning mikroskopi med lang bølgelængde multi photon fluorescens excitation implementeret for at fange høj opløsning, tre-dimensionelle, real-time videoer af tredje øjet i transgene zebrafisk embryoner, nemlig Tg (sox10:EGFP) og Tg (foxd3:GFP), som sox10 og foxd3 har været vist i adskillige dyremodeller for at regulere tidlige neurale crest differentiering og sandsynligvis repræsenterer markører for neurale crest celler. Multi photon time-lapse imaging blev brugt til at skelne den opførsel og vandrende mønstre af to neurale crest cellepopulationer bidrager til tidlig udvikling af øjet. Denne protokol giver oplysninger til at generere time-lapse videoer under zebrafisk neurale crest migration, som et eksempel, og yderligere kan anvendes til at visualisere den tidlige udvikling af mange strukturer i zebrafisk og andre modelorganismer.

Multi photon fluorescens time-lapse imaging genereret en række videoer, der afslørede migrationsmønstre kraniel neurale crest celler, der giver anledning til kraniofaciale strukturer og anterior segment af øjet i Tg (sox10:EGFP) og Tg (foxd3:GFP) zebrafisk linjer. Som et eksempel vandrer sox10 -positive neurale crest celler mellem 12 og 30 hpf fra kanten af neuralrøret i regionen craniofacial (Video 1, figur 2...

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

Multi Photon tid bortfalder Imaging til at visualisere udvikling i realtid: visualisering af overflytning neurale Crest celler i zebrafisk embryoner

En kombination af de avancerede optiske teknikker i laser scanning mikroskopi med lang bølgelængde multi photon fluorescens excitation blev implementeret for at fange høj opløsning, tre-dimensionelle, real-time imaging af neural crest migration i Tg (sox10:EGFP) og Tg (foxd3:GFP) zebrafisk embryoner.

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

Analyserer<em&gt; In vivo</em&gt; Cell Migration hjælp Cell transplantationer og Time-lapse Imaging i zebrafisk embryoner

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

Analyse af zebrafisk Kidney Udvikling med Time-lapse Imaging Ved hjælp af en dissektionsmikroskop Udstyret til Optical Sektionsinddeling

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 Måling og Real Time Visualisering af Somatisk 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 af hypoxi i levende frø og zebrafiskembryoner

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

Udvikling af et In Vitro Assay at kvantificere hæmatopoietisk stilk og stamfader celler (HSPCs) i at udvikle zebrafisk embryoner

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

Realtids Live-celle flowcytometri at undersøge Calcium tilstrømning, Pore dannelse og fagocytose af P2X7 receptorer i voksne neurale 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 lagdelt monteringsmetode til forlænget tidsforskudt Konfokal mikroskopi af hele Zebrafish-embryoner

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-time bioluminescens billeddannelse af notch signalering dynamik under murine Neurogenesis

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

Isolation, Kultur og adipogen induktion af neuralcrest original fedt-afledte stamceller fra periaortisk fedtvæv

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