The overall goal of this procedure is to detail the dissection of the intact CNS from the third Instar oph larvae. In order to study asymmetric stem cell divisions cellular differentiation and morphogenesis. This is accomplished by first ex planting the CNS from the larval body by gross dissection.
The second step is to isolate the intact brain from the peripheral tissues by fine microdissection using custom made tools. Next, the healthy brains are either mounted for live cell imaging or subjected to chemical fixation and immunofluorescence, ultimately spinning disc confocal Microscopy is used to show the events that govern neural stem cell divisions and provide mechanistic insight to the CNS development. This method can help answer key questions in the stem cell cancer and neural development fields.
By contributing to our understanding of how stem cell proliferation is regulated, we know that ABER stem cell division is associated with tumorgenesis and microcephaly, and our imaging studies reveal the mechanisms that regulate these stem cell divisions. Visual demonstration Of this method is critical because the microdissection and mounting of the brain are difficult to learn as they require steady hands, patience and practice. Begin this procedure by putting two drops of warm dissecting media on a single glass slide, one for gross dissection and the other for fine dissection.
Then transfer several larvae to one of the media drops. After that, transfer the glass slide with larvae to a dissecting microscope. Now zoom into the drop containing the larvae such that the anterior and posterior ends of the larvae are visible.
Grasp the mid region of a single larvae with a pair of forceps and grasp another region right next to it with a second pair of forceps. Then tear the larvae in half. Repeat the procedures for all the larvae on the cover, slip and dispose of the posterior halves.
Zoom into a larvae so that the anterior mouth hooks are clearly visible. After that, make a small incision or notch on the opposite side of the larval mouth hooks between the third thoracic and first abdominal CLE bands. Next, grasp the mouth hooks with the forceps.
Gently peel away the cuticle in the anterior to posterior direction and continue until the CNS is exposed. Then tear the tissues to isolate the CNS from the rest of the larvae. Be careful not to damage the CNS and discard damaged samples with a torn ventral nerve cord or distorted optic lobes.
Repeat the procedures for all the larvae on the cover slip. Then transfer the healthy explanted tissue to the other clean medium. Drop on the cover slip for fine dissection.
Use the dissecting pins to remove the peripheral tissues from the brain. Slide the scalpel between the brain and the unwanted tissue and pin the connective tissue to the glass slide. Use the hook to gently tease away the undesired tissue while simultaneously pressing the scalpel to the cover slip in Seesaw movements.
Work slowly and deliberately to remove all the discs from each brain. Use caution not to disturb the brain morphology. A healthy brain will have an intact ventral nerve cord and symmetric round optic lobes.
Now invert a 50 millimeter gas permeable culture dish with a clear film facing up deposit a small drop of medium onto the center of the dish. Next, use a hook to scoop up the brains underneath the optic lobes and transfer them to the drop of medium on the dish one by one. Collect five to 10 brains in the drop of medium.
Then push them to the bottom one by one, as many of them will be trapped at the meniscus afterward. Orient the brains to allow the neuroblast to be imaged to image the anterior ventral neuroblast. Place the brains with the ventral nerve cord facing upward align the brains such that all the ventral nerve cords point in the same direction within the XY plane.
Use a syringe or plastic transfer pipette to place four halo carbon oil drops onto the gas permeable membrane. The volume of each drop of oil should be equivalent to each other and to the drop of the medium. Once complete, the five drops will resemble the five facets on western style dice.
Next, lower a cover slip onto the assembly such that it hits all five drops at the same time. Maintaining an even pressure across the samples reduces the likelihood that they will be disrupted. Wait about three minutes.
As the oil and medium disperse under the weight of the cover slip under a dissecting scope. Lower the cover slip onto the surface of the brains further by removing excess media with a tissue paper wick until the cover slip touches, the brains do not over wick as this will cause tissue distortion or brain explosion to image anterial ventral neuroblast. The cover slip must be slightly moved in the direction of the ventral nerve cord tips.
This causes the brain to rotate, which brings the brain lobes in direct contact with the cover slip to facilitate imaging, subsequently sealed the chamber by enc. The cover slip with a small amount of halo carbon oil. Excess oil oozing from the cover slip should be minimized and blotted away with a tissue.
Prior to imaging. Add a drop of immersion oil to the cover slip just above the mounted brains to help center the objective directly above the sample. Gently position the mounted sample into the 25 degrees Celsius stage incubator and cover the chamber with its lid.
Next, locate the central brain neuroblast using transmitted light by focusing on the large round cells that reside in the central and medial areas of the optic lobes. Once in place, take quick exposures using the confocal to determine the proper location and depth of the neuroblast. Limit the depth to the first 10 to 15 micrometers.
Adjust as needed and take another test image. To eliminate axial drift, use a continuous automatic focusing device that uses an infrared LED or manually correct the focal plane. Once a neuroblast of interest is identified, optimize all the parameters before imaging.
Now image the entire neuroblast by collecting 12 to 14 images spaced by one micrometer in the Z axis. Adjust the time resolution to capture single or multiple cell cycles of the same neuroblast. Use 10 to 32nd intervals for single cell cycle imaging and one to three minute intervals for multiple cell cycles.
Next, confirm that the sample is healthy by monitoring the duration of mitosis. If mitosis takes more than 15 minutes, move on to another brain. A healthy brain will show many neuroblast within the same field of view, undergoing several rounds of mitosis.
Once the imaging is complete, disassemble the incubation chamber and discard everything but the gas permeable culturing dishes. Rinse the culture dish surface with 95%ethanol and wipe oil with a tissue. Repeat the procedure two more times.
Shown here is the live imaging of neuroblast stem cell divisions in whole mount preparations. These are the time-lapse images of a single cell cycle from a neuroblast expressing GFP Mosin for fine cellular analysis, a single neuroblast cell cycle is imaged at less than 32nd time intervals. And these are the time-lapse images of successive cell division cycles from a neuroblast expressing both G-F-P-M-O and A GFP labeled centrosome marker.
To image multiple neuroblast cell cycles. Brains are typically imaged at one to three minute time intervals over a period of two to three hours. The confocal projections of fixed stem cells from home mount preparations are shown here.
To image all the neuroblast located on both optic lobes, 20 x magnification is used. This analysis is particularly useful to examine the overall brain morphology and to quantify the neuroblast numbers. The majority of neuroblast from a single optic lobe may be visualized with 40 x magnification.
For detailed cellular analysis of a few neuroblast, 100 x magnification must be used. This mutant neuroblast shows multiple centrosomes at each spindle pole. While attempting this procedure, it's important to remember to avoid photo damage by limiting the amount of light that hits the Sample.
This technique of imaging live neuroblast divisions in whole mount preparations has paved the way for researchers in the field of stem cell biology to explore asymmetric cell division in the live native context of the developing drosophila brain.
