Stamcelle-lignende<em> Xenopus</em> Embryonale Eksplantater at studere Tidlig Neural Udviklingsmæssige Egenskaber<em> In vitro</em> Og<em> In vivo</em
Summary
In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.
Abstract
Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, cells from the roof of the blastocoel are pluripotent. These cells can be isolated, and programmed to generate various tissues through manipulation of genes expression or induction by morphogens. In this manuscript protocols are described for the use of Xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and non-autonomous properties. The blastocoel roof explants can be cultured in a serum-free defined medium and grafted into host embryos. This transplantation into an embryo allows the investigation of the long-term lineage commitment, the inductive properties, and the behavior of transplanted cells in vivo. These assays can be exploited to investigate molecular mechanisms, cellular processes and gene regulatory networks underlying neural development. In the context of regenerative medicine, these assays provide a means to generate neural-derived cell types in vitro that could be used in drug screening.
Introduction
Den hvirveldyr nervesystem fremgår neuralpladen som et homogent lag af neuroepitelceller. Forstå, hvordan udviklingsmæssige programmer induceres, kodet, og etableret under en regionalisering af den neurale plade er på nuværende tidspunkt et vigtigt mål i udviklingsmæssige biologi. Sammenlignet med andre systemer, den eksperimentelt medgørlige Xenopus foster er en model af valg for at analysere tidlige trin i neural udvikling 1,2. Det er nemt at få et stort antal af embryoner, og ekstern udvikling giver adgang til de allerførste trin i neurulation 3. Mange værktøjer til rådighed til eksperimentelt manipulere Xenopus laevis (X. laevis) fosterudviklingen. Mikro-injektion af mRNA'er eller morpholinos (MO), herunder inducerbare MOS, sammen med biokemiske og farmakologiske værktøjer, giver mulighed for kontrolleret gevinst på funktion (GOF) og tab af funktion (LOF) og specifik ændring af signalveje 4,5. Blastocoel tag ectoderm, placeret omkring dyrets pol en blastula eller en meget tidlig gastrula embryo, og benævnt "Animal Cap (AC), er en kilde til pluripotente celler, som kan programmeres ved manipulation af genekspression før eksplantater forberedelse. I dette manuskript er detaljerede protokoller til at bruge X. laevis AC eksplantater at teste in vitro og in vivo molekylære mekanismer og cellulære processer underliggende neurale udvikling.
En teknik præsenteres, så fint observation af genekspressionsmønstre i en Xenopus haletudse neuralrøret, et indledende skridt i identifikationen af skæbnen bestemmelse stikord. Ud fra følgende betragtninger observation af flade monteret væv er almindeligt anvendt i studiet af kyllingeembryoer 6, det har ikke været korrekt beskrevet i Xenopus. Manipulation af genekspression ved at injicere syntetisk mRNA eller MO ind blastomererne på 2 eller 4-celle embryoer tillader programmering af ACexplanter 4. For eksempel inhibering af knoglemorfogenetisk protein (BMP) pathway ved ekspression af anti-BMP faktor Noggin, giver en neural identitet til AC celler 3. Protokollen er beskrevet til udførelse af lokal og tidsstyret eksponering af AC eksplantater til ydre signaler via direkte kontakt med en anionbytterharpiks perle. Endelig en teknik er beskrevet til test udviklingsmæssige funktioner i neurale stamceller in vivo ved transplantation af blandede eksplantater fremstillet ud fra forskellige programmerede celler dissocieres og re-associerede.
Frøen embryo er en kraftfuld model til at studere tidlige hvirveldyr neural udvikling. Ved at kombinere manipulation af genekspression eksplanteres in vitro kulturer indeholder vigtige oplysninger i studiet af neuroepithelium regionalisering, proliferation og morfogenese 7-12. Programmeringen af AC eksplantaterne tilladte udvikling af en funktionel hjerte ex vivo 13,14. Brugenaf eksplantatet podning 15 førte til identifikation af den minimale transskriptionskontakt inducere neural crest differentieringsprogram 16. Zona limitans intrathalamica (ZLI) er en signalering center, der udskiller sonisk pindsvin (Shh) for at kontrollere væksten og regionalisering af caudale forhjernen. Når kontinuerligt udsættes for Shh, neuroepitelceller co-udtrykke tre transkriptionsfaktor gener – barH-lignende homeobox-2 (barhl2), orthodenticle-2 (otx2) og Iroquois-3 (irx3) – erhverve to karakteristika ZLI rummet: kompetence til at udtrykke shh, og evnen til at adskille fra anteriore neurale plade celler. Som et modelsystem, vil induktion af en ZLI skæbne i neuroepitelcellerne præsenteres 8.
Disse protokoller til formål at give enkle, billige og effektive værktøjer til udviklingsmæssige biologer og andre forskere til at udforske den grundlæggende mechanisms af centrale neurale celle adfærd. Disse protokoller er meget alsidig og tillade undersøgelse af en lang række ydre og indre neurale beslutsomhed signaler. Den tillader langsigtet in vivo analyser af neurale slægt engagement, induktive interaktioner og celle adfærd.
Protocol
Representative Results
Discussion
Neural udvikling orkestreret af et komplekst samspil mellem cellulære udviklingsmæssige programmer og signaler fra de omgivende væv (Anmeldt i 3,31,32). Her beskriver vi et sæt protokoller, der kan anvendes i X. laevis embryoner til at udforske ydre og indre faktorer involveret i neurale skæbne beslutsomhed og neurale morfogenese in vitro og in vivo. Disse protokoller kan anvendes som sådan på X. tropicalis embryoner dog X. tropicalis embryoner er f…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The author thanks Hugo Juraver-Geslin, Marion Wassef and Anne Hélène Monsoro-Burq for their help and advice, and the Animal Facility of the Institut Curie. The author thanks Paul Johnson for his editing work on the manuscript. This work was supported by the Centre National de la Recherche Scientifique (CNRS UMR8197, INSERM U1024) and by grants from the “Association pour la Recherche sur le Cancer” (ARC 4972 and ARC 5115; FRC DOC20120605233 and LABEX Memolife) and the Fondation Pierre Gilles de Gennes (FPGG0039).
Materials
| Paraformaldehyde | VWR | 20909.290 | Toxic |
| anion exchange resin beads | Biorad | 140- 1231 | |
| Bovine Serum Albumin | SIGMA | A-7888 | For culture of animal cappH 7.6 |
| Gentamycine | GIBCO | 15751-045 | antibiotic |
| Bovine Serum Albumin | SIGMA | A7906 | for bead preparation |
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