The overall goal of the following experiment is to generate a live time lapse recording of neural crest cell migration in order to show formation of developing cranial facial structures. This is accomplished by first mounting an anesthetized transgenic SOX 10 KD zebrafish in agarose in order to minimize movement throughout the experiment. Next, under a confocal microscope, UV photo activation is carried out to convert KD from green to red in neural crest cells.
Then focusing on the photo converted neural crest cells. A Zack is generated of the developing structure before a time-lapse video is recorded. Results are obtained that show the development of cranial neural crest cells into the zebrafish primary palate in a transgenic SOX 10 KD zebrafish embryo.
The main advantage of this technique over existing methods, such as static imaging, is that complex developmental processes such as cell migration can be visualized in real time. This makes the results more accessible to the scientific community. This method can help answer key questions in developmental biology, such as how neuro crest cell derived structures form, and how PTO genes influence their development.
To prepare for mounting embryos, use a 125 milliliter flask to mix E three water with low melting point aeros microwave to dissolve the solution. Then add trica and place the flask in a 50 degree Celsius water bath to keep warm and nest the ties brightly fluorescent transgenic soine KD zebra fish embryos at 60 hours post fertilization. Then transfer them each to a chamber slide with tissue paper.
Soak up the excess E three water and then use the agarro solution to cover the embryo. Use a micro loader tip to position the embryo perfectly dorsal, ensuring that the embryos completely submerged in agarro. After the agarro sets, use E three 0.015%trica to fill the chamber slide to carry out photo conversion.
Begin by using a 20 x air immersion objective for focusing on the embryo. Then press the L 100 light button on the microscope and select channels in the software. Open the icons.
View acquisition controls A one settings, and click the confocal setup button. Next, click auto and select DPI 4 88 and 561.9 for channels 1, 2, 3 respectively before closing the window again. Then turn off channels one and three before clicking the remove interlock button and pressing the scan button starting at one frame per second and HV 200.
Begin to focus on cells of interest. Gradually increasing the frames per second to one per 32, and reducing the voltage to 100 to 120 depending on the background noise. When the embryo is in focus, grab the green rim at the right edge of the screen and shrink it to fit the area to be photo converted before right clicking with the mouse.
Change the frames per second to one and the HV to 200 and press scan. A zoomed in image of the photo conversion area will now be visible. Next photo.
Convert cells by turning on channel one for anywhere from five to 60 seconds until the green KD signal is nearly absent. Then turn off the dappy channel. Turn on channels two and three to return to the original zoom.
Right click on the zoom window and choose return to original size to check if desired cells were photo converted adequately. Set the limits of the Zack by choosing the Capture Z Series icon in the menu bar. Adjust the lookup tables or uts to set up a time lapse.
Choose the A one simple GUI box and tick the following settings. One per 32 frames per second. Size 1024 and average four or eight x depending on the number of Z stacks that have to be taken in one loop.
Next, go to view acquisition controls ND sequence acquisition and set the timeframe. Then click continuous, and then Zack. Set the borders as demonstrated earlier.
Start the movie, add E three 0.015%trica solution to the chamber, slide throughout the day and fill the chamber completely for overnight imaging. When imaging is complete, create a high quality video from the imaging file by clicking show maximum intensity projection. Then right click on the image and create new document.
Delete unnecessary frames and adjust the L uts. In this movie showing Palato Genesis in the zebrafish, the SOX 10 K transgenic line is used to follow cranial neural crest cells as they develop to form the primary palate. The most anterior cells of the forming palate are photo converted from green to red at 60 hours post fertilization.
When the paired trabecula have met to form the primary palate, these cells are then followed until they reach their final destination. This movie demonstrates perturbed palate development leading to formation of an orofacial cleft, morino mediated knockdown of speck. One lb, A gene involved in oblique facial clefting is used as an example.
At 60 HPF unilateral photo conversion of the most anterior ethmoid plate cells shows a failure effusion between the median and the lateral ethmoid plate cells. This defect resembles the pathogenesis of oblique facial cleft development in humans where failure effusion between the lateral nasal process and maxillary prominence occurs. This technique paved the way for researchers in the field of developmental biology to explore neuro crest cell development in zebrafish.
After watching this video, you should have a good understanding of how to use this method to capture your developmental process of interest.
