This work was partly funded by a University of Nottingham Early Career award to DS. RM is funded by the Higher Committee for Education Development of Iraq.

Ethics Statement: All these experiments follow the animal care and ethical guidelines of the University of Nottingham.
1. Preparation of Heparin Beads for Grafting
<ol>
	<li>Wash heparin beads thoroughly in PBS before use. Note: Beads can be stored at 4 &#176;C as a slurry in PBS.
	<ol>
		<li>Select beads for grafting by removing them from the stock with a 20 &#181;l pipette into a 1 ml drop of PBS. Then use a micropipette set to 2 &#181;l to transfer beads into a 20 &#181;l...

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+avian+embryo+as+a+model+to+study+the+development+of+the+neural+crest:+a+long+and+still+ongoing+story.">The avian embryo as a model to study the development of the neural crest: a long and still ongoing story.</a> Mechanisms of Development. 121, 1089-1102 (2004).</li><li>Bell, E., Brickell, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication-competent+retroviral+vectors+for+expressing+genes+in+avian+cells+in+vitro+and+in+vivo.">Replication-competent retroviral vectors for expressing genes in avian cells in vitro and in vivo.</a> Molecular Biotechnology. 7, 289-298 (1997).</li><li>Abu-Elmagd, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt/Lef1+signaling+acts+via+Pitx2+to+regulate+somite+myogenesis.">Wnt/Lef1 signaling acts via Pitx2 to regulate somite myogenesis.</a> Developmental Biology. 337, 211-219 (2010).</li><li>Munsterberg, A. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+production+of+germline+transgenic+chickens+using+lentiviral+vectors.">Efficient production of germline transgenic chickens using lentiviral vectors.</a> EMBO Reports. 5, 728-733 (2004).</li><li>Valasek, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+molecular+investigations+into+the+development+of+the+pectoral+girdle.">Cellular and molecular investigations into the development of the pectoral girdle.</a> Developmental Biology. 357, 108-116 (2011).</li><li>Tickle, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+contribution+of+chicken+embryology+to+the+understanding+of+vertebrate+limb+development.">The contribution of chicken embryology to the understanding of vertebrate limb development.</a> Mechanisms of Development. 121, 1019-1029 (2004).</li><li>Riddle, R. 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S., Wilting, J., Christ, B., Birchmeier, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scatter+factor/hepatocyte+growth+factor+(SF/HGF)+induces+emigration+of+myogenic+cells+at+interlimb+level+in+vivo.">Scatter factor/hepatocyte growth factor (SF/HGF) induces emigration of myogenic cells at interlimb level in vivo.</a> Developmental Biology. 179, 303-308 (1996).</li><li>Mok, G. F., Cardenas, R., Anderton, H., Campbell, K. H. S., Sweetman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+FGF18+and+retinoic+acid+regulate+differentiation+of+chick+embryo+limb+myoblasts.">Interactions between FGF18 and retinoic acid regulate differentiation of chick embryo limb myoblasts.</a> Developmental Biology. 396, 214-223 (2014).</li><li>Abou-Elhamd, A., Cooper, O., M&#252;nsterberg, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Klhl31+is+associated+with+skeletal+myogenesis+and+its+expression+is+regulated+by+myogenic+signals+and+Myf-5.">Klhl31 is associated with skeletal myogenesis and its expression is regulated by myogenic signals and Myf-5.</a> Mechanisms of Development. , 126-852 (2009).