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
<ol>
	<li>Anesthetize a sexually mature male in 0.2% Tricaine (Table 1) for 30-40 min until the animal is unresponsive to claw pinch.</li>
	<li>Use blunt forceps to pull the skin away from the body wall. Make a horizontal incision in the skin across the lower abdomen using surgical scissors. Make a parallel incision in the underlying body wall, and then a third, perpendicular incision through the body wall up through the rib cage.
	<ol>
		<li>Fold back the resulting flaps, locate the ventricle of the heart and clip the base to exsanguinate and ensure death.</li>
	</ol>
	</li>
	<li>Pull the yellow fat bodies out of the abdomen with blunt forceps and locate the testes on each side, underlying the base of the fat bodies. Remove each testis by clipping it away from the fat bodies and any attached viscera using surgical scissors.</li>
	<li>Transfer testes to a Petri dish containing 1x Modified Barth&#39;s Solution (MBS) (Table 1). Rinse and remove any adherent blood vessels or attached viscera.
	<ol>
		<li>Transfer one testis to a 35 mm Petri dish containing testis buffer (Table 1) for immediate use and store the other in a 10 mL conical tube filled with testis buffer for later use. Testis should be stored at 4 &#176;C when not used and will remain viable for at least two weeks. 
		&#8203;NOTE: Photographs of testes isolation can be found in Reference 15.</li>
	</ol>
	</li>
</ol>
2. Collection and Fertilization of X. laevis Eggs
NOTE: Frogs should be handled with vinyl gloves or hands washed before and after handling the frogs. Latex gloves, lotions and detergent residues on human hands will damage the frogs&#39; fragile skin.
<ol>
	<li>The evening before spawning, inject 0.3 mL (1200 IU) of human chorionic gonadotropin (Table 1) into the dorsal lymph sac of two or three mature X. laevis females using a 1 mL syringe fitted with a 26 G needle. Use a new needle for each injection.</li>
	<li>After injection, place the females in a sealed container (not air-tight) in an 18 &#176;C biological incubator. Spawning generally begins 14-16 h later. 
	NOTE: Plastic drawers or plastic containers with air holes cut into the lid work well for holding females overnight. Be sure the lid is on firmly to prevent frogs from escaping and desiccating in the incubator.</li>
	<li>Hold the female over a 100 mm Petri dish containing 1x MBS and apply gentle pressure to the frog&#39;s abdomen to expel eggs into the dish.</li>
	<li>Cut a small piece of the testis (~1/5) using a razor blade and transfer it into a 1.5 mL tube containing ~500 &#181;L of 1x MBS. Crush with a pellet pestle or tissue homogenizer. Store the resulting sperm suspension on ice or at 4 &#176;C for subsequent fertilization on the same day.</li>
	<li>Tilt the 100 mm Petri dish to pour off as much of the 1x MBS as possible, and remove the remainder using a plastic transfer pipette. Apply approximately 100 &#181;L of sperm suspension, add about 1 mL of 0.1x MBS and swirl to mix.
	<ol>
		<li>After 5 min, flood the dish with dechlorinated tap water or 0.1x MBS and let stand for 30 min. 
		NOTE: Water can be dechlorinated using a filter or municipal tap water can be left standing uncovered for at least 24 h to allow the chlorine to dissipate in regions where conventional chlorine is added to the water. In areas where chloramine is added to disinfect drinking water, the water must instead be treated with a commercially available conditioner that can break down chloramine. 
		NOTE: Eggs are collected in a high salt solution (1x MBS) to prevent the formation of a jelly coat, which would provide a barrier to fertilization. After fertilization, the low salt solution (dechlorinated water or 0.1x MBS) allows for jelly coat formation and the eggs will adhere to each other and the dish. Eggs that have been fertilized will rotate such that the pigmented animal pole faces upward within 30 min. If this does not happen, and the eggs look healthy under the microscope, sperm viability is suspect&#160;and it may be necessary to harvest new testes.</li>
	</ol>
	</li>
	<li>Pour off the solution covering the eggs and fill the Petri dish with freshly prepared 2% cysteine solution (Table 1). Swirl the solution in the dish for 3-5 min until the jelly coat is dissolved and the eggs are no longer sticking to each other or the dish.
