Name: facial transplants in xenopus laevis embryos
Uploaded: 2014-03-26T13:00:00
Description: Watch this Scientific Journal Video about Facial Transplants in Xenopus laevis Embryos at JoVE.com

Vi takker Radek Sindelka for hans hjælp, og Cas Bresilla for at bistå med frø dyrehold og forberedelse foster. Dette arbejde blev finansieret af NIH via tilskud R01DE021109 til HLS Laura Jacox blev finansieret af Herschel Smith Graduate Fellowship på Harvard University og en F30 individuel stipendium F30DE022989-01 gennem NIDCR.

1.. Forberedelse reagenser <ol><li> 10x MBS: Forbered 1 L 10x Modificeret Barths Saline (MBS) løsning 17. Se Tabel 1, reagenser, ingredienser, og instruktioner. Brug destilleret vand til alle løsninger. Bland i et bæger, ved hjælp af en omrører, indtil fuld opløsning. Alle opløsninger bør foretages ved stuetemperatur. </li><li> 1x MBS: Fortynd 100 ml 10x MBS løsning i 900 ml destilleret vand til at gøre 1 L 1X MBS. Tilføj 0,7 ml 1 M CaCl2-opløsning. </li><li> 0.1x MBS...

<ol>
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Kritiske stadier og Begrænsninger: EAD ansigt transplantation procedure er tid og arbejde intensivt. Det kræver praksis, konstant hænder, og fingerfærdighed at perfektionere. Ansigtet transplantation protokol bygger på forskerens evne til at fjerne effektivt og transplantation af væv. Hvis man tager for lang tid at indsætte transplantation i værtens ansigt, vil værten ansigt begynde at kontrakten og helbrede. Tang kan bruges til forsigtigt udvide ansigtet regionen. Men hvis betydelige sår sammentræknin...

For at forstå mekanismerne bag kraniofaciale misdannelser 1-2, vigtige væv og deres signalering bidrag i løbet af kraniofaciale udvikling skal identificeres. I frøen Xenopus laevis del af ansigtet, herunder munden og næsebor form fra &quot;Extreme Anterior Domain&quot; (EAD), hvor ektoderm og endoderm direkte sammenstillet 3-4. EAD fungerer også som en signalering center til at påvirke omgivende væv, herunder kranie neurale våbenskjold, som danner kæberne og andre ansigtstræk regioner 5. Til at identificere gener, der bidrager til EAD funktion, en &#39;ansigt transplantation&#39; teknik udviklet, hvor væv transplanteres fra en donor til en værtsembryo efter fjernelse af den tilsvarende vært regionen. Efter transplantationen, medfører ansigtet udvikling vurderes. Således er virkningerne af tab af funktion (LOF) eller øget funktion (GOF) for et specifikt gen i EAD analyseret lokalt, hvor resten af ​​head og krop består af vildtype væv. Den gensidige transplantation kan udføres, hvor vildtype væv er transplanteret ind i embryoner med global LOF eller GOF i specifikke gener. Transplantation er ofte blevet brugt i Xenopus og chick studies 6. For eksempel har Xenopus transplantation behandlet homogenetic neural induktion, linse og neurale kompetence, og neuralkam migration 7-10. Quail-chick kimære podning har analyseret udviklingen af den forreste neurale plade, anterior neural højderyg, neurale våbenskjold, og kranieknogler 11-14. Dette er den første transplantation teknik til undersøgelse af kraniofaciale udvikling i Xenopus. Denne teknik har vist en hidtil ukendt rolle for Wnt inhibitorer Frzb1 og Crescent i reguleringen basalmembran dannelse i den formodede munden 5. Xenopus laevis er en ideel model for undersøgelse af kraniofaciale udvikling embryoer er store, udvikle eksternt, etnd ansigtet er umiddelbart synlige, så mikromanipulering og billeddannelse af udvikling. Mekanismerne bag facial udvikling ser bevaret, hvilket indikerer, at konstateringer i frøen give indsigt i den menneskelige udvikling 4,15-16.