</li><li>Schmidt, M., Tanaka, M., Munsterberg, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+(beta)-catenin+in+the+developing+chick+myotome+is+regulated+by+myogenic+signals.">Expression of (beta)-catenin in the developing chick myotome is regulated by myogenic signals.</a> Development. 127, 4105-4113 (2000).</li><li>Stavridis, M. P., Lunn, J. S., Collins, B. J., Storey, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+discrete+period+of+FGF-induced+Erk1/2+signalling+is+required+for+vertebrate+neural+specification.">A discrete period of FGF-induced Erk1/2 signalling is required for vertebrate neural specification.</a> Development. 134, 2889-2894 (2007).</li><li>Martinez-Morales, P. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF+and+retinoic+acid+activity+gradients+control+the+timing+of+neural+crest+cell+emigration+in+the+trunk.">FGF and retinoic acid activity gradients control the timing of neural crest cell emigration in the trunk.</a> Journal of Cell Biology. 194, 489-503 (2011).</li><li>Olivera-Martinez, I., Storey, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+signals+provide+a+timing+mechanism+for+the+FGF-retinoid+differentiation+switch+during+vertebrate+body+axis+extension.">Wnt signals provide a timing mechanism for the FGF-retinoid differentiation switch during vertebrate body axis extension.</a> Development. 134, 2125-2135 (2007).</li><li>Logan, M., Simon, H. G., Tabin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+regulation+of+T-box+and+homeobox+transcription+factors+suggests+roles+in+controlling+chick+limb-type+identity.">Differential regulation of T-box and homeobox transcription factors suggests roles in controlling chick limb-type identity.</a> Development. 125, 2825-2835 (1998).</li><li>Borycki, A. G., Mendham, L., Emerson, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+somite+patterning+by+Sonic+hedgehog+and+its+downstream+signal+response+genes.">Control of somite patterning by Sonic hedgehog and its downstream signal response genes.</a> Development. 125, 777-790 (1998).</li><li>Sweetman, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF-4+signaling+is+involved+in+mir-206+expression+in+developing+somites+of+chicken+embryos.">FGF-4 signaling is involved in mir-206 expression in developing somites of chicken embryos.</a> Developmental Dynamics. 235, 2185-2191 (2006).</li><li>Chapman, S. C., Collignon, J., Schoenwolf, G. C., Lumsden, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+method+for+chick+whole-embryo+culture+using+a+filter+paper+carrier.">Improved method for chick whole-embryo culture using a filter paper carrier.</a> Developmental Dynamics. 220, 284-289 (2001).</li><li>Sweetman, D., Wagstaff, L., Cooper, O., Weijer, C., M&#252;nsterberg, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+migration+of+paraxial+and+lateral+plate+mesoderm+cells+emerging+from+the+late+primitive+streak+is+controlled+by+different+Wnt+signals.">The migration of paraxial and lateral plate mesoderm cells emerging from the late primitive streak is controlled by different Wnt signals.</a> BMC Developmental Biology. 8, 63 (2008).</li><li>Scott, B. B., Lois, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+tissue-specific+transgenic+birds+with+lentiviral+vectors.">Generation of tissue-specific transgenic birds with lentiviral vectors.</a> Proceedings of the National Academy of Sciences of the U.S.A. 102, 16443-16447 (2005).</li><li>Seidl, A. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+quail+as+a+model+for+research+in+the+avian+nervous+system:+a+comparative+study+of+the+auditory+brainstem.">Transgenic quail as a model for research in the avian nervous system: a comparative study of the auditory brainstem.</a> Journal of Comparative Neurology. 521, 5-23 (2013).</li></ol>

