Isolering og karakterisering af enkeltceller fra zebrafisk embryoer
Summary
This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.
Abstract
The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.
Introduction
De fleste nuværende studier af cellebiologi og molekylær biologi er baseret på befolkningstal gennemsnit. Dog kan vigtige biologiske begivenheder blive maskeret af disse traditionelle populationsbaserede analyser, da mindre befolkninger kan spille vigtige roller i biologiske processer og resultater sygdom. Forståelse genekspression i heterogene populationer på enkelt celle niveau kan (og har) føre til relevante biologiske og kliniske indsigt 1,2. Af bekymring for embryonale udviklingsstudier, i en større population af celler, stamceller er ofte underrepræsenteret, hvilket gør det udfordrende at opdage subtile ændringer i genekspression, som i sidste ende initierer celle skæbne beslutninger 3. Tilsvarende kan en enkelt celletype have forskellige udtryk profiler i afhængighed af mikromiljøet 4. For eksempel residente endotelceller i samme organ eller i forskellige organer (f.eks., Aorta eller nyrer) udviser signifikant heterogenitet trods deling fælles MORPhological og funktionelle egenskaber 5. Desuden kan cancerceller befolker samme tumor også have varierende molekylære profiler eller mutationer på enkelt celle niveau 6.
I modelsystemer har transcriptomics i enkeltceller med succes identificeret nye cellepopulationer, kendetegnet mellemliggende tilstande, der opstår under celledifferentiering og afslørede differentielle cellulære responser på stimuli 7,8,9. Sådanne indsigter ville have været maskeret i konventionelle populationsbaserede studier. Zebrafisk embryoner er en uhyre underudnyttet kilde til stamceller, stamfader, og differentiere celler til at udforske spørgsmål om enkelt celle heterogenitet og molekylær regulering af cellulære identiteter under udvikling. Deres yderst stereotype, ex vivo udvikling og nem genetisk manipulation gør dem til et fremragende modelsystem for denne fremgangsmåde 10,11. Specielt har en væsentlig begrænsning for fortolkning af enkelt celle gene udtryk data er, at pålidelig identifikation af nye mellemliggende celle stater under udvikling kræver meget omhyggelig timing af væv samling 9. Dette er nødvendigt for at sikre, at heterogenitet mellem opfangede celler repræsenterer heterogenitet inden et væv på et enkelt tidspunkt i stedet for heterogenitet i genekspression præsenteret af aldersbetinget celledifferentiering. Sammenlignet med mus, kan zebrafisk embryo udvikling præcist synkroniseret på et stort antal embryoner 12. Derudover, med stor kobling størrelser, zebrafisk embryoer kan anvendes som en rigelig kilde til stamceller og progenitorceller.
Denne protokol beskriver en fremgangsmåde til isolering af celler fra zebrafisk embryoner og fange enkeltceller ved anvendelse af et kommercielt tilgængeligt integreret kredsløb mikrofluidik (IFC) chip og autopræp for QRT-PCR-genekspression analyse. Denne protokol kan være hurtigt overføres til enhver high throughput multiplexing analyser, herunder heletranskriptom sekvensering, der giver mere omfattende analyse af cellulær heterogenitet 13. Det giver også flere fordele til traditionelle genekspression assays. Den enkelt celle isolation protokol giver høj levedygtighed efter FACS, som nedsætter andelen af inficerede celler, der er inkluderet i efterfølgende anvendelser. Ved at bruge en IFC kan indfangede celler direkte observeret at evaluere opsamlingsprocent og vurdere celle sundhed morfologisk. Desuden denne protokol er bredt anvendelig til zebrafisk forskningsverden, som kun kræver en mærket transgene fisk linje og adgang til mikrofluide celle capture teknologi.
Som bevis på princippet blev enkelte celler afledt af hjerte-progenitorer isoleret og fanget på en IFC-chip, og derefter den relative forekomst af hjerte- differentieringsmarkører blev målt ved QRT-PCR. Genekspression analyse på enkelt celle niveau viser, at hjerte-stamfædre sameksistere med deres Forskelntiating afkom. Den indsigt erfaringer fra encellede profilering af hjerte stamfædre kan kaste lys over heterogenitet i genekspression mønstre blandt hjerte-stamceller under hvirveldyr udvikling, som kan være blevet maskeret i traditionelle populationsbaserede analyser.
Protocol
Representative Results
Discussion
Den heri beskrevne fremgangsmåde anvender ekspression af et fluorescerende protein under kontrol af en celletypespecifik promotor til berigelse af en population af kardiale progenitorceller fra zebrafisk embryoner til anvendelse i mikrofluid assisteret enkelt celle capture system til at vurdere ekspression af en undergruppe af kardiale gener i enkelt celler. Forudsat at FACS laser excitation og emission kapaciteter er forenelige med fluoroforen (e) valg, kan denne fremgangsmåde anvendes til enhver fluorescerende repor…
Declarações
The authors have nothing to disclose.
