Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

The overall goal of this methodology is to show how to perform quantitative analyses of anatomical traits. The quantification of phenotypes affecting morphology, size and position of nuclei with the striated muscles of drosophila larvae is demonstrated. Understanding the molecular mechanism controlling the morphology, size and distribution of intracellular organelles is one of the most important questions of modern biology.

In the past, morphological variations among and within experimental groups were only subjected to qualitative analysis. Here we propose, in quantitative analysis that combines the drosophila genetics with morphometric quantitative analysis to identify genes controlling size, shape and distribution of nuclei within the muscles. Helping with the demonstration of the procedure will be Leire Ledahawski, a graduate student from my laboratory.

After dissecting and staining the larval neuromuscular junctions as per the text protocol, use forceps to remove the samples from the 1.5 milliliter microcentrifuge tube, and lay them down on a processing slide. Using micro dissection scissors, cut the head and the tail off the filets and keep their internal surfaces up. Now, prepare a mounting slide by wrapping three strips of cellulose tape around a clean slide, one centimeter apart.

Rest the cover slip over this provided gap so it will not flatten the samples. Then, deposit about 20 microliters of the mounting medium between the cellulose tape strips and spread the medium around with clean forceps. Now remove the extra tissue from the preps.

Drag the dissected larvae from the processing slide onto the mounting slide and into the mounting medium, keeping the internal surface up. Next, gently apply a cover slip being careful to avoid trapping air. Then seal the slide with transparent nail varnish and let the slide dry for at least 10 minutes before imaging.

For this analysis, use confocal images displaying larval body wall muscles stained with DeVAP antibodies, lamin antibodies, and a nuclear marker. Then drag and drop the confocal Z stack images into the arena. Double click on the images to automatically open them in the Surpass view, accessed by a toolbar icon.

The Surpass view has three main work space panels. View area, Object List and Object Properties area. Next, click on the 3D View icon to create a volume rendered three channel image.

Then select Add New Measurement Points from the Objects toolbar and follow the Creation Wizard in the Object Properties area. Select the Edit tab first and then, from Specific Channel, select either the nuclear marker or the lamin channel to highlight the nuclei. Next, set the pointer to the Select mode by pressing the Escape tab, and then adjust the size of the 3D cursor box with the mouse wheel to contain a given nucleus in the image.

Add a measurement point by holding the Shift key and left clicking on the same nucleus. Then, add the second point on a nearby nucleus of the same muscle using the same technique. A line will automatically be drawn between the two points and the distance between the two nuclei gets displayed and recorded as a statistical variable.

Repeat the procedure for all nuclei surrounding a given nucleus. Then, from all the collected measurement points, found under Statistics Detailed Distance Data, select the shortest distance. Under the Object Properties area, select the option Export Statistics with Tab Display to File available.

This saves the data to a spreadsheet. For this analysis, use confocal images of body wall muscles stained with lamin and a nuclear marker to visualize the nuclei. To evaluate the shape of the myonuclei, measure their sphericity, which compares the nuclear surface area to that of a sphere with the same volume.

Alternatively, measure ellipticity which distinguishes prolate, oblate or ellipsoids and spheroids. Open the image as previously described. Then, click on the Objects toolbar icon Add New Surfaces.

In the Creation Wizard that appears in the Object Properties area, select the nuclear marker staining as the source channel to display the nuclei. Set the option Absolute Intensity as the threshold. Make sure that most of the nuclei show a smooth, non overloaded rendering by changing the value on the threshold curve.

At the same time, avoid the presence of holes or incomplete masks of any nuclei. Next, use the Filter tool to remove noise from the surface rendering. Under the Edit tab of the newly created surface layer, split the nuclei surfaces that are incorrectly rendered.

Another option is to merge the nuclei surfaces that are incorrectly rendered. The ellipticity and sphericity values of the surface rendered nuclei are available under the Statistics tab. Export this data for analysis.

For this protocol, use confocal images reporting body wall muscles stained with a nuclear marker and with antibodies specific to lamin and DeVAP. Open the image of interest from the Main Menu item Edit then Crop 3D. Follow the Surface Creation Wizard using the lamin channel as described in the previous section.

Once the surface is created click on Edit in the Objects Properties area, and the select Mask All to isolate a signal inside the nucleus. Next, select the DeVAP signal. From the Select Channel drop down menu click on the option Set Voxels Ouside the Surface and make that value zero.

This creates a new masked channel available in the Display Adjustment window. To visualize the presence of signal inside the nucleus, create a contour plane by clicking on the icon Add New Clipping Plane from the Objects toolbar. Then, interactively adjust the angle of the clipping plane and its position to visualize the distribution of the signal inside the nucleus.

Missense mutations in the human VAMP associated protein B are implicated in ALS pathogenesis. Using the described method, striated muscles expressing the fly ortholog gene DeVAP harboring the ALS causing mutation V2601 were compared to controls, expressing comparable levels of wild type DeVAP, or higher levels of wild-type DeVAP. Nearest neighbor analysis was used to evaluate the distribution of nuclei along the muscle fibers.

Compared to control muscles expressing the mutated DeVAP transgene, or those over expressing the wild-type protein, the mutation of interest caused a dramatic reduction in the average shortest distance between the nuclei. Next, the sphericity of the nuclei was examined. It was found that transgenes that reduced nuclei spacing also enlarged nuclei volume.

3D reconstructions and volume renderings of the nuclei revealed that muscles expressing the mutation of interest form clusters that were localized in the nuclei. This method can also be extended to the quantitative analysis of morphological features associated with other organelles, such as mitochondria. Changes in nuclear architecture, position and size have been associated with aging and a number of neurodegenerative disorders, such as Parkinson's disease and ALS.

Understanding the molecular mechanisms underlying dyspraxisis may lead to the identification of novel pharmacological targets for effective therapeutic interventions.