Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

The overall goal of this methodology is to visualize the secretion and diffusion of fluorescently tagged ligands in an intact epithelium using expression in xip pos lavis. This method can help answer key questions about the establishment of morpho in gradients that are known to be essential in the specification of cell lineages during early embryonic development. The main advantage of this technique is that it's a simple method to visualize ligand distribution in an intact epithelium.

An essential part of the protocol is generating biologically active ligands that are normally processed and secreted and able to elicit a response in receiving cells despite having a fluorescent tag attached. Confocal microscopy is an ideal way to study the localization of fluorescently tag proteins within three dimensional tissues using the independent lasers and detectors enables clean analysis within these complex tissue types and enablings robust data to be collected. After generating mRNA coating for fluorescently tagged ligands and acquiring XUS lavis embryos according to the written protocol, inject the mRNA together with a lineage tracer such as membrane RFP, into a single cell at the 16 to 32 cell stage if desired, a modulator can be co injected with a fluorescent ligand and the lineage marker to analyze its effects.

Sometimes it is useful to also inject a neighboring cell Here, a second cell is injected adjacent to the previously injected cell. One cell is injected with mRNAs coding for the GFP tagged ligand and a lineage tracer such as membrane RFP, while the other cell is injected with mRNAs coding for the modulator and a different lineage tracer such as membrane cerulean transfer embryos to a 12.5 degree Celsius incubator and culture to new coop. In Faber stage eight, the following day, when embryos have reached stage eight fill dishes coated with 1%aros with NAM two and transfer embryos using a cutoff glass pipette using tungsten needles.

Dissect circular explan from the animal pole of injected and control embryos, taking care not to include any mesoderm transfer animal explan to 55 millimeter Petri dishes, coated with 1%aros and culture embryos for four hours. To allow the fluorescent proteins to mature to generate relief. Slides apply two layers of PVC insulation tape onto microscope slides completely flattening them down.

Cut a 14 by 10 millimeter rectangle out of the tape for mounting animal caps. Next, pipette two separate droplets of NM two into the relief slide. Then using a cutoff P 20 tip transfer up to 15 animal explan to each slide and with forceps, orient the X explants so that the apical side faces upwards.

Using a diamond pen score, a glass cover slip and break it to create a smaller cover slip. To fit the relief slide gently lower a glass cover slip onto the relief slide using a bent 19 gauge needle. Compressing the apical layer of animal cap cells.

Use nail varnish to seal the slide dry in the dark for 20 minutes and image within four to six hours. To ensure healthy animal caps, Animal caps must be correctly orientated in the custom slides and then cover slips. Load onto the slides gently.

It's vital not to use too much nail varnish to seal the slides as this will cause the tape to wrinkle and then break the seal on the slide later on. Once mastered, you'll have to inject about 25 embryos in order to take 20 good animal caps per condition that you're interested in. These can then be analyzed by confocal microscopy the following day.

To carry out imaging, use a low magnification objective to find X explan. Then switch to a 63 x 1.4 oil objective for higher resolution imaging. Switch on the required lasers including 405 for MIAN 488 for GFP and 561.

For MRFP, optimize the laser powers so that all fluoro fours can be detected. Using the 32 detector array while minimizing the amount of voltage and gain required, then use Lambda mode and select the 405 nanometers 488 and 561 nanometer lasers. To permit a wider field of view, zoom out on the image to 0.6 x.

Next, set the frame size to the desired image size. This provides sufficient resolution for the analysis set averaging to four to eight to increase the signal to noise ratio and optimize the pinhole for this data set. One area unit, according to the 561 nanometer laser was used using 405 and 4 88 5 61.

Diic mirrors collect the light being emitted from membrane Saru, GFP and membrane RFP. In this experiment, light was collected about every 10 nanometers from 415 to 720.Nanometers. Carry out image analysis according to the text protocol shown here is the distribution of GFP tagged sonic hedgehog in an animal cab X explan under control conditions sonic hedgehog GFP is secreted and diffuses outside the region expressing the injected mRNAs.

However, in the presence of the modulator, SULF one, Sonic Hedgehog GFP is more restricted in its distribution. And in this sample only a few foci of sonic Hedgehog GFP can be seen a short distance from the clone of cells producing it. This figure shows the characterization of two fluorescently tagged wit ligands injected into cells as indicated here, both wind eight A and win 11 B-H-A-E-G-F-P accumulate on the membrane of animal cap cells with win eight A-H-A-E-G-F-P accumulating more efficiently than wind 11 B.This figure shows the distribution of wind eight A-H-A-E-G-F-P expressed in a clone of cells marked with membrane tethered cerulean.

The fluorescently tagged ligand is seen to diffuse away from the source cells and into the surrounding cells of the epithelium. Image analysis software is used to extract data on the distribution of fluorescently tagged wind ligands in multiple experimental samples as shown here. The distance from the mem Saru expressing clone and the level of GFP fluorescence is measured to determine the range of wind diffusion and the shape of the gradient.

After measuring the distribution of a ligand, the output of a signaling pathway can also be assessed by combining this protocol with another assay to monitor the response of receiving cells. And in this way, you can attempt to determine the threshold range of am morphogen.