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><tbody><tr><td>&amp;szlig;-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, ...

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

Research

JoVE Journal

Developmental Biology

2.4K Views.  University of Utah. Dit protocol kan worden gebruikt om het proces te bestuderen waarbij een breder scala aan gescheiden precursoreiwitten, waaronder TGF-bèta-familieleden, worden omgezet in actieve eiwitten na proteolytische splitsing. Het belangrijkste voordeel van dit protocol is dat het zeer snelle en goedkope methoden biedt om hooggeconcentreerd TGF-bètasplitsingsproduct in vivo te verkrijgen onder gevisualiseerde omstandigheden. Om te beginnen, na de injectie op de volgende dag, verwijder Ficoll-oplossin...

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

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 ...

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 ...

Transcriptional Analysis by Nascent RNA FISH of In Vivo Trophoblast Giant Cells or In Vitro Short-term Cultures of Ectoplacental Cone Explants

The placenta derives from one extra-embryonic lineage, the trophectoderm. In the peri-implantation murine blastocyst, mural trophectoderm cells differentiate into primary trophoblast giant cells (TGCs) while the polar trophectoderm overlying the inner cell mass continues to proliferate later differentiating into secondary TGCs. TGCs play a key role in developing placenta and are essential for a successful pregnancy. Investigation of transcriptional regulation of specific genes during post-implantation development can give insights into TGCs development. Cells of the ectoplacental cone (EPC) from embryos at 7-7.5 days of gestation (E7-7.5), derived from the polar trophectoderm, differentiate into secondary TGCs1. TGCs can be studied in situ, on cryostat sections of embryos at E7 although the number of TGCs is very low at this stage. An alternative means of analyzing secondary TGCs is to use short-term cultures of individual EPCs from E7 embryos. We propose a technique to investigate the transcriptional status of genes of interest both in vivo and in vitro at the single-cell level using fluorescent in situ hybridization (RNA FISH) to visualize nascent transcripts. This technique provides a direct readout of gene expression and enables assessment of the chromosomal status of TGCs, which are large endoreplicating cells. Indeed, a key feature of terminal differentiation of TGCs is that they exit the cell cycle and undergo multiple rounds of endoreplication.This approach can be applied to detect expression of any gene expressed from autosomes and/or sex chromosomes and can provide important information into developmental mechanisms as well as placental diseases.

Transcriptional  Analysis by Nascent RNA FISH of In Vivo Trophoblast Giant Cells or In Vitro Short-term Cultures of Ectoplacental Cone Explants

Trophoblast giant cells (TGCs) play a key role in the placenta to ensure a healthy pregnancy. We present a protocol for assessing the transcriptional status of genes in TGCs by nascent fluorescent in situ hybridization on cryostat sections of post-implantation embryos or short-term cultures of embryonic day 7 ectoplacental cones.

The placenta derives from one extra-embryonic lineage, the trophectoderm. In the peri-implantation murine blastocyst, mural trophectoderm cells ...

Tracing Gene Expression Through Detection of &#946;-galactosidase Activity in Whole Mouse Embryos

The Escherichia coli LacZ gene, encoding &#946;-galactosidase, is largely used as a reporter for gene expression and as a tracer in cell lineage studies. The classical histochemical reaction is based on the hydrolysis of the substrate X-gal in combination with ferric and ferrous ions, which produces an insoluble blue precipitate that is easy to visualize. Therefore, &#946;-galactosidase activity serves as a marker for the expression pattern of the gene of interest as the development proceeds. Here we describe the standard protocol for the detection of &#946;-galactosidase activity in early whole mouse embryos and the subsequent method for paraffin sectioning and counterstaining. Additionally, a procedure for clarifying whole embryos is provided to better visualize X-gal staining in deeper regions of the embryo. Consistent results are obtained by performing this procedure, although optimization of reaction conditions is needed to minimize background activity. Limitations in the assay should be also considered, particularly regarding the size of the embryo in whole mount staining. Our protocol provides a sensitive and a reliable method for &#946;-galactosidase detection during the mouse development that can be further applied to the cryostat sections as well as whole organs. Thus, the dynamic gene expression patterns throughout development can be easily analyzed by using this protocol in whole embryos, but also detailed expression at the cellular level can be assessed after paraffin sectioning.