The use of bead grafts applied directly to developing tissues in ovo is a powerful tool to dissect the role of growth factor signaling during development giving unparalleled control over the developmental stage at which they are applied and the duration of exposure.
The choice of bead for each type of molecule is important. Small hydrophobic molecules, such as the inhibitors described here and retinoic acid, usually bind well to derivatized AG 1-X2 beads although it is necessary to test the ef...

Avian embryos have provided a powerful tool for the study of development for many years1. One of their most useful characteristics is that they are relatively easy to manipulate. External development makes it possible to open the egg to access the embryo and perform various micromanipulations including examples such as the classic quail-chick chimera system for studying cell fate2,3, the injection of retroviruses for overexpression in specific tissues during development4,5 and explant culture to identify developmental signaling sources6. More recently chimeras generated between unlabeled hosts with grafts from a transgenic chicken line expressing GFP has shown that the combination of classical grafting and genetic modification can provide important insights into development7,8.
The ease with which the avian embryo can be manipulated has made it an excellent model for studying limb development9. Application of specific growth factors to developing limbs in vivo has been instrumental in identifying factors that alter limb patterning10,11 and continues to provide insights into this process12. This approach has also been used to study the factors that regulate muscle development and has uncovered roles for numerous signals such as Wnts13, BMPs14 and HGF15.
Recently this technique has been used to investigate the signals controlling myogenic gene expression in the limb bud and has shown that interactions between FGF18 and retinoic acid can control the timing of MyoD expression16. Using a combination of growth factors and small molecules that can be loaded onto beads and then grafted directly into specific tissues at defined developmental stages gives the opportunity to intervene at almost any time and region during development. This has been used to investigate many processes including somite patterning17,18, neural specification19, neural crest migration20 and axis extension21.
Here we describe a method for grafting beads soaked in either growth factors or inhibitors into developing chicken limbs. This has been used to determine the effects of these signals on myogenesis by analyzing muscle specific gene expression with in situ hybridization. We describe grafts using either heparin soaked beads, which are used for growth factors, or AG 1-X2 beads for small hydrophobic molecules such as retinoic acid or small molecule inhibitors of specific signaling pathways. However other beads are also available which have been used to deliver both FGFs22 and Shh23.

<table>
	<tbody>
		<tr>
			<td>Heparin acrylic beads</td>
			<td>Sigma</td>
			<td>H5263</td>
			<td>The acrylic-heparin beads used have been discontinued. However a replacement product is available, Heparin agarose beads, cat no H6508. These are transparent so harder to work with but can be stained with phenol red in the same way as AG 1-X2 beads,</td>
		</tr>
		<tr>
			<td>AG 1-X2 beads</td>
			<td>Bio-Rad</td>
			<td>140-1231</td>
			<td></td>
		</tr>
		<tr>
			<td>Affi Gel blue beads</td>
			<td>Bio-Rad</td>
			<td>153-7301; 153-7302</td>
			<td>These beads have been used with a range of growth factors including Shh and FGFs and can be used to replace heparin beads</td>
		</tr>
		<tr>
			<td>FGF18</td>
			<td>Peprotech</td>
			<td>100-28</td>
			<td>Resuspend in PBS with 0/1% BSA, prepare single use aliquots of 0.5-1ul and store at -80&#176;C. Batches and suppliers can vary so different concentrations should be tested to determine an effective dose.</td>
		</tr>
		<tr>
			<td>U0126</td>
			<td>Cell Signalling</td>
			<td>9903</td>
			<td>Make to 20mM stock in DMSO and store in single use aliquots at -80&#176;C. Protect from light.</td>
		</tr>
		<tr>
			<td>BMS-493</td>
			<td>Tocris Biosciences</td>
			<td>3509</td>
			<td>Resuspend in DMSO and store in single use aliquots at -80&#176;C. Protect from light.</td>
		</tr>
		<tr>
			<td>Black Indian ink</td>
			<td>Windsor and Newton</td>
			<td>5012572003384 (30ml)</td>
			<td>While alternatives to this product are available care should be taken as some inks are toxic to embryos</td>
		</tr>
		<tr>
			<td>Tungsten wire, 0.1mm dia. 99.95%</td>
			<td>Alfa Aesar</td>
			<td>10404</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin / streptomycin</td>
			<td>Sigma</td>
			<td>P0781</td>
			<td>Dilute 100X in PBS/ink and PBS/FCS</td>
		</tr>
	</tbody>
</table>

grafting of beads into developing chicken embryo limbs to identify signal transduction pathways affecting gene expression

Using chicken embryos it is possible to test directly the effects of either growth factors or specific inhibitors of signaling pathways on gene expression and activation of signal transduction pathways. This technique allows the delivery of signaling molecules at precisely defined developmental stages for specific times. After this embryos can be harvested and gene expression examined, for example by in situ hybridization, or activation of signal transduction pathways observed with immunostaining.
In this video heparin beads soaked in FGF18 or AG 1-X2 beads soaked in U0126, a MEK inhibitor, are grafted into the limb bud in ovo. This shows that FGF18 induces expression of MyoD and ERK phosphorylation and both endogenous and FGF18 induced MyoD expression is inhibited by U0126. Beads soaked in a retinoic acid antagonist can potentiate premature MyoD induction by FGF18.
This approach can be used with a wide range of different growth factors and inhibitors and is easily adapted to other tissues in the developing embryo.