	<ol>
		<li>Tilt the dish and carefully pour off the cysteine solution, or remove with a plastic pipette. Replace with 1x DeBoer&#39;s pond water (Table 1). Swirl to rinse and decant.</li>
		<li>Repeat one wash with DeBoer&#39;s solution, one with 0.1x MBS, and refill the dish with 0.1x MBS. After removing the jelly coat, the eggs become more fragile and must not be exposed to the air-liquid interface.</li>
	</ol>
	</li>
	<li>Move the fertilized eggs to a 16 &#176;C biological incubator or leave them on the bench at room temperature. If left at room temperature, the first cleavage will occur 1-1.5 h after fertilization, and subsequent cleavages will occur approximately every 30 min. By contrast, if embryos are incubated at 15-16 &#176;C, cleavages will occur roughly every hour and&#160;it will be possible to inject a larger number of embryos from a given spawn.</li>
</ol>
3. Microinjection of X. laevis Embryos
<ol>
	<li>Heat and pull glass capillaries to a fine point using a micropipette puller with the following settings: Two-step pull; Heat 1: 67.4 &#176;C; Heat 2: 62 &#176;C. 
	NOTE: These settings are specific for the micropipette puller and glass capillaries used here (Table of Materials).</li>
	<li>Under a dissecting microscope, clip off the tip of a pulled needle close to the end using Dumont #5 forceps. The needle should be sharp but not so thin that the tip bends when it contacts the embryo. Figure 1 shows an example of an appropriately pulled needle that has not been clipped for microinjection (A) and one that has been clipped&#160;(B).</li>
	<li>Insert the needle into a needle holder connected to a microinjector. Attach the needle holder to a micromanipulator. Place 2-4 &#181;L of capped synthetic RNA encoding the Tgf&#223; precursor protein to be analyzed on a small piece of parafilm under a dissecting microscope.
	<ol>
		<li>Using the micromanipulator, lower the needle into the drop and use the fill function on the microinjector to aspirate RNA into the needle. Be sure to keep the needle below the surface of the drop, and watch progress under the microscope to avoid aspirating air into the needle. 
		NOTE: If antibodies that recognize the prodomain and/or ligand domain of the Tgf&#223; precursor to be analyzed are not available, sequence encoding an epitope tag (e.g., V5, myc, Flag) can be inserted in-frame into the cDNA that will be used as a template for RNA transcription. The in vivo bioactivity of the tagged protein should be compared with that of untagged protein to ensure that the epitope does not interfere with proper folding, cleavage or activity.</li>
	</ol>
	</li>
	<li>Eject a drop of the RNA solution into the air and measure the drop size using a micrometer mounted on the injection stage or in the eyepiece of the microscope. Adjust the drop size to approximately 10 nL utilizing the time and pressure controls on the microinjector. 
	NOTE: RNA concentrations in the range of 5-20 ng/&#181;L are appropriate to deliver 50-200 pg of RNA into each embryo. This amount of RNA is generally sufficient to detect cleavage products in aspirates from 10-20 embryos. It is best to aim for the lowest dose of RNA that gives a detectable signal on immunoblots. Higher amounts of RNA can cause gastrulation defects, which interfere with the expansion of the blastocoele.</li>
	<li>Move embryos to an injection dish or tray containing 5% Ficoll in 0.1x MBS (Table 1) when they reach the 2-4 cell stage. A 35 mm Petri dish fitted with a piece of nylon mesh in the bottom can serve as an inexpensive injection dish that will keep the embryos immobilized. Insert the needle near the animal pole and inject 10 nL of RNA into one cell of each embryo. 
	NOTE: Embryos can easily be injected at any time between the one and 16 cell stages. Injecting the embryo after it has cleaved ensures that the egg was fertilized and that the embryo is healthy. The exact location of the injection is not essential. A more detailed description of spawning, culture and microinjection of RNAs into X. laevis embryos can be found in Reference 15.</li>
	<li>Transfer the injected embryos into a 35 mm Petri dish filled with 5% Ficoll in 0.1x MBS (Table 1). Place the small dishes inside a 150 mm Petri dish and cover loosely with a lid to prevent the culture media from evaporating. Culture the embryos overnight at 15-16 &#176;C. 
