The overall goal of this twin-spot MARCM protocol is to conduct high resolution cell lineage analyses and better gene function studies. Brain tissue is comprised of a vast number of diverse neurons, it's challenging to study the mechanism underlying how neurons assemble together during development. The main advantage of this technique is to label neurons derived from common neuron progenitors in two distinct colors.
Which permit the retrieval of the birth of the information of individual neurons. Therefore this technique can help researchers to explore the cellular and molecular mechanism underlying neural development. Demonstrating the procedure today will be Pei-Chi Chung.
A technician from Professor Yu's laboratory. Begin by combining twin-spot MARCM ready males and females in a fly food vial with fresh yeast in the vial. Maintain the crossed twin-spot MARCM ready flies at 25 degrees Celsius for two days to enhance the chance of mating before collecting fertilized eggs.
To avoid overcrowding when the larvae hatch, tap down the crossed twin-spot MARCM ready flies, and transfer into freshly yeasted vials. Culture the twin-spot MARCM offspring, until they reach the desired stage of development. Here, flies at the larval stage will be heat shocked to induce flippase expression.
Transfer the fly vials to a 37 degrees Celsius water bath for 10, 20, 30, 40, and 50 minutes to determine the optimal heat shock for flippase expression. To generate the twin-spot MARCM clones of interest. The timing of the heat shock determines the stage of clearing generation, while the heat shock period defines how many clearings will be generated.
Culture the heat shocked twin-spot MARCM animals at 25 degrees Celsius, until the desired developmental stage. Then proceed to staining and mounting. Add PBS to the forceps protection dishes.
And then, transfer the twin-spot MARCM animals to the dishes. While looking through a dissection microscope, use forceps to dissect the brains from the twin-spot MARCM animals. First, remove the head from the body of the fly.
Then gently remove the cuticle without damaging the brain. Next, carefully remove tracheae which appear as white colored fibers. Transfer the brains to a glass spot plate, Containing 4%formaldehyde and PBS.
And fix at room temperature for 20 minutes. Begin the staining procedure by rinsing the fly brains three times with 1%PBT. And then washing with 1%PBT three times.
With shaking for 30 minutes each time. Incubate the fly brains with a mixture of primary antibodies overnight at four degrees Celsius or for four hours at room temperature. Rinse and wash the fly brains three times each with 1%PBT, with shaking as before.
Then incubate the fly brains with a mixture of secondary antibodies for four hours at room temperature or overnight at four degrees Celsius. After rinsing and washing the brains as before, mount the brains on a microslide with an anti-quenching reagent. And then place a cover glass to cover and protect the fly brains.
Seal the edges of the micro cover glass and microslide using clear nail polish. Finally, capture fluorescent images of twin-spot MARCM clones from the samples using the confocal microscopy system of choice. This figure shows results from the twin-spot MARCM clone, a VL2p antero dorsal olfactory projection neurons derived by the induction of flippase.
To mediate mytotic recombination in the adPN neuro blast at the embryonic stage. This first diagram shows the neurogenesis pattern. In this confocal microscope image of the brain, The VL2p adPNs are shown in green and are associated with multi cellular neuro blast adPNs in magenta.
This image shows the cells in the lateral horn. The neuronal morphology of the antennal lobe is seen here. This image shows the data for DL1 adPNs.
As seen here, the DL1 adPNs appear later in development than VL2p adPNs. Induction of twin-spot MARCM clones of DC3 adPNs demonstrate that this neuronal type appears later in development. This image shows that twin-spot MARCM event occurs in a GAL4 negative neuron within the adPN lineage.
Once mastered, this technique can help researchers to analyze neural lineage and provide different markers to distinguish neurons from neuroblast clone. Furthermore twin-spot MARCM can produce homozygous mutant clones to explore molecular mechanism. In a heterozygous genetic background.
