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

Imaging di espianti cerebrali vivi da larve di Drosophila melanogaster terzo stadio

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.

Autore in primo piano: Indagine sulle dinamiche di divisione cellulare asimmetrica: un protocollo per l'imaging dal vivo di espianti di cervello larvale di Drosophila 

Imaging delle cellule vive di Drosophila melanogaster Terzo stadio Larvale Cervelli

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  

Dissezione del cervello larvale di Drosophila melanogaster Third Instar per l'imaging di cellule vive

Inizia pipettando circa 400 microlitri di dissezione e mezzo di imaging in ciascun pozzetto di un piatto di dissezione a tre pozzetti. Lavorando al microscopio a dissezione, lavare le larve sperimentali in dissezione e mezzo di imaging per rimuovere i residui di cibo. Mentre continui a lavorare al microscopio a dissezione, tieni le larve per i ganci della bocca usando una serie di pinzette.Con un'altra serie di pinzette, tagliare con cura circa 1/3 della larva dal lato posteriore. Successivamente, mentre si tiene la larva per i ganci della bocca usando una serie di pinzette, utilizzare l'altra serie di pinzette per spazzolare delicatamente la cuticola verso i ganci della bocca. Contemporaneamente, spingere verso l'interno con le pinzette che tengono i ganci della bocca fino a quando l'intera larva non viene capovolta.Capovolgere la larva per esporre il sistema nervoso centrale e altri tessuti mantenendo la loro connessione con la cuticola. Localizzare il sistema nervoso centrale, o SNC, per evitare la rimozione accidentale. Rimuovere delicatamente i tessuti non del SNC usando una pinzetta, mantenendo solo il SNC e il cervello attaccati alla cuticola.Per liberare il cervello dalla cuticola recidendo le connessioni assonali, usando le forbici da microdissezione, tagliare delicatamente sotto i lobi cerebrali. Quindi, tagliare le connessioni sotto il cordone nervoso ventrale. Sezionare le larve in lotti per mantenere il tempo di dissezione inferiore a 20 minuti.Trasferire il cervello sezionato in un pozzo contenente il mezzo di dissezione e imaging. Per immagini che durano più di tre ore, integrare il mezzo con corpi grassi.

dissection-drosophila-melanogaster-third-instar-larval-brain-for-live

Research

JoVE Journal

Developmental Biology

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

522 Views.  University of Washington. Inizia pipettando circa 400 microlitri di dissezione e mezzo di imaging in ciascun pozzetto di un piatto di dissezione a tre pozzetti. Lavorando al microscopio a dissezione, lavare le larve sperimentali in dissezione e mezzo di imaging per rimuovere i residui di cibo. Mentre continui a lavorare al microscopio a dissezione, tieni le larve per i ganci della bocca usando una serie di pinzette.Con un'altra serie di pinzette, tagliare con cura circa 1/3 della larva dal lato posteriore. Succ...

Video: 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 differentiating ganglion mother cell (GMC), which will undergo one additional division to give rise to two neurons or glia. Studies in NBs have uncovered the molecular mechanisms underlying cell polarity, spindle orientation, neural stem cell self-renewal, and differentiation. These asymmetric cell divisions are readily observable via live-cell imaging, making larval NBs ideally suited for investigating the spatiotemporal dynamics of asymmetric cell division in living tissue. When properly dissected and imaged in nutrient-supplemented medium, NBs in explant brains robustly divide for 12-20 h. Previously described methods are technically difficult and may be challenging to those new to the field. Here, a protocol is described for the preparation, dissection, mounting, and imaging of live third-instar larval brain explants using fat body supplements. Potential problems are also discussed, and examples are provided for how this technique can be used.

Here, we discuss a workflow to prepare, dissect, mount, and image live explant brains from Drosophila melanogaster third instar larvae to observe the cellular and subcellular dynamics under physiological conditions.

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

Ricerca

Biologia dello sviluppo

﻿Staging and Collecting Drosophila melanogaster Third Instar Larvae

Staging and Collecting Drosophila melanogaster Third Instar Larvae  

Begin by crossing one to five-day-old female virgin flies with one to seven-day-old adult male flies to produce progeny with the desired genotype. To do so, place the flies in a fly cage with a meal cap. Use 10 to 15 female virgins with five to 10 males to get optimal yield.Incubate the fly cage containing flies at 25 degrees Celsius. Change the meal cap daily to prevent overcrowding of larvae which reduces the quality of the dissected brains. If the meal cap has more than 30 larvae, split it into half and replace one portion with a fresh meal cap cut in half.Incubate the meal caps with larvae at 25 degrees Celsius until the larvae reached the desired age.

