Name: analysis of transforming growth factor &#223; family cleavage products secreted into the blastocoele of xenopus laevis embryos
Uploaded: 2021-07-21T15:00:00
Description: Watch this Scientific Journal Video about Analysis of Transforming Growth Factor ÃŸ Family Cleavage Products Secreted Into the Blastocoele of Xenopus laevis Embryos at JoVE.com

We thank Mary Sanchez for excellent animal care. The authors&#39; research is supported by the National Institute of Child Health and Human Development of the National Institutes of Health (NIH/NICHD) grants R01HD067473-08 and R21 HD102668-01 and by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK/NIH) grant R01DK128068-01.

All procedures described are approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Utah. Frogs are housed in an IACUC approved facility. Male frogs are euthanized by immersion in tricaine following by clipping the ventricle of the heart. Female frogs are housed in the laboratory for a maximum of 24 h following hormonal induction of spawning to allow for egg collection and then returned to the care facility.
1. Collection of X. laevis Testes
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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nodal+stability+determines+signaling+range.">Nodal stability determines signaling range.</a> Current Biology. 15 (1), 31-36 (2005).</li><li>Tilak, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+rather+than+ordered+cleavage+of+two+sites+within+the+BMP4+prodomain+leads+to+loss+of+ligand+in+mice.">Simultaneous rather than ordered cleavage of two sites within the BMP4 prodomain leads to loss of ligand in mice.</a> Development. 141 (15), 3062-3071 (2014).</li><li>Neugebauer, J. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+cleavage+of+BMP4+by+furin,+PC6,+and+PC7.">Site-specific cleavage of BMP4 by furin, PC6, and PC7.</a> Journal of Biological Chemistry. 284 (40), 27157-27166 (2009).</li><li>Birsoy, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=XPACE4+is+a+localized+pro-protein+convertase+required+for+mesoderm+induction+and+the+cleavage+of+specific+TGFbeta+proteins+in+Xenopus+development.">XPACE4 is a localized pro-protein convertase required for mesoderm induction and the cleavage of specific TGFbeta proteins in Xenopus development.</a> Development. 132 (3), 591-602 (2005).</li><li>Mimoto, M. S., Christian, J. 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A., Whitman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Analysis of Growth Factor Signaling in Embryos. , 38-55 (2006).</li><li>Kim, H. S., McKnite, A., Christian, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteolytic+Activation+of+Bmps:+Analysis+of+Cleavage+in+Xenopus+Oocytes+and+Embryos.">Proteolytic Activation of Bmps: Analysis of Cleavage in Xenopus Oocytes and Embryos.</a> Methods in Molecular Biology. 1891, 115-133 (2019).</li><li>Degnin, C., Jean, F., Thomas, G., Christian, J. 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The main advantages to the protocol described here are that it can be completed relatively quickly, does not require expensive reagents and provides a source of concentrated Tgf&#223; cleavage products under physiologic conditions. Another advantage is that it allows one to analyze epitope-tagged proteins and thus circumvent the shortage of commercially available antibodies that recognize most Tgf&#223; family prodomain. Although it is also possible to analyze the cleavage of epitope-tagged Tgf&#223; precursor proteins i...

