Name: visualizing neuroblast cytokinesis during c. elegans embryogenesis
Uploaded: 2014-03-12T18:05:00
Description: Watch this Scientific Journal Video about Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis at JoVE.com

Forfatterne vil gerne anerkende, at dette arbejde blev støttet af naturvidenskab og teknik Forskningsråd Canada (NSERC) tilskud.

1.. Fremstilling af plader til Opretholdelse Worm Stammer og Performing RNAi <ol><li> Nematodevækst Media Plates <ol><li> Plader <ol><li> Forbered nematodevækst Media (NGM) plader for at opretholde ormen stammer og til at udføre genetiske kors. Kombiner 3 g NaCl, 17 g agar og 2,5 g bactopepton med 1 liter destilleret vand i en 2 liters kolbe og tilsættes et metal omrørerstav. </li><li> Autoklaver kolben for at opløse agaren og at sterilisere mediet. Derefter anbringes kolben på en omrøringsplade og lade medier...

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Denne protokol beskriver brugen af ​​forskellige typer af mikroskopi til billede celledelinger under midten embryogenese. Især denne protokol fremhæver, hvordan billedet delingen af ​​neuroblasts, celler, der kan fremme epidermal morfogenese. Celle-celle-kommunikation er vigtig for vævsdannelse under metazo udvikling og C. elegans er en fremragende model til at studere vævsdannelse in vivo. En begivenhed, der pænt skildrer samspillet af væv er epidermal morfogenese, der dækker foster i e...