	&#8203;NOTE: Culturing embryos in Ficoll helps to heal the injection site. If fine needles are used, culturing in 2-3% Ficoll is sufficient, whereas 5% Ficoll is preferred if apparent damage is observed. Keeping the embryos in Ficoll solution for 1-2 h after injection is enough to aid healing, but overnight incubation does not damage the embryo as long as the solution is changed before the onset of gastrulation.</li>
</ol>
4. Blastocoele Extraction and Analysis of Tgf&#223; Cleavage Products
<ol>
	<li>The following morning after injection, remove the Ficoll solution and remove any dead or dying embryos. Rinse the embryos once or twice with 0.1x MBS and culture the embryos in 0.1x MBS on the bench at room temperature. 
	NOTE: Embryos can also be returned to the 16 &#176;C biological incubator to slow down development.</li>
	<li>Heat and pull glass capillaries to a fine point using a micropipette puller with the following settings: Two step pull; Heat 1: 67.4 &#176;C; Heat 2: 62 &#176;C. 
	NOTE: These settings are specific for the puller and glass capillary used here (Table of Materials).</li>
	<li>Under a dissecting microscope, clip off the tip of a pulled needle using forceps. To prevent clogging, the opening of a needle used for blastocoele aspiration should be larger than that in a needle used for microinjection. An example of a good pulled and clipped needle to be used for blastocoele aspiration is shown in Figure 1C. Insert the aspiration needle into needle holder connected to a microinjector and attach the needle holder to a micromanipulator. 
	NOTE: The optimal needle diameter is determined empirically by trial and error. Needles with a large diameter fill too quickly and make it more likely that cellular debris will enter the needle. By contrast, very small diameter needles are likely to become clogged. Once an appropriate needle is generated, it&#39;s useful to generate multiple needles clipped at the same position.</li>
	<li>Place embryos in an injection tray or dish filled with 0.1x MBS when they reach the early to the mid-gastrula stage (stage 10-11).</li>
	<li>Insert the needle below the surface of the embryo near the animal pole. Press the fill button on the microinjector while looking through the dissecting microscope to keep an eye on the needle and the embryo. Over a few seconds, the level of clear fluid will rise in the needle and the embryo will collapse and become concave.
	<ol>
		<li>If cloudy white matter enters the needle, let go of the fill button and pulse the inject button one or more times to eject any cloudy matter, which consists of cellular debris that is likely to contain proteases. 
		NOTE: If the blastocoele collapses and cell debris enters the needle as soon as the fill button is pressed, this indicates that the opening in the needle is too large. If the blastocoele fails to collapse but white matter enters the needle immediately, this shows that the needle was inserted too deeply and is touching the blastocoele floor. Eject the white matter, and move to the next embryo. The aspiration rate can be more carefully controlled by pulsing the fill button rather than pressing it continuously. This is particularly important if the opening in the needle is larger than optimal.</li>
	</ol>
	</li>
	<li>Repeat step 4.5 until the fluid has been aspirated from the blastocoele of 10-20 embryos. 
	NOTE: Generally, blastocoele fluid aspirated from 10-20 embryos is sufficient to detect cleavage products on immunoblots. However, this can vary depending on antibody quality and must be determined empirically.</li>
	<li>Place a piece of paraffin on the injection tray and pipette 1 &#181;L of nuclease-free water onto it. Submerge the needle under the drop of water and press the inject button to dispel the blastocoele fluid into the water.
	<ol>
		<li>Alternatively, eject the fluid directly onto the parafilm, but in this case, pulse the inject button rather than holding it down. This expels the fluid under lower pressure, preventing it from flattening out on the parafilm, making it difficult to draw up in a pipette.</li>
	</ol>
	</li>
