Analysis of Transforming Growth Factor ß Family Cleavage Products Secreted Into the Blastocoele of Xenopus laevis Embryos

This protocol can be used to study the process by which a wider range of segregated precursor proteins, including TGF-beta family members, are converted to active proteins following proteolytic cleavage. The main advantage of this protocol is that it provides very rapid and inexpensive methods to obtain highly concentrated TGF-beta cleavage product in vivo under visualized condition. To begin with, after the injection on the following day, remove Ficoll solution and any dead or dying embryos, then rinse the embryos once or twice with MBS and culture the embryos in MBS on the bench at room temperature or at 16 degrees Celsius in the incubator to slow down the development.

Heat and pull the glass capillaries to a fine point using a micropipette puller with the desired settings. Using forceps, clip off the tip of a pulled needle. To prevent clogging, the opening of the blastocele aspiration needle should be larger than the microinjection needle.

Insert the aspiration needle into the needle holder connected to the microinjector and attach the needle holder to the micromanipulator. Place early to mid gastrula stage embryos in an MBS-filled injection tray or dish. Insert the needle below the embryo surface near the animal pole.

Press the fill button on the microinjector while observing the needle and the embryo through a dissecting microscope. Over a few seconds, the level of clear fluid rises in the needle and the embryo collapses and becomes concave. Pulse the inject button one or more times to eject any cloudy white matter entering the needle containing debris or proteases.

To detect the cleavage products on immunoblots, aspirate the blastocele fluid of 10 to 20 embryos or more depending on antibody. Pipette one microliter of nucleus-free water onto a paraffin piece placed on the injection tray. Submerge the needle in the water drop and press the inject button to dispel the blastocele fluid into the water.

Alternatively, to eject the fluid directly onto the parafilm and prevent it from flattening out, pulse the inject button to expel the fluid under lower pressure. Transfer the harvested blastocele fluid into a sterile microcentrifuge tube on ice and add nucleus-free water to adjust the final volume to 30 microliters. To detect TGF-beta precursor proteins, transfer the blastocele fluid depleted embryos to a separate tube on ice.

Remove excess MBS and add 200 microliters of pre-chilled embryo lysate buffer. To fully homogenize the embryos, pipette up and down 10 to 20 times until no clumps remain. Centrifuge the homogenized embryos in a refrigerated microcentrifuge at 10, 000 times G for 10 minutes, then remove 160 microliters of the supernatant using a P200 pipette and transfer to a new tube on ice, being careful to avoid the white yolk proteins and other cellular debris in the bottom half of the tube.

Repeat the microcentrifugation once and transfer 128 microliters of the clear supernatant to a new tube on ice. At this point, cleared embryo lysates and the blastocele fluid collected can be stored at minus 80 degrees Celsius for as long as desired. Deglycosylate the cleaved proteins present in blastocele fluid modified through the trans-Golgi network with PNGase F by following the manufacturer's instructions.

The deglycosylated products would migrate as a more condensed band on SDS gels, which can aid in accurate identification. To assess the prodomain fragment monomers and unfolded proteins in the blastocele and lysate, analyze the proteins under reducing conditions by adding five microliters of reducing 4X sample buffer to 15 microliters blastocele fluid and 15 microliters of clarified embryo lysate. To assess the formation of cleaved homodimeric or heterodimeric ligands in the blastocele, analyze the proteins under non-reducing conditions by adding five microliters of non-reducing 4X sample buffer to the remaining 15 microliters of blastocele fluid, then heat it for five minutes and place it on ice.

After using this protocol, the proteins were separated by SDS-PAGE and the immunoblots were probed with antibodies that recognized the myc epitope tag. Under the reducing conditions, in the lysates from embryos expressing only Bmp4 or Bmp7, a single band corresponding to cleaved Bmp4 monomers and a slower migrating band corresponding to cleaved Bmp7 monomers were detected. Both bands were detected in embryos co-expressing Bmp4 and Bmp7.

When the proteins were separated under non-reducing conditions, a single mature Bmp4 and 7 heterodimer band of intermediate mobility were detected along with a trace amount of Bmp4 homodimer in the embryos co-expressing Bmp4 and Bmp7. Bmp4 and Bmp7 heterodimer formation was also observed when Bmp7 protein levels were high. However, in this case, the excess Bmp7 formed homodimers and embryos co-expressed Bmp4 and Bmp7.

These results demonstrated that Bmp4 and 7 preferentially form heterodimers when co-expressed in Xenopus laevis embryos. In this experiment, collecting pure blastocele is the most important step which requires precise needle handling experience. So just keep trying and fixing errors.

This procedure tests whether BMP heterodimers form and which amino acids are important for this. Next step, knockin mouse can be generated to ask whether these amino acids are functionally important during mammalian development.