Acknowledgements
We thank Dr. C. Geoffrey Burns for fish stock. The authors are grateful to UNC Flow Cytometry Core Facility, UNC-CGIBD AAC core for resources enabling this project, and the ZAC facility for animal care. L.S. is supported by NIH T32 grant HL069768-13 (PI, Nobuyo Maeda). N.F. is supported by NSF Graduate Research Fellowship NSF-DGE-1144081. This study was supported by NIH P30DK034987 grant (to UNC Advanced Analytics Core), American Heart Association Scientist Development Grant 13SDG17060010 and Ellison Medical Foundation New Scholar Grant AG-NS-1064-13 (to Dr. Qian), and NIH R00 HL109079 grant (to Dr. Liu).
Materials
| Supplies | |||
| Dumont #5 forceps | Fine Science Tools | 11254-20 | For removing the chorion from embryos |
| Microcentrifuge tube 2 mL | GeneMate | C-3261-1 | |
| 40 um cell strainer | Biobasic | SP104151 | |
| 35 mm culture dish | Falcon | 351008 | |
| FACS tubes topped with 35 um cell strainer | Falcon | 352235 | |
| P1000 and tips | Rainin | 17005089 | |
| P20 and tips | Rainin | 17005091 | |
| IFC chip manufacturer's protocol | Fluidigm | 100-6117 | Version 100-6117 E1 was used in representative experiment |
| Wide bore pastuer glass pippette | VWR | 14673-010 | For transferring embryos |
| Adult wild type zebrafish | N/A | We used AB line | |
| Adult transgenic zebrafish | N/A | We used Tg(nkx2.5:ZsYellow) | |
| Name | Company | Catalog Number | Comments |
| Reagents for cell dissociation | |||
| Double distilled water | N/A | ||
| Instant Ocean Sea Salt | Instant Ocean | SS15-10 | |
| NaCl | FisherScientific | S271-3 | make stock in water and use for de-yolking buffer |
| KCl | Sigma Aldrich | P5405 | make stock in water and use for de-yolking buffer |
| NaHCO3 | Sigma Aldrich | S6014 | make stock in water and use for de-yolking buffer |
| Leibovitz's L-15 | Gibco | 21083-027 | |
| FBS | FisherScientific | 03-600-511 | Heat inactivate; any brand of FBS should be fine |
| Cell Dissociation Reagent 1 -TrypLE | Life Technologies | 12605-010 | Store at room temperature. |
| Cell Dissociation Reagent 2 – FACSmax | Genlantis | T200100 | Store -20; thaw on ice; bring to room temp before use |
| pronase (optional) | Sigma | P5147 | |
| L/D Dye – Sytox Blue | Life Technologies | S34857 | Any live/dead stain suitable for flow cytometry will work |
| Trypan Blue | Gibco | 15250-061 | |
| Name | Company | Catalog Number | Comments |
| Reagents for IFC plate use and qRT-PCR | |||
| Gene-specific probes | Probes will vary by experiment | ||
| TaqMan Probe ef1a | Life Technologies | Dr03432748_m1 | |
| TaqMan Probe gata4 | Life Technologies | Dr03443262_g1 | |
| TaqMan Probe nk2-5 | Life Technologies | Dr03074126_m1 | |
| TaqMan Probe myl7 | Life Technologies | Dr03105700_m1 | |
| TaqMan Probe vmhc | Life Technologies | Dr03431136_m1 | |
| TaqMan Probe isl1 | Life Technologies | Dr03425734_m1 | |
| TaqMan Gene Expression Master Mix | Life Technologies | 4369016 | |
| Reagents listed in IFC manufacturer's protocol | |||
| C1 Reagent Kit | Fluidigm | 100-5319 | Reagents for loading cells onto IFC plate |
| Ambion Single Cell-to-CTTM | Life Technologies | 4458237 | Reagents for reverse transcription and pre-amplification steps |
| Molecular Biology Quality Water | Corning | 46-000CM | |
| C1 IFC for PreAmp (5-10 um) | Fluidigm | 100-5757 | IFC plate for small cells |
| Name | Company | Catalog Number | Comments |
| Equipment | |||
| C1 AutoPrep machine | Fluidigm | 100-5477 | For IFC plate use |
| Hemocytometer | Sigma Aldrich | Z359629 | For counting cells and assessing cell size |
| Dissecting microscope | For removing embryos from chorion | ||
| Tissue culture microscope | For assessing single cell digestion | ||
| FACS machine | For isolating cells of interest |
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