Name: Dissection of Drosophila melanogaster Third Instar Larval Brain for Live-Cell Imaging
Uploaded: 2023-06-23T12:40:00
Description: Watch this Scientific Journal Video about Dissection of Drosophila melanogaster Third Instar Larval Brain for Live-Cell Imaging at JoVE.com

dissection of drosophila melanogaster third instar larval brain for live-cell imaging

Avbildning av levende hjerneeksplantasjoner fra Drosophila melanogaster Third Instar larver

Asymmetric cell division (ACD) is the process by which subcellular components such as RNA, proteins, and organelles are partitioned unequally between daughter cells1,2. This process is commonly seen in stem cells, which undergo ACD to give rise to daughter cells with different developmental fates. Drosophila NBs divide asymmetrically to produce one NB, which retains its stemness, and one ganglion mother cell (GMC). The GMC undergoes further divisions to produce differentiating neurons or glia3. Asymmetrically dividing NBs are abundant in the developing brains of third-instar larvae, which are readily observed via microscopy. At the third instar larval stage, there are roughly 100 NBs present in each central brain lobe3,4,5,6.
Asymmetric cell division is a highly dynamic process. Live-cell imaging protocols have been used to measure and quantify the dynamics of cell polarity7,8,9,10, spindle orientation11,12,13, the dynamics of the actomyosin cortex14,15,16,17,18, microtubule and centrosome biology19,20,21,22,23,24,25,26,27, and membrane10,28 and chromatin dynamics29. Qualitative and quantitative descriptions of ACD rely on robust methods and protocols to image dividing NBs in intact living brains. The following protocol outlines methods to prepare, dissect, and image third instar larval brains for live-cell imaging in vivo using two different mounting approaches. These methods are best suited for researchers interested in the spatiotemporal dynamics of stem cell divisions, as well as divisions in other brain cells, as they allow for short- and long-term observations of cellular events. Additionally, these techniques are readily accessible to newcomers to the field. We demonstrate the effectiveness and adaptability of this approach with larval brains expressing fluorescently tagged microtubule and cortical fusion proteins. We additionally discuss methods of analysis and considerations for application in other studies.

Forfatter Spotlight: Undersøkelse av asymmetrisk celledelingsdynamikk: En protokoll for levende avbildning av Drosophila-larvehjerneeksplantater 

Live-Cell Imaging av Drosophila melanogaster Tredje Instar Larval Brains

Our research focuses on studying the impact of asymmetric cell division or ACD, on stem cell proliferation and differentiation. We employ Drosophila neural stem cells or neuroblasts as a model to understand the mechanisms, regulations, and impacts of asymmetric cell division on development and neurogenesis. The recent developments in artificial intelligence have been making major strides in image quantification and analysis, which has significantly advanced the field of life cell imaging.Maintaining sample quality for a long-term movie is a long-standing challenge. While it's insightful to observe a sample for eight to 10 hours, logistical constraints, like the sample's imaging requirements and available imaging tools, make it difficult. Our protocol addresses this by demonstrating long-term imaging without impacting the sample's quality.Our technique could be easily adopted and applied to the field by newcomers. With some time and practice, one can feasibly generate short and long-term movies, which have the potential to yield insightful and meaningful data.

Watch this Scientific Journal Video about Dissection of Drosophila melanogaster Third Instar Larval Brain for Live-Cell Imaging at JoVE.com

Dissection of Drosophila melanogaster Third Instar Larval Brain for Live-Cell Imaging  

Disseksjon av Drosophila melanogaster Tredje instar Larval Brain for Live-Cell Imaging

Begynn med å pipettere rundt 400 mikroliter disseksjons- og bildebehandlingsmedium i hver brønn i en tre-brønns disseksjonsfat. Arbeid under et disseksjonsmikroskop, vask de eksperimentelle larver i disseksjon og bildebehandlingsmedium for å fjerne matrester. Mens du fortsetter å jobbe under disseksjonsmikroskopet, hold larvene ved munnkrokene ved hjelp av ett sett med pinsett.Med et annet sett med pinsett, kutt forsiktig av ca. 1/3 av larven fra bakre side. Deretter, mens du holder larven ved munnkrokene ved hjelp av ett sett pinsett, bruk det andre settet med pinsett til å børste neglebåndet mot munnkrokene forsiktig. Skyv samtidig innover med pinsetten som holder munnkrokene til hele larven er vendt innvendig ut.Inverter larven for å eksponere sentralnervesystemet og annet vev samtidig som de opprettholder forbindelsen til kutikula. Finn sentralnervesystemet, eller CNS, for å unngå utilsiktet fjerning. Fjern forsiktig ikke-CNS-vev ved hjelp av pinsett, og hold bare CNS og hjernen festet til neglebåndet.For å frigjøre hjernen fra neglebåndet ved å kutte aksonale forbindelser, ved hjelp av mikrodisseksjon saks, kutt forsiktig under hjernelappene. Deretter kutter du forbindelsene under den ventrale nervestrengen. Dissekere larver i grupper for å holde disseksjonstiden under 20 minutter.Overfør den dissekerte hjernen til en brønn som inneholder disseksjons- og bildemediet. For avbildning som varer over tre timer, suppler mediet med fete kropper.

Research

JoVE Journal

Developmental Biology

Dissection of Drosophila melanogaster Third Instar Larval Brain for Live-Cell Imaging

Live-Cell Imaging of Drosophila melanogaster Third Instar Larval Brains

Drosophila neural stem cells (neuroblasts, NBs hereafter) undergo asymmetric divisions, regenerating the self-renewing neuroblast, while also forming a ...

﻿Staging and Collecting Drosophila melanogaster Third Instar Larvae

Staging and Collecting Drosophila melanogaster Third Instar Larvae  

Iscenesettelse og innsamling av Drosophila melanogaster Tredje instar larver

Fat Body Collection from Drosophila melanogaster Third Instar Larvae

Fat Body Collection from Drosophila melanogaster Third Instar Larvae  

Fat Body Collection fra Drosophila melanogaster Tredje instar larver

Mounting and Live-Cell Imaging of Drosophila melanogaster Third Instar Larval Brain

Mounting and Live-Cell Imaging of Drosophila melanogaster Third Instar Larval Brain  

Montering og live-celleavbildning av Drosophila melanogaster Tredje instar Larval Brain