Live-celle billeddannelse af Drosophila melanogaster tredje stjerne larvehjerner
Summary
Her diskuterer vi en arbejdsgang til at forberede, dissekere, montere og afbilde levende explant hjerner fra Drosophila melanogaster tredje instar larver for at observere den cellulære og subcellulære dynamik under fysiologiske forhold.
Abstract
Drosophila neurale stamceller (neuroblaster, NB’er i det følgende) gennemgår asymmetriske opdelinger, regenererer den selvfornyende neuroblast, samtidig med at der dannes en differentierende ganglionmodercelle (GMC), som vil gennemgå en yderligere opdeling for at give anledning til to neuroner eller glia. Undersøgelser i NB’er har afdækket de molekylære mekanismer, der ligger til grund for cellepolaritet, spindelorientering, neurale stamcellers selvfornyelse og differentiering. Disse asymmetriske celledelinger kan let observeres via levende cellebilleddannelse, hvilket gør larve-NB’er ideelle til at undersøge den rumlige temporale dynamik i asymmetrisk celledeling i levende væv. Når NB’er i eksplantathjerner dissekeres korrekt og afbildes i næringsstofsuppleret medium, deler de sig robust i 12-20 timer. Tidligere beskrevne metoder er teknisk vanskelige og kan være udfordrende for dem, der er nye på området. Her beskrives en protokol til forberedelse, dissektion, montering og billeddannelse af levende tredje-instar larvehjerneeksplanter ved hjælp af fedtkropstilskud. Potentielle problemer diskuteres også, og der gives eksempler på, hvordan denne teknik kan bruges.
Introduction
Asymmetrisk celledeling (ACD) er den proces, hvorved subcellulære komponenter såsom RNA, proteiner og organeller fordeles ujævnt mellem datterceller 1,2. Denne proces ses almindeligvis i stamceller, som gennemgår ACD for at give anledning til datterceller med forskellige udviklingsmæssige skæbner. Drosophila NB’er deler sig asymmetrisk for at producere en NB, som bevarer sin stamme, og en ganglionmodercelle (GMC). GMC gennemgår yderligere opdelinger for at producere differentierende neuroner eller glia3. Asymmetrisk delende NB’er er rigelige i de udviklende hjerner hos tredjestjernelarver, som let observeres via mikroskopi. På det tredje stjernelarvestadie er der omkring 100 NB’er til stede i hver central hjernelap 3,4,5,6.
Asymmetrisk celledeling er en meget dynamisk proces. Live-celle imaging protokoller er blevet brugt til at måle og kvantificere dynamikken i cellepolaritet 7,8,9,10, spindel orientering 11,12,13, dynamikken i actomyosin cortex 14,15,16,17,18, mikrotubuli og centrosombiologi 19,20,21,22,23,24,25,26,27 og membran 10,28 og kromatin dynamik 29. Kvalitative og kvantitative beskrivelser af ACD er afhængige af robuste metoder og protokoller til billedopdeling af NB’er i intakte levende hjerner. Følgende protokol skitserer metoder til at forberede, dissekere og afbilde tredje stjernelarvehjerner til levende cellebilleddannelse in vivo ved hjælp af to forskellige monteringsmetoder. Disse metoder er bedst egnet til forskere, der er interesseret i den rumlige temporale dynamik i stamcelledelinger samt opdelinger i andre hjerneceller, da de giver mulighed for kort- og langsigtede observationer af cellulære begivenheder. Derudover er disse teknikker let tilgængelige for nybegyndere på området. Vi demonstrerer effektiviteten og tilpasningsevnen af denne tilgang med larvehjerner, der udtrykker fluorescerende mærkede mikrotubuli og kortikale fusionsproteiner. Vi diskuterer desuden analysemetoder og overvejelser til anvendelse i andre undersøgelser.
Protocol
Representative Results
Discussion
Denne protokol skitserer en tilgang til billeddannelse af levende explant hjerner fra Drosophila melanogaster larver. Protokollen beskrevet her gør det muligt at observere explant-hjerner i 12-20 timer under de rigtige eksperimentelle betingelser. Der skal tages særligt hensyn til forberedelsen af prøver og udformningen af de ønskede forsøg. Som nævnt ovenfor er en af de mest kritiske faktorer, der bestemmer kvaliteten af det dissekerede væv, larvernes sundhed. For at opnå den højest mulige kvalitet ska…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Denne forskning støttes af R35GM148160 (CC) og et National Institutes of Health (NIH) Training Grant T32 GM007270 (RCS)
Materials
| 0.22 µm polyethersulfone (PES) Membrane | Genesee | 25-231 | Vacuum-driven filters |
| Agar | Genesee | 20-248 | granulated agar |
| Analytical Computer | Dell | NA | Intel Xeon Gold 5222 CPU with two 3.80 GHz processors running Windows 10 on a 64-bit operating system |
| Bovine Growth Serum | HyClone | SH30541.02 | |
| Chambered Imaging Slides | Ibidi | 80826 | |
| Confocal Microscope | Nikon | NA | |
| Custom-machined metal slide | NA | NA | See Cabernard and Doe 2013 (Ref. 34) for specifications |
| Dissection Dishes | Fisher Scientific | 5024343 | 3-well porcelain micro spot plate |
| Dissection Forceps | World Precision Instruments | Dumont #5 | |
| Dissection Microscope | Leica | NA | |
| Dissection Scissors | Fine Science Tools (FST) | 15003-08 | |
| Embryo collection cage | Genesee | 59-100 | |
| Flypad with access to CO2 to anesthetize adult flies | Genesee | 59-172 | |
| Gas-permeable membrane | YSI | 98095 | Gas-permeable membrane |
| Glass Cover Slides | Electron Microscopy Sciences | 72204-03 | # 1.5; 22 mm x 40 mm glass coverslips |
| Imaris | Oxford Instruments | NA | Alternatives: Fiji, Volocity, Aivia |
| Imaris File Converter | Oxford Instruments | NA | |
| Instant Yeast | Saf-Instant | NA | |
| Molasses | Genesee | 62-117 | |
| Petri dish | Greiner Bio-One | 628161 | 60 mm x 15 mm Petri dish |
| Petroleum Jelly | Vaseline | NA | |
| Schneider's Insect Medium with L-glutamine and sodium bicarbonate liquid | Millipore Sigma | S0146 | |
| SlideBook acquisition software | 3i | NA | |
| Vacuum-Driven Filtration Unit with a 0.22 µµm PES membrane filter | Genesee Scientific, GenClone | 25-231 |
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