Our research studies tissue morphogenesis, the formation of complex three-dimensional tissue structures in development. We are interested in the genes and the molecules that regulate morphogenesis and seek to understand the physical principles underlying morphogenesis, for example, how mechanical forces are generated and how they drive tissue revitalization. Contractile forces generated by filamentous actin and nonmuscle myosin II, also known as actomyosin contractivity, is one of the most important forces that drive tissue morphogenesis.
Our current research addresses how actomyosin contractivity mediates the folding of blood epithelial cell sheets, a fundamental tissue construction mechanism in development. An in-depth understanding of the role of actomyosin contractivity in epithelial folding and answerable for genetic processes requires approaches that can quickly inactivate actomyosin at mass limited time and location and record the immediate impact of tissue behavior and properties. However, this is difficult to achieve using conventional genetic approaches.
The approach described in this protocol is designed to address how cells and tissues respond to sudden change in mechanical forces that normally drive tissue remodeling. This is an important question, but has been difficult to tackle due to limited approaches that can quickly alter mechanical forces or intact tissues. In this study, we developed an optogenetic tool to inactivate actomyosin at specific tissue regions in Drosophila embryos quickly.
We found they combined these two with laser ablation to investigate the direct impact of actomyosin contractivity on tissue mechanics in epithelial folding. Our findings provide new insights into the mechanical mechanism and a new constriction media to epithelial folding. With manner modifications, the protocol can be easily adapted to study the function of actomyosin contractivity in a wide range of morphogenetic processes.
To begin collecting the Drosophila embryos, change the apple juice plate from the cup and label the plate. Cover the surface of the plate with a thin layer of halocarbon oil 27. Next, position an orange red plastic shield on the stage of an upright stereoscope.
Place the apple juice plate on the orange red shield. Turn on the transmitted light of the stereo microscope to illuminate the sample. Collect five to 15 embryos from the apple juice plate using a pair of tweezers.
Gently blot the embryos on a paper towel, sized approximately 1.5 by 1.5 centimeters. Then add several drops of freshly prepared 40%bleach to a new paper towel using plastic transfer pipette to cover the paper towel with a thin layer of bleach. Transfer the embryo from the dry paper towel to the bleach-soaked paper towel and ensure the embryos are soaked in the bleach.
Wait two minutes for the embryo to become dechorionated. After dechorionation, using tweezers, blot the paper towel on a large piece of tissue paper to remove the excess bleach. Rinse the embryos with deionized water eight times to remove residue bleach.
Using an eyelash tool, transfer the embryo from the paper towel to a 35-millimeter glass bottom dish, then add deionized water to the dish to cover the embryos completely. Finally, fine-tune the position and orientation of the embryos using the eyelash tool. Place the dish with the embryos inside a lightproof black box to protect the sample from light exposure during the transfer process.
To begin, turn off the room light. Select ocular under the ocular panel in the FlowView software. Change the cube turret to 4TRTC and turn off the touch panel controller by clicking off on the backlight of touch panel controller in the software, then turn off the computer screen.
Open the front side of the black cloth cover on the microscope. Take the 35-millimeter glass bottom dish containing the embryos from the black box and place it on the microscope stage. Then, turn on the fluorescence illumination unit and use the eyepiece to identify the embryo of interest.
Bring it into focus. Close the black cloth cover to protect the sample from light. Turn on the computer screen to access the software that controls the microscope.
Change the ocular to LSM in the software for image acquisition. Perform laser ablation in control unstimulated embryos using a 25 times magnification water immersion objective. Click Bright Z, sequence manager, and LSM stimulation from the tool window.
Set the scanner type as galvano and the scan size as 512 by 512. Turn on channel one and channel three under the PMT setting panel to allow the use of the 1040-nanometer laser and click live four times speed to visualize the embryo. Rotate the embryo using the rotation function to make the anterior posterior-axis vertically oriented and set the zoom to three.