	<li>Transfer the blastocoele fluid into a sterile microcentrifuge tube on ice. Expect to harvest approximately 0.3-0.5 &#181;L of blastocoele fluid per embryo. Add nuclease-free water to adjust the final volume to 30 &#181;L.</li>
	<li>Transfer the blastocoele fluid-depleted embryos to a separate tube on ice. Remove excess 0.1x MBS and add 200 &#181;L of chilled (4 &#176;C) embryo lysate buffer (10 &#181;L per embryo) (Table 1). Pipette the embryos up and down 10-20 times using a micropipette until they are fully homogenized and no clumps remain.</li>
	<li>Centrifuge the homogenized embryos in a refrigerated microcentrifuge at 10,000 x g for 10 min. Remove 160 &#181;L 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.
	<ol>
		<li>Repeat the microcentrifugation once and transfer 128 &#181;L of the clear supernatant to a new tube on ice. At this point, cleared embryo lysates and blastocoele fluid can be stored at -80 &#176;C for as long as desired. 
		NOTE: With a few exceptions, Tgf&#223; precursor proteins are not secreted into the blastocoele and will only be detected in the depleted embryo lysates. By contrast, Tgf&#223; family cleavage products are primarily seen in the blastocoele fluid. It is helpful to harvest and analyze both the embryo lysates and the blastocoele fluid for most purposes. At a minimum, this provides a positive control to ensure that the precursor was robustly translated and stable. In addition, it allows one to compare relative ratios of cleavage products to precursors among different wild type or mutant proteins, to identify mutations that enable intact precursors to be secreted into blastocoele fluid (e.g., mutations within the PC consensus motif that allow for proper folding and secretion without cleavage, in which case the intact precursor is detected in both the embryo and the blastocoele fluid) or mutations that generate misfolded, non-cleavable proteins (in which case the precursor will be detected in the embryo lysate, but cleavage products will be absent in the blastocoele fluid).</li>
	</ol>
	</li>
	<li>Deglycosylate proteins present in blastocoele fluid with PNGase F at this point by following the manufacturer&#39;s instructions. 
	NOTE There is no need to deglycosylate precursor proteins present in the embryo lysate since these represent endoplasmic reticulum (ER)--resident forms of the precursor that lack the complex N-linked oligosaccharides removed by PNGase F. By contrast, cleavage products present in blastocoele fluid have moved through the trans-Golgi network (TGN) where carbohydrates are further modified and can now be removed by PNGase F. Following deglycosylation, cleavage products migrate as a more tightly condensed band on SDS gels, which can aid in visualizing, quantitating and accurately identifying cleavage products. This is not strictly required, but it can be helpful, for example, if you are trying to distinguish between homodimers and heterodimers based on migration patterns or if a cleavage product contains heterogeneous carbohydrates that cause it to migrate as a broad fuzzy band.</li>
	<li>Add 5 &#181;L of reducing 4x sample buffer (Table 1) to 15 &#181;L of blastocoele fluid and 15 &#181;L of clarified embryo lysate. Add 5 &#181;L of non-reducing 4x sample buffer (Table 1) to the remaining 15 &#181;L of blastocoele fluid. Heat to 100 &#176;C for 5 min and place on ice. 
	NOTE: Analyzing proteins under non-reducing conditions is essential to analyze the formation of cleaved homodimeric or heterodimeric ligands but is not needed if only analyzing cleavage sites or levels of cleaved proteins. With a few exceptions, prodomain fragments are secreted as monomers. Furthermore, precursor proteins present in the embryo lysate are primarily unfolded, ER resident monomers. Thus, there is no need to analyze these products under non-reducing conditions.</li>
	<li>Resolve precursor proteins and cleavage products by electrophoresis on 15% SDS/polyacrylamide gels.</li>
	<li>Transfer proteins from the gel onto a PVDF membrane using electrophoretic transfer.</li>
	<li>Incubate membranes with appropriate antibodies that recognize the prodomain and the ligand domain (or epitope tags inserted into those domains), wash and incubate with proper secondary antibodies. Visualize proteins with chemiluminescence or fluorescent imaging.</li>
</ol>