Here we describe the standard protocol for the detection of &#946;-galactosidase activity in early whole mouse embryos and the method for paraffin sectioning and counterstaining. This is an easy and quick procedure to monitor gene expression during development that can also be applied to tissue sections, organs or cultured cells.

The Escherichia coli LacZ gene, encoding &#946;-galactosidase, is largely used as a reporter for gene expression and as a tracer in cell lineage studies. ...

Imaging Dpp Release from a Drosophila Wing Disc

The transforming Growth Factor-beta (TGF-&#946;) superfamily is essential for early embryonic patterning and development of adult structures in multicellular organisms. The TGF-&#946; superfamily includes TGF-&#946;, bone morphogenetic protein (BMPs), Activins, Growth and Differentiation Factors, and Nodals. It has long been known that the amount of ligand exposed to cells is important for its effects. It was thought that long-range concentration gradients set up embryonic pattern. However, recently it has become clear that the timing of exposure to these ligands is also important for their downstream transcriptional consequences. A TGF-&#946; superfamily ligand cannot have a developmental consequence until it is released from the cell in which it was produced. Until recently, it was difficult to determine when these ligands were released from cells. Here we show how to measure the release of a Drosophila BMP called Decapentaplegic (Dpp) from the cells of the wing primordium or wing disc. This method could be modified for other systems or signaling ligands.

Imaging Dpp Release from a Drosophila Wing Disc

The timing of exposure to ligands may impact their developmental consequences. Here we show how to image release of a Drosophila bone morphogenetic protein (BMP) called Dpp from cells of the wing disc.

The transforming Growth Factor-beta (TGF-&#946;) superfamily is essential for early embryonic patterning and development of adult structures in ...

Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing (Frog) Embryo

Characterization of molecular events as cells give rise to tissues and organs raises a potential to better understand normal development and design efficient remedies for diseases. Technologies enabling accurate identification and quantification of diverse types and large numbers of proteins would provide still missing information on molecular mechanisms orchestrating tissue and organism development in space and time. Here, we present a mass spectrometry-based protocol that enables the measurement of thousands of proteins in identified cell lineages in Xenopus laevis (frog) embryos. The approach builds on reproducible cell-fate maps and established methods to identify, fluorescently label, track, and sample cells and their progeny (clones) from this model of vertebrate development. After collecting cellular contents using microsampling or isolating cells by dissection or fluorescence-activated cell sorting, proteins are extracted and processed for bottom-up proteomic analysis. Liquid chromatography and capillary electrophoresis are used to provide scalable separation for protein detection and quantification with high-resolution mass spectrometry (HRMS). Representative examples are provided for the proteomic characterization of neural-tissue fated cells. Cell-lineage-guided HRMS proteomics is adaptable to different tissues and organisms. It is sufficiently sensitive, specific, and quantitative to peer into the spatio-temporal dynamics of the proteome during vertebrate development.

Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing Frog Embryo

Here we describe a mass spectrometry-based proteomic characterization of cell lineages with known tissue fates in the vertebrate Xenopus laevis embryo.

Characterization of molecular events as cells give rise to tissues and organs raises a potential to better understand normal development and design ...