Draw a region of interest, or ROI, using the shape tool under scan settings and set the ROI size in the reference panel. Next, set the ROI as 512 pixels in width and 100 pixels in height. To set the acquisition parameters for the pre-ablation Z-stack, register the embryo's surface as zero under the Z section.
Set the start as zero and the end as 100 micrometers. Set the step size as two micrometers and activate the Z acquisition mode by checking Z under the series tab. Using the Bright Z function, set the 1040-nanometer laser intensity to increase linearly from 3%to 7%Save the current imaging setting as the first task of the pipeline by clicking LSM in sequence manager.
To set acquisition parameters for the pre-ablation movie, set an ROI of 512 by 512 pixels near the embryo's ventral surface, as previously demonstrated. Set the 1014-nanometer laser intensity to 3%check time, and uncheck Z under the series panel. Keep the interval as free run under the timelapse panel and set the cycle as 10.
Save the current setting as the next task of the pipeline by clicking LSM in sequence manager. To set the parameters for laser ablation, define a 3D region immediately below the vitelline membrane. Set the start of the Z-stack as the plane and the end as 20 micrometers deeper.
Set the step size as 1.5 micrometers. Turn on channel two and channel four under the PMT setting panel to allow the use of the 920-nanometer laser. Set the intensity of the laser to 30%and set image acquisition with the laser for a single Z-stack within the defined 3D region.
Save the current setting as the next task of the pipeline by clicking LSM in sequence manager. To set acquisition parameters for the post-ablation movie, set image acquisition for a 100-frame single Z-plane post-ablation movie using the 1040 and 920 nanometer lasers. Set the intensity of the lasers to 3%and 0.3%Save the current setting as the next task of the pipeline by clicking LSM in sequence manager.
Select sequence under acquire. Change the data saving path and file name as needed. Click ready and wait for the software to initialize the pipeline, then click start to execute the pipeline.
To perform laser ablation in stimulated embryos, set acquisition parameters for the pre-ablation Z-stack, as demonstrated for the unstimulated embryos. Save the current setting as the first task of the pipeline by clicking LSM in sequence manager. To set parameters for optogenetic stimulation within a defined ROI, change the zoom to one and select an ROI that covers the embryo's ventral surface.
Turn off the channel one to channel four detectors, click LSM stimulation, uncheck continuous within duration, and type 12 seconds. Save the current setting as the next task of the pipeline by clicking stimulation in sequence manager. Set a three minute wait time after stimulation to ensure total inactivation of myosin and apical F-actin disassembly and achieve a static tissue morphology before laser ablation.
Next, set acquisition parameters for the single Z-plane pre-ablation movie as demonstrated for the unstimulated embryos, except that the 1040 and 920-nanometer lasers are used for image acquisition. Turn on the channel one to channel four detectors. Set the intensity of the lasers to 3%and 0.3%Save the current setting as the next task of the pipeline by clicking LSM in sequence manager.
Set parameters for laser ablation as demonstrated for the unstimulated embryos. Save the current setting as the next task of the pipeline by clicking LSM in sequence manager. Set acquisition parameters for the single Z-plane post-ablation movie as demonstrated for the unstimulated embryos.
Save the current setting as the next task of the pipeline by clicking LSM in sequence manager. Select sequence under acquire. Change the data saving path and file name as needed.
Click ready and wait for the software to initialize the pipeline, then click start to execute the pipeline. In the unstimulated embryos undergoing apical constriction, spaghetti squash mCherry became enriched at the medial apical region, whereas CRY2 row one dominant negative mCherry was cytosolic. In the stimulated embryos, the CRY2 row one dominant negative mCherry signal became plasma membrane localized, whereas the medial apical signal of spaghetti squash mCherry completely disappeared.
Laser ablation of the unstimulated embryos within the constriction domain led to a rapid tissue recoil along the anterior-posterior axis, whereas laser ablation in the stimulated embryos did not result in noticeable tissue recoil. The ablation was quantified and shown here.