Mens der er protokoller, der beskriver, hvordan billedet celledeling i begyndelsen C. elegans embryo, denne protokol beskriver, hvordan billedet celledeling i et væv under midten embryogenese. En af de store udfordringer i billeddannelse organismer under udvikling har været deres følsomhed over for fototoksicitet. Imidlertid har øget adgang til spinning disk konfokal mikroskoper eller fejes felt mikroskoper tilladt mere udbredte billedbehandlingsprogrammer. Begge systemer bruger solid state lasere og lysspredning, begrænse niveauerne af UV at organismerne udsættes for. Dog kan Widefield stande stadig bruges til billeddannelse in vivo, især hvis de er udstyret med kameraer med høj følsomhed (f.eks EMCCD), blænde kontrol og lysstyring (f.eks lysdioder eller justerbare kviksølv pærer). Denne protokol beskriver, hvordan man bruge enten en konfokal baseret system eller et vidvinkel-system til billede celledeling indenfor udviklingen af C. elegans &lt;/ Em&gt; embryoner. Som et eksempel beskriver vi, hvordan billedet neuroblast celledeling. Neuroblasts kan lette epidermal morfogenese ved at give kemiske eller mekaniske signaler til de overliggende epidermale celler, og giver et glimrende eksempel på, hvor vigtigt det intercellulære kommunikation i dannelsen af ​​væv. Caenorhabditis elegans er en ideel model organisme til mikroskopi baserede undersøgelser, på grund af sin åbenhed og enkle væv organisation 1. Endvidere C. elegans er modtagelig for genetiske metoder og RNAi, og da mange af dens gener har humane homologer, kan den anvendes til at identificere konserverede mekanismer til vævsdannelse 2-5. I C. elegans, dannelse af overhuden sker i midten embryogenese, når fosteret har&gt; 300 celler. Epidermal morfogenese består af flere vigtige faser, hvorunder foster er indesluttet i et lag af epidermale celler, snøre og udvide til at omdanne embryo fra en ægformet form til den aflange form af en orm 6. Ventral kabinet beskriver en af disse morfogenetiske begivenheder, når ventrale epidermale celler migrere til den ventrale midterlinie til at dække den ventrale overflade af embryo (figur 1). Først to par forreste placeret forkant celler migrere til den ventrale midterlinjen hvor de klæber og sikring med deres kontralaterale 6 naboer. Dette efterfølges af migrationen af de posteriore placeret lomme celler, som danner kile lignende figurer skaber en ventral lomme 6-7. Den mekanisme, som lukker lommen er ikke godt forstået. En mulighed er, at en supracellular actin myosin kontraktile struktur binder pocket celler sammen i en pung streng som mode, svarende til sårheling 8. Interessant er migration af nogle af pocket-celler medieres af specifikke delmængder af underliggende neuroblaster 9 (neuronale prækursorer, der er fundet under epidermis; Figur 1B). Tidligere undersøgelser viste, at de neuroblaster regulere ventrale epidermal cellemigration og ventrale kabinet. VAB-1 (Ephrin receptor) og VAB-2 (Ephrin ligand) er stærkt udtrykt i neuroblaster og lette sorteringen af forreste og bageste neuroblaster fra hinanden, og mutationer i VAB-1 eller VAB-2 årsag ventrale kabinet Fænotypers 10-13. Imidlertid viste promotor redning eksperimenter, VAB-1 også er påkrævet i de overliggende ventrale epidermale celler og receptorer for andre vejledende tidskoder udskilles af neuroblaster udtrykkes i ventrale epidermisceller 9. Selvom mutationer i nogen af disse receptorer forårsager ventrale kabinet fænotyper, er det ikke klart, om fejl opstår på grund af problemer i neuroblast positionering eller på grund af svigt af ventrale epidermale celler til at reagere på vejledning tidskoder 14. Ændring af opdelingen af ​​neuroblasts without påvirker deres evne til at udskille vejledning tidskoder kunne kaste lys over den rolle af neuroblasts og deres evne til at levere mekanisk input under epidermal morfogenese. For nylig blev det konstateret, at en celledeling gen, ani-1 (anillin) er overudtrykt i de neuroblasts (figur 2a) og udtynding forårsager neuroblast division defekter. Interessant, disse embryoner vise ventrale kabinet fænotyper (Fotopoulos, Wernike og Piekny, upublicerede observationer). Anillin er nødvendig for celledeling, og især for cytokinese, der beskriver den proces, hvor en mor celle fysisk deler sig i to datterceller. Cytokinese er drevet af dannelsen af ​​et actomyosin kontraktile ring, som skal stramt kontrolleret i rum og tid til at sikre, at det er korrekt kombineret med søsterkromatid adskillelse. Føreren regulator af metazoan cytokinese er RhoA (RHO-1 i C. elegans), en lille GTPase, der er enctive i GTP-bundne form,. GEF Ect2/ECT-2 aktiverer RhoA, hvorefter RhoA-GTP interagerer med nedstrøms effektorer, der danner den kontraktile ringen og formidler sin indtrængen 15.. Anillin er et multi-domæne, der binder til RhoA via sin C-terminale ende og actin og myosin via sin N-terminus. Anillin er nødvendig for at stabilisere positionen af den kontraktile ringen i pattedyrs-eller Drosophila-S2-celler 16. Anillin udtømning forårsager kontraktile ringe til at gennemgå laterale svingninger, og cytokinese sidst fejler danner multinucleate celler 17-19. Interessant, selvom C. elegans ani-1 koordinater actomyosin kontraktilitet i det tidlige embryon, er det ikke afgørende for cytokinese. Men som beskrevet ovenfor, er ani-1 kræves for neuroblast cytokinese i midten af embryogenese (Fotopoulos, Wernike og Piekny, upublicerede observationer). Cytokinese fiasko ville ændre på antallet og placeringen af ​​neuroblasts og kan påvirke locning af kemiske vejledning tidskoder, eller det kan ændre de mekaniske egenskaber af vævet. Begge modeller fremhæve nonautonomous rolle neuroblasts for ventrale kabinet, og betydningen af ​​væv-væv kommunikation under fosterudviklingen. Denne forsøgsprotokol beskriver, hvordan man billedet celledeling under C. elegans midten embryogenese ved hjælp af fluorescensmikroskopi. De fleste eksperimenter studere mekanismer celledeling blev udført i enkelte celler i dyrkningsskåle (f.eks. HeLa-eller S2-celler) eller tidlige embryoner med et begrænset antal celler (fx C. elegans en celle embryon Xenopus eller echinoderm embryoner). Men det er også vigtigt at undersøge celledeling i væv, som der er eksterne signaler, der kan påvirke timing og placering af divisionen flyet. Desuden kan celler giver kemiske eller mekaniske signaler at påvirke udviklingen af ​​tilstødende vævs, og det er vigtigt at forstå, hvordan intercellulær kommunikation hjælper væv dannes under udvikling.

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			<td height="21" style="height:21px;width:291px;">Agar</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>BioShop Canada Inc.</td>
			<td>#GLY001.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IPTG (isopropylthio-&#946;-galactoside)</td>
			<td>Bio Basic Inc.</td>
			<td>#367-93-1</td>
			<td>Store powder and dissolved IPTG at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KH2PO4 (potassium phosphate, monobasic)</td>
			<td>BioShop Canada Inc.</td>
			<td>#PPM666.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">K2HPO4 (potassium phosphate, dibasic)</td>
			<td>BioShop Canada Inc.</td>
			<td>#PPD303.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L4440&#160; (feeding vector)</td>
			<td>Addgene</td>
			<td>#1654</td>
			<td>Keep as glycerol stock at -80 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgSO4&#160;&#160; (magnesium sulfate)</td>
			<td>BioShop Canada Inc.</td>
			<td>#MAG511.500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl (sodium chloride)</td>
			<td>Bio Basic Inc.</td>
			<td>#7647-14-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2HPO4 (sodium phosphate, dibasic)</td>
			<td>Bio Basic Inc.</td>
			<td>#7558-79-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Normal Donkey Serum (NDS)</td>
			<td>Wisent Bioproducts</td>
			<td>#035-110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">n-propyl 3.4.5-trihydroxybenzoate (propyl gallate)</td>
			<td>Alfa Aesar</td>
			<td>#A10877</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-L-lysine</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>#P8920</td>
			<td>For optimal results coat microscope slides three times&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptomycin</td>
			<td>BioShop Canada Inc.</td>
			<td>#STP101.50</td>
			<td>Store powder at 4 &#176;C and dissolved streptomycin at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tetracyclin</td>
			<td>BioShop Canada Inc.</td>
			<td>#TET701.10</td>
			<td>Store powder at 4 &#176;C and dissolved tetracycline at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween-20</td>
			<td>Bio Basic Inc.</td>
			<td>CAS#9005-64-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tryptone</td>
			<td>BioShop Canada Inc.</td>
			<td>#TRP402.500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast Extract</td>
			<td>Bio Basic Inc.</td>
			<td>#8013-01-2</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualizing neuroblast cytokinesis during c. elegans embryogenesis

