Quantification of Orofacial Phenotypes in Xenopus

The overall goal of the following procedure is to describe oral facial phenotypes of xap Levis embryos in a quantitative statistical manner. This is achieved by first mounting detached xip embryo heads in a clay mold for photo documentation using the images. Facial dimensions are measured to provide information about the size of the oral facial region.

Next landmarks are applied via geometric morphometrics to assess the shape of the oral facial region. The results show significant size and shape changes in the Opus oral facial region based on statistical analyses of facial dimensions and relative landmark position. Though this method can provide insight into opus or facial development, it can also be applied to other vertebrate morphology studies, such as comparisons of closely related species for evolutionary or ecological purposes.

This section outlines how to make clay line dishes to hold embryo heads for their photo documentation. First, flatten some modeling clay into a dish any size will do, and smooth it out to cover the entire plate. Then lightly press a straight teasing needle into the clay at a 45 degree angle.

Hold the needle firmly and slowly move the dish to create a depressed line. Next, use a glass pipette to make shallow circular depressions along each line. In each depression, an embryo head can be placed before loading.

The heads filled the dish with PBT. Details on culturing zap piss are outlined in the text protocol as well as details on fixing embryos. For this protocol, fix embryos between stages 42 and 45.Accordingly.

First, place the fixed embryos in a 150 millimeter Petri dish containing approximately 100 milliliters of PBT. Now, sever their heads. First, hold an embryo with a pair of forceps and use a scalpel to make an incision on the posterior side of the gut.

Second, make an incision on the anterior side of the gut near the heart to completely remove the head. Using a pipette transfer the heads to the clay line dish. It's critical that embryo heads are placed in the clay in the exact same orientation for photography.

To ensure success, use the rows drawn in the clay as guides. It's also beneficial if the same person captures all the images in the same day. For frontal view, photographs position the heads posterior side down in the depressions.

Use forceps to manipulate the heads and the surrounding clay so that the heads are fixed facing the camera and are not tilted for lateral view. Photographs position the heads so their sides are all facing the same direction. Manipulate the heads and the surrounding clay, so cement glands are all pointing down at the same angle.

Now using any dissection microscope with image capture capabilities, collect images of the tadpole heads. Use several lights to prevent excessive shadowing and use the highest possible magnification. Photograph each head the same way.

Then save the images uncompressed and at the highest possible resolution. From the images, measure the oral facial dimensions. First, determine the face width.

Identify the points at which the ventral part of each eye meets the peripheries of the face, and measure the distance between these points. Details on measuring the face height and oral facial area can be found in the text protocol. Pay particular attention to determining the snout length On lateral images, make a vertical line that marks the anterior of the eye.

Then measure the horizontal distance from the anterior most point where the face meets the dorsal edge of the cement gland to the vertical line marking the anterior of the eye. Other dimensions like mouth width and roundness are explained in the text protocol for this analysis, facial landmark coordinates should be standardized and saved to a file. The landmarks should capture the shape of the image and be usable in all images.

So do not use structures that may not exist in all samples. Now open the first image file. To assess, select the plugins tab on the main toolbar and choose point picker.

From the dropdown menu, select the add points tab and position the landmark points on the image. They appear as multicolored. Crosses switch between landmark points using the move crosses tool.

Once all are placed, select the display results tab on the main toolbar and choose the show option. This displays the landmark coordinates. Next, copy the coordinates into a spreadsheet program in an empty row above the pasted data type LM equals followed by the number of landmarks in the sample in the row below.

The pasted data type ID equals followed by the name identifying the sample. Each sample must have a unique identifier. Repeat this process until all the sample images have been mapped and entered into the spreadsheet.

Next, copy the columns with a sample identification and coordinates of each landmark and save it into a text document. Then open the morphometric software program. In this software, select create new dataset from the main menu.

Name the file and dataset in the pop-up screen and import the file as a TPS. Now select a text file of coordinates to create the data set. Next, perform a procrustes fit and alignment.

First, highlight the data set in the project tree tab. Then choose preliminaries on the main toolbar and new procrustes fit from the dropdown menu. From the selections available, choose aligned by principle axis.

Then select the option to perform crusty's fit back in the preliminaries dropdown menu, select generate Covance matrix and select Execute. Now from the results tab, view the correlations of the Covance matrix. To distinguish experimental and control group samples, create a classifier file by assigning each sample in the dataset to the appropriate classifier variable.

In a spreadsheet, create a column labeled ID and create a column labeled treatment. Then list each sample in the ID column and assign it to a group. Under the treatment column, save this as a text file and in the morphometric software program, import it through the choose match by identifier option.

Now under the comparison tab, create a discriminant function analysis transformation in the pop-up menu. Under this function, ensure that the appropriate data set is selected and the type of data is the procrustes fit coordinates. Highlight the pairs of groups to be compared and then run a 1000 permutation test.

To view the vector map of the coordinates, go to the graphics menu and select the shape difference tab. If the image is inverted, bring up the pop-up menu in the plot space to flip the diagram to the correct orientation. Also, in the popup menu, change the graph to a transformation grid to superimpose the vectors onto a grid details on creating a principle component analysis.

Scatterplot principle component analysis. Transformation grids CVA, scatterplot and CVA transformation grids are all provided in the text protocol. Embryos were treated with one micromolar retinoic acid receptor or RAR inhibitor from developmental stage 24 to stage 30, and then fixed at stage 42 and photographed control embryos treated with DMSO developed.

Normally, the RAR inhibitor treatment resulted in a narrowing of the face eye abnormalities and a malformed triangular shaped mouth. After performing a geometric morphometric analysis variance was assessed via principle component analysis. Treated embryos were clearly distinguished from controls.

Along the PC one axis, the arrows identify statistical outliers from within a group. Morphological differences were next assessed by a discriminant function analysis. Dramatic shifts in the position of lateral landmarks indicate a narrowing of the face shape.

A slight outward shift in nostril position is noted by the arrows. There is also notable warping in the midface region. After watching this video, you should have a good understanding of how to use a combination of traditional size measurements and geometric morphometrics to assess size and shape of Theus embryonic face.