Stadiazione e raccolta di Drosophila melanogaster Larve del terzo stadio

Fat Body Collection from Drosophila melanogaster Third Instar Larvae

Fat Body Collection from Drosophila melanogaster Third Instar Larvae  

Begin by pipetting around 400 microliters of dissection and imaging medium into each well of a three-well dissection dish. Under a microscope, wash 10, well-fed, wild-type larvae by gently holding them with dissection forceps and dipping them in and out of the dissection medium in the bottommost well to remove all food particulates. While continuing to work under the dissection microscope, transfer the clean larvae to the middle well.Hold the larva by the mouth hooks using one set of tweezers. With another set of tweezers, rupture one side of the larva's cuticle to spill out the fat bodies. Using forceps, collect as much fat body from each larva as possible and transfer it to the topmost well containing 400 microliters of dissection medium at room temperature.

Raccolta di corpi grassi da larve di terza stella di Drosophila melanogaster

Begin by pipetting around 400 microliters of dissection and imaging medium into each well of a three-well dissection dish. Working under a dissection microscope, wash the experimental larvae in dissection and imaging medium to remove food residues. While continuing to work under the dissection microscope, hold the larvae by the mouth hooks using one set of tweezers.With another set of tweezers, carefully cut off approximately 1/3 of the larva from the posterior side. Next, while holding the larva by the mouth hooks using one set of tweezers, use the other set of tweezers to brush the cuticle towards the mouth hooks gently. Simultaneously, push inward with the tweezers holding the mouth hooks until the entire larva is turned inside out.Invert the larva to expose the central nervous system and other tissues while maintaining their connection to the cuticle. Locate the central nervous system, or CNS, to avoid accidental removal. Gently remove non-CNS tissues using tweezers, keeping only the CNS and brain attached to the cuticle.To release the brain from the cuticle by severing the axonal connections, using microdissection scissors, cut gently beneath the brain lobes. Then, cut the connections under the ventral nerve cord. Dissect larvae in batches to keep the dissection time under 20 minutes.Transfer the dissected brain into a well containing the dissection and imaging medium. For imaging lasting over three hours, supplement the medium with fat bodies.

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

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

To perform live cell imaging of the third instar larval brain, begin by collecting the dissected brains and the isolated fat bodies in the last well of a three well dissection dish containing the dissection and imaging medium. Place a gas permeable membrane on the back of a slide and press the split ring into the center. Using a 200 microliter micro pipette, transfer up to 10 dissected brains with maximum possible fat bodies and 130 to 140 microliters of the medium onto the center of the membrane.Orient the brains according to the neural stem cell population to be imaged and the microscope type, ensuring the samples are close to the microscope's objective. For example, to image neural stem cells in the central brain lobes, ensure the brain lobes are closest to the objective. Then gently place a glass cover slip over the solution on the membrane to spread the solution with the brain's and fat bodies throughout the membrane.Hold the laboratory tissue at the cover slip edge to blot excess solution until the brains touch the cover slip without being squashed. Next, immobilize the cover slip by applying molten petroleum jelly along its edges using a paintbrush and allow the jelly to solidify. Add 400 microliters of imaging medium to a well in a multi well micro slide.Transfer the previously dissected fat bodies to this well. Then, place up to 10 brains in a cluster near the center of the well. Orient the brains according to the neural stem cell population to be imaged and the microscope type.Allow the brains to settle in the well for two to five minutes. Cover the micro slide with the slide cover before transferring it to the microscope. Start the acquisition process using the lowest laser power and exposure time to minimize photo bleaching.Imaging of larval brains expressing UAS driven Cherry-Jupiter and androgynously tagged pins GFP using multi-well imaging slides and without fat body supplementation revealed that the pins formed a pronounced apical crescent in dividing neuroblasts to which the mitotic spindles consistently aligned during mitosis. In those samples, the cell cycle length increased with increasing imaging time. Samples that were imaged in a fat body supplemented medium on a membrane-bound slide did not show an increase in cell cycle length.Furthermore, neuroblasts with four divisions were observed on the 10 hour membrane-bound slide.