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

Imaging Live Brain Explants from Drosophila melanogaster Third Instar Larvae

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.

Author Spotlight: Investigating Asymmetric Cell Division Dynamics: A Protocol for Live-Imaging of Drosophila Larval Brain Explants 

Live-Cell Imaging of Drosophila melanogaster Third 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  

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

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.

Research

JoVE Journal

Developmental Biology

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