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

mounting and live-cell imaging of drosophila melanogaster third instar larval brain

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 Mounting and Live-Cell Imaging of Drosophila melanogaster Third Instar Larval Brain at JoVE.com

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.

Research

JoVE Journal

Developmental Biology

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