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			<td height="20" style="height:20px;width:195px;">Pasteur pipette</td>
			<td rowspan="3" style="width:101px;">VWR</td>
			<td rowspan="3" style="width:136px;">14672-400</td>
			<td style="width:128px;">Lime Glass&#160;</td>
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			<td height="20" style="height:20px;width:195px;">Size 5 3/4&#8217;&#8217;</td>
			<td style="width:128px;">Cotton Plugged</td>
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			<td style="width:128px;">Disposable</td>
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		<tr height="20">
			<td height="41" rowspan="2" style="height:41px;width:195px;">Graduated Transfer Pipette</td>
			<td rowspan="2" style="width:101px;">VWR</td>
			<td rowspan="2" style="width:136px;">16001-180</td>
			<td style="width:128px;">Disposable&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Polyethylene</td>
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		<tr height="80">
			<td height="100" rowspan="2" style="height:100px;width:195px;">#5/45 forceps</td>
			<td rowspan="2" style="width:101px;">Fine Science Tools by Dupont medical</td>
			<td rowspan="2" style="width:136px;">11251-35</td>
			<td rowspan="2" style="width:128px;">Angled 45 degrees</td>
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		<tr height="40">
			<td height="81" rowspan="3" style="height:81px;width:195px;">Standard Pattern Forceps</td>
			<td rowspan="3" style="width:101px;">Fine Science Tools</td>
			<td rowspan="3" style="width:136px;">11000-20</td>
			<td style="width:128px;">Straight; serrated tip</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:128px;">Stainless Steel;</td>
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			<td height="21" style="height:21px;width:128px;">20cm long</td>
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			<td height="81" rowspan="2" style="height:81px;width:195px;">Capillary Tubing (for needles)</td>
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			<td rowspan="2" style="width:136px;">30-30-1</td>
			<td style="width:128px;">Borosil 1.0mm OD x 0.5mm ID/Fiber</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:128px;">100mm each</td>
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			<td height="20" style="height:20px;width:195px;">Cover slip&#160;</td>
			<td rowspan="4" style="width:101px;">VWR</td>
			<td style="width:136px;">48393 252&#160;</td>
			<td style="width:128px;">24x60mm&#160;</td>
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			<td height="20" style="height:20px;width:195px;">micro cover glass</td>
			<td style="width:136px;">or&#160;</td>
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			<td height="20" style="height:20px;width:195px;">(for glass bridges)</td>
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			<td style="width:128px;">24x40mm</td>
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			<td height="21" style="height:21px;width:195px;">No.1.5</td>
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			<td height="41" style="height:41px;width:195px;">Ficoll 400</td>
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			<td style="width:136px;">F9378</td>
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			<td height="60" style="height:60px;width:195px;">Needle Puller&#160;</td>
			<td style="width:101px;">Sutter Instrument Co</td>
			<td rowspan="3" style="width:136px;">Needle Puller: discontinued Filament: FB300B</td>
			<td rowspan="3" style="width:128px;">The most similar, currently available&#160; needle puller is the P-97. For filaments, use Sutter 3.00mm square box filaments, 3.0mm wide.</td>
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		<tr height="80">
			<td height="80" style="height:80px;width:195px;">Model P-80</td>
			<td style="width:101px;">Flaming / Brown micropipette puller</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:195px;">(discontinued)</td>
			<td style="width:101px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:195px;">Stereomicroscope</td>
			<td rowspan="2" style="width:101px;">Zeiss</td>
			<td rowspan="2" style="width:136px;"></td>
			<td rowspan="2" style="width:128px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Zeiss Stemi 1000</td>
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		<tr height="61">
			<td height="61" style="height:61px;width:195px;">Stereomicroscope Lighting by Fostec</td>
			<td style="width:101px;">Fostec</td>
			<td style="width:136px;"></td>
			<td style="width:128px;">Use a light box with 2 fiberoptic arms.&#160;&#160;</td>
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		<tr height="60">
			<td height="81" rowspan="2" style="height:81px;width:195px;">Nickel Plated Pin Holder</td>
			<td rowspan="2" style="width:101px;">Fine Science Tools</td>
			<td rowspan="2" style="width:136px;">26018-17</td>
			<td style="width:128px;">Jaw Opening Diameter: 0 to 1mm</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Length: 17cm</td>
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		<tr height="60">
			<td height="101" rowspan="3" style="height:101px;width:195px;">Moria Nickel Plated Pin Holder</td>
			<td rowspan="3" style="width:101px;">Fine Science Tools</td>
			<td rowspan="3" style="width:136px;">26016-12</td>
			<td style="width:128px;">Jaw opening Diameter: 0 to 1mm</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:128px;">Length: 12cm</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:128px;"></td>
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		<tr height="40">
			<td height="60" rowspan="2" style="height:60px;width:195px;">Tungsten Needles</td>
			<td rowspan="2" style="width:101px;">Fine Science Tools</td>
			<td rowspan="2" style="width:136px;">10130-05</td>
			<td rowspan="2" style="width:128px;">0.125mm Rod diameter</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:195px;">Van Aken Plastalina</td>
			<td rowspan="2" style="width:101px;">Blick&#160;</td>
			<td rowspan="2" style="width:136px;">#33268-2981</td>
			<td rowspan="2" style="width:128px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:195px;">Modeling Clay- white, red or yellow</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:195px;">mMessage mMashine SP6 or T7 Kit</td>
			<td style="width:101px;">Ambion</td>
			<td style="width:136px;">AM1340</td>
			<td style="width:128px;"></td>
		</tr>
	</tbody>
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facial transplants in xenopus laevis embryos

