﻿staging and collecting drosophila melanogaster third instar larvae

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 ﻿Staging and Collecting Drosophila melanogaster Third Instar Larvae at JoVE.com

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.

Research

JoVE Journal

Developmental Biology

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

Fat Body Collection from Drosophila melanogaster Third Instar Larvae

Fat Body Collection from Drosophila melanogaster Third Instar Larvae  

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

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

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

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