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The authors have nothing to disclose.

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 ÃƒÅ¸ 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, ...

analysis-transforming-growth-factor-family-cleavage-products-secreted

Research

JoVE Journal

Developmental Biology

2.4K Views.  University of Utah. When Transforming Growth Factor ß 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.

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

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq

The recruitment of chromatin regulators and the assignment of chromatin states to specific genomic loci are pivotal to cell fate decisions and tissue and organ formation during development. Determining the locations and levels of such chromatin features in vivo will provide valuable information about the spatio-temporal regulation of genomic elements, and will support aspirations to mimic embryonic tissue development in vitro. The most commonly used method for genome-wide and high-resolution profiling is chromatin immunoprecipitation followed by next-generation sequencing (ChIP-Seq). This protocol outlines how yolk-rich embryos such as those of the frog Xenopus can be processed for ChIP-Seq experiments, and it offers simple command lines for post-sequencing analysis. Because of the high efficiency with which the protocol extracts nuclei from formaldehyde-fixed tissue, the method allows easy upscaling to obtain enough ChIP material for genome-wide profiling. Our protocol has been used successfully to map various DNA-binding proteins such as transcription factors, signaling mediators, components of the transcription machinery, chromatin modifiers and post-translational histone modifications, and for this to be done at various stages of embryogenesis. Lastly, this protocol should be widely applicable to other model and non-model organisms as more and more genome assemblies become available.

Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq

The question of how chromatin regulators and chromatin states affect the genome in vivo is key to our understanding of how early cell fate decisions are made in the developing embryo. ChIP-Seq&#8212;the most popular approach to investigate chromatin features at a global level&#8212;is outlined here for Xenopus embryos.

The recruitment of chromatin regulators and the assignment of chromatin states to specific genomic loci are pivotal to cell fate decisions and tissue and ...

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

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the large size of their cells in early embryos, make Xenopus laevis a useful model for visualising GFP-tagged ligands. Synthetic mRNAs are efficiently translated after injection into early stage Xenopus embryos, and injections can be targeted to a single cell. When combined with a lineage tracer such as membrane tethered RFP, the injected cell (and its descendants) that are producing the overexpressed protein can easily be followed. This protocol describes a method for the production of fluorescently tagged Wnt and Shh ligands from injected mRNA. The methods involve the micro dissection of ectodermal explants (animal caps) and the analysis of ligand diffusion in multiple samples. By using confocal imaging, information about ligand secretion and diffusion over a field of cells can be obtained. Statistical analyses of confocal images provide quantitative data on the shape of ligand gradients. These methods may be useful to researchers who want to test the effects of factors that may regulate the shape of morphogen gradients.

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

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, cell culture has been widely used in developmental biology studies. However, it is important to determine whether in vitro cultured cells truly represent in vivo cell types. Grafting cells into embryos, followed by an assessment of their contribution during development is a useful method to determine the potential of in vitro cultured cells. In this study, we describe a method for grafting cells into a defined site of early postimplantation mouse embryos, followed by ex vivo culture. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing precise localization and adjustment of the number of cells receiving exogenous DNA with both high transfection efficiency and low cell death. These techniques, which do not require any specialized equipment, render experimental manipulations of the gastrulation and early organogenesis-stage mouse embryo possible, allowing analysis of commitment in cultured cell subpopulations and the effect of genetic manipulations in situ on cell differentiation.

