Voorbereiding van de ontwikkeling van de perifere olfactorische weefsel voor moleculaire en analyse van Immunohistochemical in Drosophila

The overall goal of staging and dissecting developing pupil and adult periphery olfactory tissues in Drosophila species is to generate samples for molecular and developmental analyses of the peripheral olfactory tissues. This method can help answer key questions in the olfactory system development and evolution field such as the expression dynamics levels and patterns of specific genes in the developing peripheral olfactory tissues. Through this method we can obtain insights into the developing peripheral olfactory system in Drosophila species.

It can also be applied to other sensory systems, such as the peripheral taste and visual systems. For an RNA sequencing experiment, plan to collect between 100 to 200 antennal discs or antenna for four different stages of development. For immunohistochemistry, plan to dissect 10 to 20 flies.

Clean all of the materials involved in the dissection using 70%ethanol. Then, further clean the materials with RNase neutralizers. And make all the solutions using nuclease-free or DEPC-treated water.

To collect pupae, monitor the larvae on 30 minute intervals. And collect pupa at the zero hour APF prepupal stage. The larvae will be immobile and surrounded by a very light, almost white colored pupal case.

At this stage, transfer the larvae to a small two inch petri dish with a moist filter paper. For zero hour APF stage dissections, immediately proceed to harvest the prepupal antennal disc. Otherwise, cover the dish with paraffin film and allow the animals to develop at 25 degrees Celsius, until they are at the developmental stage of interest.

To stage pupal development of other Drosophila species, collect zero hour prepupal larvae and sort them into a fresh vial. Then record the number of days from prepupa to adulthood. And simply scale this time to the timeline for melanogaster.

To begin, chill 150 microliters of RNA isolation solution in a RNase-free 1.5 millimeter tube. Next, deposit several separate droplets of PBS on the dissection pad. Into each drop of PBS, place one pupa to be dissected.

For the zero to two hour pupae, use forceps to grip the mouth hooks and pull on these structures to tear out and expose the brains and imaginal discs. For the eight hour pupae, first gently peel the pupal case from the most anterior quarter of the pupa, to expose the developing head. Then detach the head at the neck joint.

The antennal discs are in the head by this stage of development. Now transfer the tissues containing the antennal discs to another droplet of PBS. Next, identify the eye antennal disc connected to the brain at the optic lobes.

Using forceps, disconnect the eye antennal disc and transfer it to a new droplet. In eight hour pupae, the eye discs cup the antennal discs and both are anterior to the brain. In the next droplet, divide the eye and the antennal discs using forceps.

Then using a P20 pipet tip, gently collect the dissected tissue with a minimal amount of liquid. Deposit the disc into the RNA isolation solution reagent and continue dissecting antennal discs until at least 100 are collected. By 40 hours after pupa formation, the adult antenna has taken it's final shape, but is still transparent.

At least 50 prepupae at this stage are needed for RNA sequence collection. First prepare 150 microliters of chilled RNA isolation solution. On the dissection pad, place a pupae into a droplet of PBS.

Then stabilize the body with one pair of forceps, and gently peel off the pupal case from the most anterior quarter to expose the head. Then, carefully remove the head at the neck joint. Now gently transfer the head into a clean droplet of PBS.

There to remove the membrane surrounding the head, pipet the head up and down in the PBS. With this cleaning, the antenna should become visible. Now disconnect the antenna from the membrane between the second and third segments at the segmental joint.

The third segment contains the antennal olfactory receptors. Then use a pipet to gently transfer the dissected tissue into the RNA isolation solution minimizing the amount of liquid transferred. For RNA extraction, dissect adults while they're anesthetized on a CO2 pad.

First, treat the CO2 pad and the forceps with RNase inhibitors. Then anesthetize the adults and view the pad under a dissection microscope. Using two pair of forceps, stabilize the body and gently pull or pinch out the third antennal segment from the second antennal segment to collect the tissues.

Transfer the antennae into a tube of RNA isolation solution by dipping the forceps into the liquid and releasing. The antenna must be submerged. If the antenna gets stuck to the side wall, the RNA will degrade prematurely.

After collecting enough antennae, proceed with the RNA extraction. Or store the collection tube at 80 degrees Celsius indefinitely. If the antenna are intended for immunohistochemistry, perform this dissection on a dissection pad with the fly submerged in PBS with or without Triton.

The dissected developing olfactory tissue can be used for RNA extraction followed by RT-PCR to assess gene expression. For example, the expression of surface receptor transcripts and olfactory receptors transcripts can be studied in this manner. Alternatively, the tissues can be used for immunohistochemistry to determine the expression pattern and the subcellular localization of genes of interest.

For example, Rn-EGFP transgenic flies can be stained with anti-GFP and anti-Laminin antibodies. The analysis shows that at 40 hours after pupa formation, the Or67d gene is active in specific cells in the antenna, driving expression of GFP under the GAL4-UAS system. Once mastered, dissections can be done in one hour if they are performed properly.

While attempting this procedure, it's important to remember to keep time and appropriately scheduled picking, aging and dissecting, based upon the pupal development time frame for each Drosophila species. Following this procedure, other methods like qRT-PCR, immunohistochemistry, as well as other in vivo staining and transcriptional profiling techniques can be performed in order to answer additional questions regarding pattern and profiles of transcription at the systems level within a developing tissue. After it's development, this technique will pave the way for researchers in the field of evolution and development of sensory systems to explore transcriptional dynamics and novel gene discovery in other sensory systems in Drosophila species.

Don't forget that working with sharp forceps, some RNA extraction solutions and formaldehyde can be extremely hazardous. Precautions such as previous practice with dissections, to develop fine motor skills, high attentiveness and wearing appropriate lab clothes should always be taken while performing this procedure.