Kraniofaciale misdannelser forekommer i 1 ud af hver 700 levendefødte, men Ætiologien sjældent kendt på grund af begrænset forståelse af kraniofaciale udvikling. At identificere, hvor signalveje og væv handle under mønstret af ansigtet udvikle, har en &#39;ansigt transplantation&#39; teknik er udviklet i embryoner fra frøen Xenopus laevis. En region af formodede ansigtsservietter (den &quot;Extreme Anterior Domain&quot; (EAD)) fjernes fra en donor embryo på tailbud scenen, og transplanteres til en værtsembryo af samme fase, hvorfra den tilsvarende region er blevet fjernet. Dette kan bruges til at generere et kimært ansigt, hvor værten eller donorvæv har et tab eller gevinst af funktion i et gen, og / eller omfatter en slægt etiket. Efter healing, er resultatet af udviklingen overvåges, og angiver roller signalvejen inden donor eller de omkringliggende værtsvævene. Xenopus er en værdifuld model for ansigt udvikling, da ansigtet regionen er stor og let enccessible for mikromanipuleringer. Mange embryoner kan analyseres i løbet af en kort tidsperiode, da udvikling sker hurtigt. Fund i frøen er relevante for menneskelig udvikling, da kraniofaciale processer vises konserveret mellem Xenopus og pattedyr.

Transplanteret væv skal sættes helt ind i værten hovedet efter transplantation som vist i figur 3A, og har et glas bro passende placeret på fosterets ansigt, som vist i figur 2bc. Det transplanterede donor væv skal være korrekt dimensioneret til værten åbning for transplantation at være vellykket. EAD-vævet rager ud fra hovedet, på nogen måde, som det ses i figur 3B og 3C. Desuden bør ansigt transplantation ikke drejes i forhold til dens po...

Watch this Scientific Journal Video about Facial Transplants in Xenopus laevis Embryos at JoVE.com

Facial Transplants in Xenopus laevis Embryos

Facial Transplantation i<em&gt; Xenopus laevis</em&gt; Embryoner

En teknik til omplantning &quot;Extreme Anterior Domain&quot; facial væv mellem Xenopus laevis embryoner er blevet udviklet. Væv kan flyttes fra en genekspression baggrund til en anden, hvilket tillader undersøgelse af lokale krav til kraniofaciale udvikling og til signalering interaktioner mellem ansigtet regioner.

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...

Research

JoVE Journal

Biology

Facial Transplants in Xenopus laevis Embryos

Dissection of Organizer and Animal Pole Explants from Xenopus laevis Embryos and Assembly of a Cell Adhesion Assay

Plastic Embedding and Sectioning of Xenopus laevis Embryos

Obtaining Eggs from Xenopus laevis Females

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Tissue Determination Using the Animal Cap Transplant (ACT) Assay in Xenopus laevis

Tissue Determination Using the Animal Cap Transplant ACT Assay in Xenopus laevis

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...