Chemistry

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later stages of embryonic development, and to define how enhancers and promoters regulate gene expression within the embryo. The protocol demonstrated here can be used to efficiently produce transgenic Xenopus laevis embryos. This transgenesis approach involves three parts: 1. Sperm nuclei are isolated from adult X. laevis testis by treatment with lysolecithin, which permeabilizes the sperm plasma membrane. 2. Egg extract is prepared by low speed centrifugation, addition of calcium to cause the extract to progress to interphase of the cell cycle, and a high-speed centrifugation to isolate interphase cytosol. 3. Nuclear transplantation: the nuclei and extract are combined with the linearized plasmid DNA to be introduced as the transgene and a small amount of restriction enzyme. During a short reaction, egg extract partially decondenses the sperm chromatin and the restriction enzyme generates chromosomal breaks that promote recombination of the transgene into the genome. The treated sperm nuclei are then transplanted into unfertilized eggs. Integration of the transgene usually occurs prior to the first embryonic cleavage such that the resulting embryos are not chimeric. These embryos can be analyzed without any need to breed to the next generation, allowing for efficient and rapid generation of transgenic embryos for analyses of promoter and gene function. Adult X. laevis resulting from this procedure also propagate the transgene through the germline and can be used to generate lines of transgenic animals for multiple purposes.

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

Biology

Generation of Na&#239;ve Blastoderm Explants from Zebrafish Embryos

Due to their optical clarity and rapid development, zebrafish embryos are an excellent system for examining cell behaviors and developmental processes. However, because of the complexity and redundancy of embryonic signals, it can be challenging to discern the complete role of any single signal during early embryogenesis. By explanting the animal region of the zebrafish blastoderm, relatively na&#239;ve clusters of embryonic cells are generated that can be easily cultured and manipulated ex vivo. By introducing a gene of interest by RNA injection before explantation, one can assess the effect of this molecule on gene expression, cell behaviors, and other developmental processes in relative isolation. Furthermore, cells from embryos of different genotypes or conditions can be combined in a single chimeric explant to examine cell/tissue interactions and tissue-specific gene functions. This article provides instructions for generating zebrafish blastoderm explants and demonstrates that a single signaling molecule - a Nodal ligand - is sufficient to induce germ layer formation and extension morphogenesis in otherwise na&#239;ve embryonic tissues. Due to their ability to recapitulate embryonic cell behaviors, morphogen gradients, and gene expression patterns in a simplified ex vivo system, these explants are anticipated to be of great utility to many zebrafish researchers.

Zebrafish blastoderm explants are generated by isolating embryonic cells from endogenous signaling centers within the early embryo, producing relatively na&#239;ve cell clusters easily manipulated and cultured ex vivo. This article provides instructions for making such explants and demonstrates their utility by interrogating roles for Nodal signaling during gastrulation.

Due to their optical clarity and rapid development, zebrafish embryos are an excellent system for examining cell behaviors and developmental processes. ...

Protocol for Human Blastoids Modeling Blastocyst Development and Implantation

A model of the human blastocyst formed from stem cells (blastoid) would support scientific and medical advances. However, its predictive power will depend on its ability to efficiently, timely, and faithfully recapitulate the sequences of blastocyst development (morphogenesis, specification, patterning), and to form cells reflecting the blastocyst stage. Here we show that na&#239;ve human pluripotent stem cells cultured in PXGL conditions and then triply inhibited for the Hippo, transforming growth factor- &#946;, and extracellular signal-regulated kinase pathways efficiently undergo morphogenesis to form blastoids (&gt;70%). Matching with developmental timing (~4 days), blastoids unroll the blastocyst sequence of specification by producing analogs of the trophoblast and epiblast, followed by the formation of analogs of the primitive endoderm and the polar trophoblasts. This results in the formation of cells transcriptionally similar to the blastocyst (&gt;96%) and a minority of post-implantation analogs. Blastoids efficiently pattern by forming the embryonic-abembryonic axis marked by the maturation of the polar region (NR2F2+), which acquires the specific potential to directionally attach to hormonally stimulated endometrial cells, as in utero. Such a human blastoid is a scalable, versatile, and ethical model to study human development and implantation in vitro.

A protocol outlining the formation of human blastoids that efficiently, timely, and sequentially generate blastocyst-like cells.

A model of the human blastocyst formed from stem cells (blastoid) would support scientific and medical advances. However, its predictive power will depend ...

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.

The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

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

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