Denne protokol beskriver anvendelsen af fluorescensmikroskopi billedet delende celler under udvikling af Caenorhabditis elegans embryoner. Især denne protokol fokuserer på, hvordan billedet dividere neuroblasts, som findes under de epidermale celler og kan være vigtige for epidermal morfogenese. Tissue dannelse er afgørende for metazoan udvikling og er afhængig af eksterne signaler fra omkringliggende væv. C. elegans er en fremragende model organisme for at studere vævsmorfogenese in vivo på grund af dets gennemsigtighed og enkel organisation gøre sine væv nemt at studere via mikroskopi. Ventral kabinettet er den proces, hvor den ventrale overflade af embryoet er dækket af et enkelt lag af epitelceller. Denne begivenhed menes at være lettet af de underliggende neuroblasts, som giver kemisk vejlednings stikord til at mægle migration af de overliggende epitelceller. Men neuroblaster er yderst proliferativ og kan også fungeresom en mekanisk substrat for ventrale epidermale celler. Undersøgelser ved hjælp af denne forsøgsplan kunne afdække betydningen af ​​intercellulær kommunikation under vævsdannelse og kunne anvendes til at afsløre de roller gener involveret i celledeling i udviklingslandene væv.

Denne forsøgsprotokol beskriver, hvordan man billedet celledeling i C. elegans embryoner under midten embryogenese. Især det beskriver, hvordan billed neuroblasts, der kan lette epidermal morfogenese. Epidermal morfogenese opstår på grund af en kombination af epidermal celle form ændringer, migration og adhæsion, men også er afhængig af kemiske eller mekaniske signaler fra de underliggende neuroblasts (figur 1b). De neuroblaster udskiller vejledning signaler, der modtages af receptorer ...

Watch this Scientific Journal Video about Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis at JoVE.com

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

Denne protokol beskriver, hvordan billedet dividere celler i et væv i Caenorhabditis elegans embryoner. Mens flere protokoller beskriver, hvordan du billedet celledeling i begyndelsen foster, denne protokol beskriver, hvordan billedet celledeling i et udviklingsland væv under midten sent embryogenese.

Visualisering neuroblast cytokinese Under<em&gt; C. elegans</em&gt; Embryogenesen

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this ...

Research

JoVE Journal

Neuroscience

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

Assaying &beta;-amyloid Toxicity using a Transgenic C. elegans Model

Assaying &#946;-amyloid Toxicity using a Transgenic C. elegans Model

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant ...

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment ...

In vivo Laser Axotomy in C. elegans

In vivo Laser Axotomy in C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron ...

Automated Quantification of Synaptic Fluorescence in C. elegans

Automated Quantification of Synaptic Fluorescence in C. elegans

Automatiseret Kvantificering af Synaptic Fluorescens in<em&gt; C. elegans</em

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall ...

C. elegans Tracking and Behavioral Measurement

C. elegans Tracking and Behavioral Measurement

C. elegans Tracking og adfærdsmæssige måling

We have developed instrumentation, image processing, and data analysis techniques to quantify the locomotory behavior of C. elegans as it crawls on the ...

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

En Molekylær Udlæsning af Langsigtet olfaktorisk Adaptation in<em&gt; C. elegans</em

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps ...

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Hippocampale Insulin Mikroinjektion og<em&gt; In vivo</em&gt; Mikrodialyse Under Spatial Memory Test

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance ...

In vivo Neuronal Calcium Imaging in C. elegans

In vivo Neuronal Calcium Imaging in C. elegans

In vivo Neuronal Calcium Imaging i C. elegans

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

In vivo billeddannelse af Dauer-specifik neuronal Remodeling i C. elegans

The mechanisms controlling stress-induced phenotypic plasticity in animals are frequently complex and difficult to study in vivo. A classic example of ...

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

En analyse til måling virkningerne af ethanol på Locomotion hastighed<em&gt; Caenorhabditis elegans</em

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the ...