We demonstrate a method for grafting cultured cells into defined sites of early mouse embryos to determine their in vivo potential. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing the precise delivery of exogenous DNA into a few cells in the embryos.

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, ...

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).

This protocol presents a method to perform quantitative, single-cell in situ analysis of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for collection of blastocysts, whole-mount immunofluorescent detection of proteins, imaging of samples on a confocal microscope, and nuclear segmentation and image analysis are described.

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We quantitatively assessed follicle survival, measured follicle and oocyte diameters, and examined ultrastructure of the oocytes produced from the system. We observed decreased follicle survival, downregulation of expressions of proliferating cell nuclear antigen and growth differentiation factor 9, as indicators for the development of granulosa cells and oocytes, respectively, and oocyte ultrastructural abnormalities under the simulated microgravity condition. The simulated microgravity experimental setup needs to be optimized to provide a model for investigation of the mechanisms involved in the oocyte/follicle in vitro development.

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

A highly promising technique to generate tissue constructs without using matrix is to culture cells in a simulated microgravity condition. Using a rotary culture system, we examined ovarian follicle growth and oocyte maturation in terms of follicle survival, morphology, growth, and oocyte function under the simulated microgravity condition.

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We ...

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The efficient in vivo visualization of blood vessels and the reliable quantification of their complexity are key to understanding the biology and disease of the vascular network. Here, we provide a detailed method to visualize blood vessels with a commercially available fluorescent dye, human plasma acetylated low density lipoprotein DiI complex (DiI-AcLDL), and to quantify their complexity in Xenopus tropicalis. Blood vessels can be labeled by a simple injection of DiI-AcLDL into the beating heart of an embryo, and blood vessels in the entire embryo can be imaged in live or fixed embryos. Combined with gene perturbation by the targeted microinjection of nucleic acids and/or the bath application of pharmacological reagents, the roles of a gene or of a signaling pathway on vascular development can be investigated within one week without resorting to sophisticated genetically engineered animals. Because of the well-defined venous system of Xenopus and its stereotypic angiogenesis, the sprouting of pre-existing vessels, vessel complexity can be quantified efficiently after perturbation experiments. This relatively simple protocol should serve as an easily accessible tool in diverse fields of cardiovascular research.

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

This protocol demonstrates a fluorescence-based method to visualize the vasculature and to quantify its complexity in Xenopus tropicalis. Blood vessels can be imaged minutes after the injection of a fluorescent dye into the beating heart of an embryo after genetic and/or pharmacological manipulations to study cardiovascular development in vivo.

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 and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis.
This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

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 mouse embryonic, dermal, and cardiac fibroblasts can be reprogrammed into functional induced-cardiomyocyte-like cells (iCMs) through overexpression of cardiogenic transcription factors including GATA4, Hand2, Mef2c, and Tbx5 both in vitro and in vivo. However, these previous studies have shown relatively low efficiency. In order to restore heart function following injury, mechanisms governing cardiac reprogramming must be elucidated to increase efficiency and maturation of iCMs.
We previously demonstrated that inhibition of pro-fibrotic signaling dramatically increases reprogramming efficiency. Here, we detail methods to achieve a reprogramming efficiency of up to 60%. Furthermore, we describe several methods including flow cytometry, immunofluorescent imaging, and calcium imaging to quantify reprogramming efficiency and maturation of reprogrammed fibroblasts. Using the protocol detailed here, mechanistic studies can be undertaken to determine positive and negative regulators of cardiac reprogramming. These studies may identify signaling pathways that can be targeted to promote reprogramming efficiency and maturation, which could lead to novel cell therapies to treat human heart disease.

Here we present a robust method to reprogram primary embryonic fibroblasts into functional cardiomyocytes through overexpression of GATA4, Hand2, Mef2c, Tbx5, miR-1, and miR-133 (GHMT2m) alongside inhibition of TGF-&#946; signaling. Our protocol generates beating cardiomyocytes as early as 7 days post-transduction with up to 60% efficiency.

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