Summary

Analyse av Neural Crest Migrasjon og differensiering av Cross-arter Transplantation

Published: February 07, 2012
doi:

Summary

En tilnærming for å analysere migrasjon og eventuell skjebne av aviær neural crest cellene i vaktel-chick chimeric embryoer er beskrevet. Denne metoden er en enkel og grei teknikk for sporing neural crest cellene under trekket og differensiering som ellers er vanskelig å skille innenfor en unmanipulated kylling embryo.

Abstract

Avian embryoer gir en unik plattform for å studere mange virveldyr utviklingsprosesser, på grunn av enkel tilgang av embryoene i egget. Chimeric avian embryo, der vaktel donor vev blir transplantert inn en kylling embryo i ovo, kombinere kraften uutslettelige genetisk merking av celle populasjoner med enkel manipulasjon presenteres av aviær embryo.

Quail-chick chimeras er et klassisk verktøy for å spore vandrende neural crest cellene (NCCer) 1-3. NCCer er en forbigående vandringer populasjon av celler i embryoet, som stammer i den dorsale delen av å utvikle nevralrøret 4. De gjennomgår en epitelial til mesenchymale overgang og deretter migrere til andre regioner av embryoet, hvor de differensiere i ulike celletyper, inkludert 5-13 brusk, melanocytter 11,14-20, nevroner og gliaceller 21-32. NCCer er multipotent, og deres endelige skjebne er innflytelsepåvirket av 1) den delen av nevralrøret hvor de stammer langs Rostro-kaudal aksen av embryoet 11,33-37, 2) signaler fra nabocellene som de vandrer 38-44, og 3) mikromiljøet av deres endelige destinasjon i fosteret 45,46. Følge disse cellene fra deres synspunkt opprinnelse i nevralrøret, til sin endelige posisjon og skjebne innenfor fosteret, gir viktig innsikt i de utviklingsprosesser som regulerer mønster og organogenesen.

Transplantasjon av komplementære områder av donor nevralrøret (homotopic pode) eller ulike regioner av donor nevralrøret (heterotop pode) kan avsløre forskjeller i pre-spesifikasjon av NCCer langs Rostro-kaudal aksen 2,47. Denne teknikken kan videre tilpasses transplantere en ensidig kupé av nevralrøret, slik at den ene siden er avledet fra donor vev, og motsatt side restene uaffisert i verten embryo, yiElding en intern kontroll innenfor samme prøve 2,47. Det kan også tilpasses for transplantasjon av hjernen segmenter i senere embryoer, etter HH10, når den fremre nevralrøret har stengt 47.

Her rapporterer vi teknikker for å generere vaktel-chick chimeras via nevralrøret transplantasjon, som gir mulighet for sporing av trekkende NCCer avledet fra en diskret del av nevralrøret. Artsspesifikk merking av donor-deriverte celler med vaktel-spesifikt QCPN antistoff 48-56 tillater forskeren å skille donor og vertsceller ved eksperimentelle endepunktet. Denne teknikken er enkel, billig, og har mange bruksområder, inkludert skjebne-mapping, celle avstamning sporing, og identifisere pre-mønster hendelser langs Rostro-kaudal aksen 45. På grunn av den enkle tilgangen til avian embryo, kan vaktel-chick pode teknikk kombineres med andre manipulasjoner, inkludert men ikke begrenset til objektiv ablasjon 40, injeksjon av hemmende molekyler 57,58, eller genetisk manipulering via electroporation av uttrykk plasmider 59-61, for å identifisere responsen av bestemte trekkende strømmer av NCCer til forstyrrelsene i embryoet utviklingstrinn program. Videre kan dette pode teknikken også brukes til å generere andre interspecific chimeric embryoer som vaktel-Duck chimeras å studere NCC bidrag til kraniofaciale morphogenesis, eller mus-chick chimeras å kombinere kraften til musen genetikk med den enkle manipulering av aviær embryoet . 62

Protocol

1. Inkuber dama og vaktler egg til ønsket scene For HH9 embryoer, typiske inkubasjonstider varierer fra 29-33 timer ved 38 ° C. 63 Vask eventuelle rester fra eggene med lunkent vann. Ordne kylling egg på brett horisontalt. Marker den siden som vender opp med blyant, og dette vil tilsvare den regionen hvor embryoet vil bli lokalisert. Inkuber vaktel egg butte enden opp. Plass i 38 ° C fuktet inkubator. Slå på gyngefunksjon. <p class="j…

Discussion

Den pode av vaktel nevralrøret inn vert kyllingembryo beskrevet her er en enkel og billig teknikk for å spore spesifikke subpopulasjoner trekkende NCCer kommer fra ulike regioner langs Rostro-kaudal aksen 21,67-69. Denne teknikken tar nytte av enkel tilgang til avian embryoer (i forhold til pattedyr embryoer) og kan kombineres med andre teknikker, for eksempel vev ablasjon, injeksjon av hemmende molekyler, eller genetisk manipulasjon via electroporation av uttrykk plasmider, til eksperimentelt undersøke re…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Forfatterne takker medlemmene av Lwigale laboratorium for kritikk av manuskriptet. SLG er støttet av en Ruth L. Kirschstein NRSA Fellowship fra National Eye Institute (F32 EY02167301). PYL støttes av National Eye Institute (EY018050).

Materials

Reagent Company Catalog number
Chick eggs Various – we use Texas A&M University’s Poultry Science Department, TX.  
Quail eggs Various – we use Ozarks Egg Company, MO.  
Egg incubator (Digital Readout 1502 Sportsman Incubator w/Humidity 110-120 Volt AC) www.poultrysupply.com 1502
Dumont AA forceps, Inox Epoxy-coated Fine Science Tools 11210-10
Scotch tape Any office supply store  
Curved Iris forceps Fine Science Tools 11065-07
India ink Any art supply store  
Pen/Strep (Penicillin, Streptomycin) Solution VWR International 101447-068
Clear Packing tape Any office supply store  
Needle pulling apparatus Narashige, Japan PE-21
Pulled glass needle, made from 1.5-1.8 x 100mm borosilicate glass capillary tube Kimble chase 34500 99
Pulled glass pipette, made from 5¾” Pasteur pipette Fisher Scientific 13-678-6A
Mouth pipette apparatus (aspirator tube assembly for calibrated microcapillary pipette) Sigma-Aldrich A5177-52A
Dumont #5 forceps Fine Science Tools 11251-30
Tungsten wire, 0.1mm diameter VWR International AA10404-H2
Needle holders (Nickel-plated pin holder) Fine Science Tools 26018-17
QCPN antiserum Developmental Studies Hybridoma Bank, University of Iowa QCPN
Alexa Fluor secondary antibody (e.g., Alexa Fluor 594 goat anti-mouse IgG1) Invitrogen A21125
Ringer’s Solution (2L):
  • 14.4g NaCl
  • 0.34g CaCl2
  • 0.74g KCl
  • 0.230g Na2HPO4
  • 0.04g KH2PO4
  • ddH2O to 2L
  • Filter and autoclave
All reagents from Fisher Scientific
  • 7647-14-5
  • 10043-52-4
  • 7447-40-7
  • 7558-79-4
  • 7778-77-0

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Cite This Article
Griswold, S. L., Lwigale, P. Y. Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation. J. Vis. Exp. (60), e3622, doi:10.3791/3622 (2012).

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