Members of the Transforming Growth Factor &#223; (Tgf&#223;) superfamily are synthesized as inactive, dimerized precursor proteins. The precursors are then cleaved by members of the proprotein convertase (PC) family, either within the secretory pathway or outside of cells. This creates an active, disulfide-bonded ligand dimer and two prodomain fragments1. Although it has been known for over 30 years that the prodomain of Tgf&#223; family precursors is required to generate an active ligand2, the understanding of how prodomains contribute to ligand function is incomplete.
Although the understanding of the process of proteolytic activation of Tgf&#223; family members remains incomplete, there is increasing interest in understanding which PC consensus motif(s) are cleaved in vivo, whether the cleavage occurs in a specific subcellular or extracellular compartment, and whether the prodomain remains covalently or noncovalently associated with the cleaved ligand3. Several studies have shown that the prodomain not only guides ligand folding before cleavage4,5, but can also influence growth factor stability and range of action6,7,8,9, drive the formation of homodimers or heterodimers10, anchor the ligand in the extracellular matrix to maintain ligand latency11, and in some cases, function as a ligand in its own right to activate heterologous signaling12. Heterozygous point mutations within the prodomain of many members of the Tgf&#223; family are associated with eye, bone, kidney, skeletal or other defects in humans3. These findings highlight the critical role of the prodomain in generating and maintaining an active ligand and stress the importance of identifying and deciphering the role of cleavage products developed during proteolytic maturation of Tgf&#223; family precursors.
Here we describe a detailed protocol for aspirating cleavage products generated during maturation of Tgf&#223; family precursors from the blastocoele of X. laevis embryos and then analyzing them on immunoblots. This protocol can be used to determine whether one or more PC consensus motif(s) in a precursor protein are cleaved in vivo10,13, identify the endogenous PC(s) that cleave each motif13,14, compare in vivo formation of Tgf&#223; family homodimers versus heterodimers10 or analyze whether human disease-associated point mutations in Tgf&#223; precursors impact their ability to form functional dimeric ligands.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#223;-mercaptoethanol</td>
			<td>Fisher</td>
			<td>AC125470100</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromophenol Blue</td>
			<td>Fisher</td>
			<td>B392-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Fisher</td>
			<td>C79-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Nitrate</td>
			<td>Fisher</td>
			<td>C109-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Pellet Pestle/Tissue Grinder</td>
			<td>Fisher</td>
			<td>12-141-364</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 forceps</td>
			<td>Fine Science tools</td>
			<td>11251-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11150H</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll 400</td>
			<td>Sigma Aldrich</td>
			<td>F9378-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass capillary, 1 X 90 mm</td>
			<td>Narshige</td>
			<td>G-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher</td>
			<td>G33-4</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fisher</td>
			<td>BP310-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Human chorionic gonadotropin</td>
			<td>Sigma Aldrich</td>
			<td>CG10-10VL</td>
			<td></td>
		</tr>
		<tr>
			<td>Injection Syringe, 1 mL</td>
			<td>Fisher</td>
			<td>8881501368</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine</td>
			<td>Sigma Aldrich</td>
			<td>C7352</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate</td>
			<td>Fisher</td>
			<td>M63-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle, 26 G</td>
			<td>Fisher</td>
			<td>305111</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>15140148</td>
			<td></td>
		</tr>
		<tr>
			<td>Picoliter Microinjector</td>
			<td>Warner Instruments</td>
			<td>PLI-100A</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Puller</td>
			<td>Narashige</td>
			<td>PC-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Fisher</td>
			<td>P217-500</td>
			<td></td>
		</tr>
		<tr>
			<td>PVDF Membrane</td>
			<td>Sigma Aldrich</td>
			<td>IPVH00010</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Fisher</td>
			<td>S233-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher</td>
			<td>S271-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfate</td>
			<td>Fisher</td>
			<td>BP166-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Fisher</td>
			<td>S318-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine-S</td>
			<td>Pentair</td>
			<td>TRS5</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Fisher</td>
			<td>BP152-5</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of transforming growth factor &#223; family cleavage products secreted into the blastocoele of xenopus laevis embryos

The two arms of the Transforming Growth Factor &#223; (Tgf&#223;) superfamily, represented by Tgf&#223;/Nodal or Bone morphogenetic protein (Bmp) ligands, respectively, play essential roles in embryonic development and adult homeostasis. Members of the Tgf&#223; family are made as inactive precursors that dimerize and fold within the endoplasmic reticulum. The precursor is subsequently cleaved into ligand and prodomain fragments. Although only the dimeric ligand can engage Tgf&#223; receptors and activate downstream signaling, there is growing recognition that the prodomain moiety contributes to ligand activity. This article describes a protocol that can be used to identify cleavage products generated during activation of Tgf&#223; precursor proteins. RNA encoding Tgf&#223; precursors are first microinjected into X. laevis embryos. The following day, cleavage products are collected from the blastocoele of&#160;gastrula stage embryos and analyzed on Western blots. This protocol can be completed relatively quickly, does not require expensive reagents and provides a source of concentrated Tgf&#223; cleavage products under physiologic conditions.

The experiment&#39;s goal described below was to determine whether Bmp4 and Bmp7 form heterodimers (dimers composed of one Bmp4 ligand and one Bmp7 ligand), homodimers (composed of two Bmp4 or two Bmp7 ligands), or a mixture of each when they are co-expressed in X. laevis. Data shown in Figure 2 are extracted from a previously published study10. Figure 2A is a schematic showing cleavage products generated by proteolytic maturatio...

Watch this Scientific Journal Video about Analysis of Transforming Growth Factor ÃŸ Family Cleavage Products Secreted Into the Blastocoele of Xenopus laevis Embryos at JoVE.com

Analysis of Transforming Growth Factor &#223; Family Cleavage Products Secreted Into the Blastocoele of Xenopus laevis Embryos

When Transforming Growth Factor &#223; family precursor proteins are ectopically expressed in Xenopus laevis embryos, they dimerize, get cleaved and are secreted into the blastocoele, which begins at the late blastula to early gastrula stage. We describe a method for aspirating cleavage products from the blastocoele cavity for immunoblot analysis.

Analysis of Transforming Growth Factor &#223; Family Cleavage Products Secreted Into the Blastocoele of Xenopus laevis Embryos

The two arms of the Transforming Growth Factor &#223; (Tgf&#223;) superfamily, represented by Tgf&#223;/Nodal or Bone morphogenetic protein (Bmp) ligands, ...

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.

Research

JoVE Journal

Developmental Biology

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Technique to Target Microinjection to the Developing Xenopus Kidney

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Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

Suppression of Pro-fibrotic Signaling Potentiates Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts into Induced Cardiomyocytes

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that ...

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the ...