Studying Muscle Transcriptional Dynamics at Single-molecule Scales in Drosophila

In our team, we studied the cellular and molecular mechanisms governing muscle development and repair. In fact, the special distribution of mRNA molecules is known to regulate various cellular processes. And here we use the Drosophila as a model to investigate how the mRNA are spatially distributed within the flight muscle tissue.

Gene expression dynamic have been proven to regulate various muscle biological process. The advent of high throughput single-cell and single-nuclei RNA sequencing techniques has enabled a comprehensive exploration of the transcriptional dynamics. One notable limitation of classical omics techniques is an inability to provide the spatial distribution of mRNA molecules within the Drosophila muscle fibers.

This feature can be investigated by single-molecule fluorescence in-situ hybridization. Current methods are insufficient to determine the mRNA transcriptional dynamics and spatial distribution within Drosophila muscle syncytia. To address this limitation, we optimized a method to detect and to quantify the individual mRNA molecules in Drosophila muscle fibers with high-percent resolution and single-molecule scales.

A current challenge in the field is to visualize the muscle regeneration dynamic in real time and in living animal. That's why we are trying to develop a live imaging approach to visualize the muscle stem cell behavior in their native environment and also to address the question of their mode and range of migration, for example, during the muscle regeneration, and also their differentiation, and this with unprecedented resolution. To begin, rinse the L3-staged Drosophila larvae thrice for five minutes each in cold PBS.

Using two pairs of sharp forceps, hold the anterior part of the larva and remove approximately 2/3 of the body tissues. Hold the first 1/3 of the larva with one pair of forceps and mouth hooks with the other pair. Then retract the mouth hooks inside the larva until the larva is fully inverted.

Remove the leg discs and brain to obtain a clean inverted larval carcass with a pair of wing discs attached to the trachea. Transfer the wing discs into a one-milliliter centrifuge tube. Fix the wing discs in 4%paraformaldehyde diluted in PBS for 45 minutes at room temperature under agitation.

After fixation, wash the samples thrice for five minutes each with 70%ethanol. Remove the heads, abdomens, legs, and wings from the anesthetized flies and place the dissected thoraxes in cold PBS. Prefix the thoraxes in 4%paraformaldehyde diluted in 1%Triton for 20 minutes under agitation.

Then wash the samples thrice for five minutes each using PBT under agitation. Position the thoraxes on a double-sided tape on a glass slide, and bisect them using a sharp microtome blade to produce two hemithoraces. Fix the hemithoraces in 4%paraformaldehyde for 45 minutes under agitation.

Wash the samples twice for 20 minutes each using PBT under agitation. Place the hemithoraces in cold PBS and use forceps to carefully isolate the indirect flight muscles from the hemithoraces. Permeabilize the muscles in 70%ethanol for two to seven days at four degrees Celsius.

After permeabilization, wash the IFMs twice for 20 minutes each using buffer A.Then add 400 microliters of the hybridization buffer, and pre-hybridize the muscles for 30 minutes at 37 degrees Celsius in a thermal mixer. Add 100 microliters of freshly prepared hybridization buffer containing the probe and the primary antibody. Incubate the samples in the dark at 37 degrees Celsius for 16 hours at 300 RPM in a thermal mixer.

After incubation, wash the samples thrice using warm buffer A for 10 minutes each. Then incubate the samples in secondary antibody and DAPI diluted in buffer A at 37 degrees Celsius for one hour. Wash the samples thrice with buffer B for 20 minutes each before transferring them onto a microscope slide.

Wipe off the residual buffer B and add 30 microliters of mounting medium onto the slide. Place an 18-by-18 millimeter coverslip onto the slide and seal the coverslip using nail polish. After turning on the confocal microscope equipped with 40x and 63x oil immersion objectives, place the slide on the microscope stage.

To image the wing disc-associated muscle progenitors and indirect flight muscles, or IFMs, set DAPI with an excitation of 405 and emission of 450 nanometers. Then select Alexa Fluor 488 with an excitation of 496 and emission of 519 nanometers and Quasar 670 for the smFISH probes with an excitation of 647 and emission of 670 nanometers. Locate the sample using a DAPI signal and UV lamp.

Save the captured images as TIF files. To image the adult muscle stem cells, set the excitation and emission wavelengths for DAPI and Alexa Fluor 88 as demonstrated previously. Then set the wavelengths for Alexa Fluor 555 and Quasar 670 for the smFISH probes.

Locate the sample and save the captured images as TIF. Launch ImageJ and navigate to the Plugins menu. Select Macros, then Edit to open the macros source code.

To quantify Mef2 smFISH spots in larvae, set the log radius to three and log quality to 20. Then segment Mef2-positive nuclei with a blur of two, a nucleus scale parameter of 30, nucleus threshold of 8, and nucleus size of 300. Initiate the macro by executing the run command.

The macro will automatically load all images from the designated folder and quantify them sequentially. In muscle fibers, set the log radius to 2.5, log quality to 60, blur to two, nucleus scale parameter to 100, nucleus threshold to zero, and nucleus size to 300. Initiate the macro by executing the run command.

The macro will automatically load all images from the designated folder and quantify them sequentially. After analysis, check the results displayed in file FISH_results and file nuclei_results. The Mef2 and zfh1 transcripts were uniformly detected in the AMP population and co-localized with Mef2 and zfh1 proteins, respectively.

A higher magnification of AMPs distinguished between transcription sites'foci and mature mRNA scattered in the cytoplasm. Similarly, the transcription site and distribution of Mef2 and zfh1 mRNA were examined in differentiated adult IFMs and associated stem cells. The in-house-built ImageJ macro effectively detected and segmented Mef2 spots and muscle nuclei.

The quantitative analysis of Mef2 mRNA per nuclei in adult IFMs and the AMPs were not significantly different. The quantification of Mef2 spot distribution showed that 92%of Mef2 mRNA is present in the cytoplasm, and 8%are associated with muscle nuclei.