A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

The overall goal of this procedure is to test for mutations that affect Meno sensation in adult flies. This method can help identify key questions in the field of Meno sensation, such as the identification of the Meno sensitive channel. The main advantage of this technique is that by using mosaic animals, we are able to screen mutations that would otherwise be lethal to the organism.

By using a mosaic screen, we are able to identify mutations that may have been missed by more traditional behavioral screens. Visual demonstration of this method is critical. The behavioral testing steps are difficult to learn.

It takes a steady hand and practice to identify the relevant bristles. To begin the experiment, maintain flies on cornmeal molasses media and store them in an incubator. Obtain virgin females from a markum ready stock containing the necessary genetic elements.

Cross the markum ready virgin females to males that contain the mutation of interest and its corresponding FRT site. Next, set up the second control cross using male flies containing the same FRT site, but without a mutation. At a ratio of five females to one male, after allowing the flies to mate for at least one night, move adults into new vials with fresh media.

Let the flies lay their eggs at room temperature in a dark cabinet. Once the flies have laid eggs, transfer the crosses into new vials. Document the start and end times of the egg laying period.

Retain the vials that yield more than 20 eggs for heat shock and return them to the incubator. 85 to 100 hours after egg laying place vials into a water bath, ensure that the water level is above the height of the media, but that it does not fully submerge the vials. After one hour, remove the vials from the water bath and provide the flies a recovery period.

Following recovery, place the vials back into the water bath for one hour. Take the vials out of the water bath and store them back in the incubator following a anesthetize. The flies on ice.

Once the flies are immobile, select individuals that contain all of the desired traits. Next, carefully decapitate the selected flies with iridectomy scissors being careful to place the scissors between the head and thorax. Cut the connection without nicking the thorax.

Place the headless flies in a closed, moist environment and allow them to recover for 10 to 20 hours upon recovery. Retrieve the flies for examination. Only use flies that write themselves.

When perturbed observe decapitated flies under a fluorescence dissecting microscope to identify homozygous clones marked with GFP. At the bristle external sensory organs. Record the bristle name and whether it is on the left or right side of the fly, elicit a grooming reflex by deflecting the GFP marked bristle towards the fly body with a stiff hair or fine forceps and observe the leg response.

A sinus score of one to flies that lift their leg in response to bristle stimulation and a sinus score of zero to flies that do not move their legs. When possible. We test flies that have mosaic tissue on the post AOR bristles, which respond well to stimulation using the behavioral assay in post AOR bristles, while type flies were compared against the known mechano sensitive mutant no mechano receptor potential C or NMSI disrupted the grooming response control flies responded to 34%of their bristle stimulations.

In contrast, animals with bristles that were homozygous for NMSI exhibited a 2%response. Following this procedure and the identification of a new behavioral response gene, additional experiments can be used such as electrophysiology and protein localization. To further characterize the role of this gene, for example, we can answer the question whether the gene is involved in meno sensation transduction or the development of the menos sensory neurons.