An Efficient Protocol to Assess ERK Activity Modulation in Early Zebrafish Noonan Syndrome Models via Live FRET Microscopy and Immunofluorescence

We generate zebrafish model of complex developmental diseases, linking patient sequencing to functional genomics, to support variant interpretation and also preclinical drug testing. Despite improved FRET sensors, robust in vivo assay detecting subtle pathogenic changes of Ras-MAPK signaling with sufficient spatiotemporal sensitivity remain lacking. To begin, place the custom microinjection plate containing molded 2%agarose in E3 medium on a working platform.

Arrange and align fertilized teen zebrafish eggs in the plate to appropriate lanes to restrict egg movement during microinjection. Back load the needle with two microliters of injection material using a micro loader pipette. Adjust the pressure and time settings to calibrate each injection based on the needle and embryo quality on the microinjection device.

Inject the solution into the one-cell stage of teen zebrafish embryos. Raise microinjected teen embryos under controlled husbandry conditions. Clean out any eggs that appear cloudy or degenerated within three hours of deposition.

At approximately four hours post-fertilization, using a standard fluorescence stereo microscope, set to a wavelength range of 465-to-500 nanometers. Screen the embryos for fluorescence reporter expression. Select teen positive fish for FRET imaging and reserve negative siblings for immunohistochemical assessments.

Place embryo pools of equal numbers into different wells of a six-well plate. To start treatment, immerse the embryos in three milliliters of E3 medium containing vehicle control as DMSO or diluted MEK at the desired concentration. To begin, melt the 1.5%to-2%low-melting agarose aliquot in a ThermoMixer at 50 degrees Celsius.

Once dissolved, reduce the temperature to approximately 30 degrees Celsius. Position a single injected teen positive embryo at the center of a 35-millimeter glass bottom dish for live imaging. Remove excess E3 medium, and add a drop of low-melting agarose to immobilize the embryo.

Allow the agarose to polymerize at room temperature. Turn on the incubator controller at least one hour before acquisition and set the temperature to 28 degrees Celsius to maintain embryo health. Once the incubator stabilizes, place the imaging dish with the embryo on the sample holder and use a 10x dry objective to visualize the sample.

Switch on the argon ion laser and adjust the laser power to 50%In Hardware Settings, choose eight-bit depth resolution to acquire images. Next, in the Acquisition panel, select the spectral detection xy, gamma, lambda, z scanning mode, and set the format image to 512 by 512 pixels, scanning speed of 400 hertz, and optical zoom of 0.75. Activate the 458-nanometer laser line of the argon ion laser, and set its intensity value to 8.5%Then select a hybrid detector and set its sensitivity to a gain value of 500.

Open the dropdown menu in the detector bar to select the cyan fluorescence protein or ECFP emission curve. To display the YPET emission curve, activate a second detector. Then select the yellow fluorescence protein YFP emission curve.

To start live acquisitions, position the detection cursor in the range of the most intense YFP signal to visualize the sample. Set the start and end positions of the sample thickness in the Z-Stack LASX window. Set the gamma scan range properties to begin at 460 nanometers and end at 570 nanometers of the detection range.

Define the detection bandwidth as five nanometers and scan step size as five nanometers. Start z-stack acquisition. Insert and save two adapted reference emission spectra for CFP and YFP in the dye database available in the Configuration window to exclude spectral bleed-through.

Select the resulting spectral image file from the imaging session and open the process window. Next, in the dye separation tool, select Spectral Dye Separation, and configure the settings for the dye separation. In the dropdown lists on the left side of the dialogue, select the new CFP emission spectrum in the first position and the new YFP emissions spectrum from the spectrum database.

In the gamma scan of the images, identify the image with the greatest signal intensity. Then move along the z-scan to select the optical section that highlights the area of interest on the margin zone. Activate the ROI selection mode on the margin zone of the animal pole to define the area with the best spectrum.

Click on ROI crosshair in the display window and adjust the size of the reference ROI to 40 voxels in the measurements area. Open the newly generated file containing the two separated channels. Produce a two-dimensional projection image from the three-dimensional image series using the maximum intensity projection.

In the Process window, select crop to separate channels into two separate files, the channel one and channel two channels. In the Process window, select Combine Images, then select the channel two file and insert it in the first option. Then select the channel one file and insert it in the second option.

Set a rescale with factor five and choose the ratio operation. Click on Apply to generate the new file containing the ratio metric image. Save the file for data analysis.

Import the spectral imaging and dye separation image files into the open-source Fiji software for analysis. Configure parameters for readout measurements in Fiji using Analyze and Set Measurements. Select area, integrated density and mean gray value as the parameters of interest.

Using the polygon selection tool from the toolbar, select the region of interest corresponding to the margin of the gastrula. Sequentially, click Analyze, Tools and ROI Manager to save the ROI xy specifications. To perform measurements in a selected ROI, click on Analyze and Measure.

Organize FRET by CFP ratio values for each ROI in a worksheet with experimental groups in columns and raw values in rows. For a single biological replicate, create a column table with one grouping variable, where each group is defined by a column. At the end of gastrulation, fix the embryos through immersion in 4%paraldehyde prepared in PBS for 20 minutes at room temperature.

After fixation, wash the embryos several times with PBS. Using a pastor pipette, orient the embryos laterally in a single well of a 12-well plate containing fresh PBS. Using a stereo microscope with a 2.7x magnification objective with 0.63x scanning objective and 8.6 zoom factor, capture the images in a brightfield mode to evaluate the presence of an oval shape.

Import the captured embryo image into Fiji software. To measure the embryo's axes'length, select the straight tool from the toolbar and click on Analyze, followed by Measure. After performing selected measurements, add them to the ROI manager list and save the ROI file.

Export the data into a worksheet. Calculate the axes'ratio by dividing the length of the major axis by the length of the minor axis. Calculate the mean, the standard deviation of the mean, or the standard error of the mean of different replicates.

Our live FRET pipeline in zebrafish enables early sensitive detection of pathogenic ERK signaling activation, supporting variant interpretation, and MEK inhibitor efficiency testing.