Analyserer<em> In vivo</em> Cell Migration hjælp Cell transplantationer og Time-lapse Imaging i zebrafisk embryoner
Summary
Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.
Abstract
Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.
Introduction
I flercellede organismer, celle migration er afgørende både for udviklingen af embryo, hvor det sikrer organisering af celler i væv og organer, og til voksenlivet, hvor det tager en del til vævshomeostase (sårheling) og immunitet. Foruden disse fysiologiske funktioner, cellemigrering er også involveret i forskellige patologiske situationer, herunder især, cancermetastase.
Cell migration er blevet analyseret in vitro i årtier, giver en overordnet forståelse for de molekylære mekanismer, der sikrer celle bevægelser på flade overflader. In vivo dog celler konfronteret med et mere komplekst miljø. Det tydeligt viste sig i de seneste år, at migration inden en organisme kan påvirkes af eksterne signaler såsom den ekstracellulære matrix, tilstødende celler eller udskilles kemokiner vejledende migration, og at de mekanismer, der driver celle migration kan variere fra hvad der er blevet beskrevet <em> in vitro 1,2. De mekanismer, der sikrer in vivo celle migration har fået mindre opmærksomhed hidtil, primært på grund af den øgede tekniske vanskeligheder i forhold til in vitro-undersøgelser. In vivo analyse af cellemigration især kræver direkte optisk adgang til migrerende celler, teknikker at mærke unikke celler i for at kunne se deres dynamik og morfologi samt gevinst eller tab af funktion tilgange til at teste rollen som kandidatgener. Hidtil har kun få modelsystemer huser disse karakteristika blevet anvendt til at dissekere in vivo cellemigrering 3.
Vi har for nylig brugt migrationen af de fremadrettede prechordal plade i tidlige zebrafisk embryoner som en ny praktisk model system til at vurdere funktionen af kandidatgener i at kontrollere in vivo celle migration 4,5. Prospektiv prechordal plade (også kendt som anterior mesendoderm) er en gruppe af celler danner i starten af gastrulation på dorsale side af fosteret. Under gastrulation denne gruppe migrerer kollektivt mod dyret pol af embryonet 6-8, til dannelse af den prechordal plade, en mesendodermal fortykkelse, anterior til rygstrengen og underliggende neurale plade. Den forreste del af prechordal plade vil give anledning til udrugning kirtel, mens dens bageste del sandsynligvis bidrager til hovedet mesoderm 9. Takket være den eksterne udvikling og optisk klarhed af fisken embryo, kan cellemigrering let og direkte observeret i denne struktur.
Cell transplantation er en meget potent teknik, der giver mulighed for hurtig og nem oprettelse af mosaik embryoner 10. Udtrykker fluorescerende cytoskelet-markører i transplanterede celler resulterer i mærkning af isolerede celler, morfologi og dynamik som let kan observeres. Kombinere dette til tab eller gevinst på funktion nærmer tillader analyse af celle-autonome funktioner i en dåsedidat gen.
Den præsenterede protokol beskriver, hvordan vi vurderede funktionen af TORC2 komponent Sin1 i at kontrollere celle migration og actin dynamik in vivo fem. Men som nævnt i resultaterne, diskuteres yderligere, det kunne anvendes til at analysere den potentielle konsekvenser af ethvert kandidat-gen i at kontrollere cellemigrering in vivo.
Protocol
Representative Results
Discussion
Denne protokol viser en nem måde at studere rollen af en kandidat-gen i cellemigrering in vivo, ved at kombinere skabelse af kimære embryoner under anvendelse celletransplantation med levende billeddannelse.
Oprettelse af mosaik embryoner
Studere dynamikken i en celle kræver visualisering af sin kontur til at analysere cytoplasmatiske udvidelser. Dette kan opnås ved mærkning isolerede celler i en ellers umærket – eller forskelligt mærket – miljø,…
Declarações
The authors have nothing to disclose.
Acknowledgements
We thank F. Bouallague and the IBENS animal facility for excellent zebrafish care. Research reported in this publication was supported by the Fondation ARC pour la recherche sur le cancer, grants N° SFI20111203770 and N° PJA 20131200143.
Materials
| Glass capillaries (outside diameter 1.0 mm, inside diameter 0.58 mm) | Harvard Apparatus | 300085 | standard thickness |
| Glass capillaries (outside diameter 1.0 mm, inside diameter 0.78 mm) | Harvard Apparatus | 300085 | thin-walled |
| Penicillin-Streptomycin | Sigma-Aldrich | P4333 | 10 000 units penicillin and 10 mg streptomycin per ml |
| fine tweezers | Dumont Fine Science Tools | 11254-20 | 5F |
| glass bottom dishes | MatTek | P35G-0-10-C | |
| Air transjector | Eppendorf | 5246 | |
| Micro-forge | Narishige | MF-900 | |
| Microgrinder | Narishige | EG-44 | |
| Micromanipulator (for injection) | Narishige | MN-151 | |
| Micromanipulator (for cell transplantation) | Leica | Leica Micromanipulator | |
| Hammilton Syringe | Narishige | IM-9B | |
| Micropipette puller | David Kopf Instruments | Model 720 | |
| Transplantation mold | Adapative Science Tools | PT-1 | |
| Needle holder | Narishige | HI-7 | |
| Tube connector | Narishige | CI-1 | |
| PTFE tubing | Narishige | CT-1 |
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