At HH stage 21 MyoD is not expressed in developing limb myoblasts although staining can be seen in the myotome of the developing somites (Figure 1A). Figure 1B shows in situ hybridization for MyoD 6 hr following an FGF18 bead graft. MyoD is induced in myoblasts close to the bead while there is no expression in the contralateral limb. Co-grafting a bead soaked in U0126, a specific inhibitor of MEK, blocks FGF18 induction of MyoD (Figure 1C). Similarly grafting a ...

Watch this Scientific Journal Video about Grafting of Beads into Developing Chicken Embryo Limbs to Identify Signal Transduction Pathways Affecting Gene Expression at JoVE.com

Grafting of Beads into Developing Chicken Embryo Limbs to Identify Signal Transduction Pathways Affecting Gene Expression

By grafting beads soaked in growth factors or specific inhibitors of signaling pathways into developing embryos it is possible to directly test their effects in vivo. In this protocol beads are grafted into the limb bud to determine the effects of these molecules on gene expression and signal transduction.

Using chicken embryos it is possible to test directly the effects of either growth factors or specific inhibitors of signaling pathways on gene expression ...

The overall goal of this procedure is to show how to determine the effects of growth factors and inhibitors, in the gene expression of developing chicken embryo limbs. This method can help answer key questions in development biology, such as how signalling molecules influence the growth and differentiations of cells in vivo. The main advantage of this technique is that it provides a method to test these factors directly in the embryo.Though this method can provide insight into the limb development, it can also be applied to other systems, such as neural tissue, somites or gastrulation. Demonstrating the procedure will be Rebeea Mohammed, a graduate student from my laboratory. To begin, transfer one milliliter of the beads from the original stock solution into a fresh tube.Allow the beads to settle, then remove the liquid, and replace it with one milliliter of PBS. Repeat this step five times and store the washed beads at four degrees Celsius, as a slurry in PBS. Select the beads for grafting, by removing them from the washed stock solution using a 20 microliter pipette and place them into one milliliter of PBS.Using a stereoscope, choose beads that are around 100 microns in diameter. Transfer the selected beads in two micro-liters of liquid into a new Petri dish containing a 20 milliliter drop of PBS Next, remove the PBS from the beads. First, with 20 micro-liter pipette, and then, remove the residual liquid by capillary action using a fine watch-maker's forceps.It's important to remove as much liquid as possible so that the growth factor is not diluted when it's added to the beads. Pipette the growth factor directly onto the beads. For FGF18, add 0.5 micro-liters of recombinant FGF18, reconstituted add 0.5 milligrams per milliliter in PBS with 0.1 percent BSA.Then, place several drops of water around the edges of the dish to prevent the liquid from evaporating. When ready, incubate the beads for one hour at room temperature. Following incubation, remove the drops of water from the dish with a Pasteur pipette and place the dish on ice.Derivatize the beads, by first using a spatula to transfer the beads to a 1.5 milliliter micro-centrifuge tube. Then, add one milliliter of 0.2 normal formic acid and incubate the beads in the acid on a shaking platform for one hour. Next, allow the beads to settle, remove the formic acid and replace it with one milliliter of water.Wash the beads for one hour on the shaking platform and repeat the wash six times to remove any residual formic acid. Following the last wash, remove the remaining liquid and store the beads at four degrees Celsius. Use a spatula to remove a small palette of beads approximately five to ten micro-liters in volume from the stock, and place the beads into a 30 milliliter Petri dish.Then, add one milliliter of DMSO. Next, use a P2 pipette set to two micro-liters, to transfer beads of approximately 100 microns in diameter, to a 20 micro-liter drop of solvent in another 30 milliliter Petri dish. Remove the bulk of the solvent with a 20 micro-liter pipette and any residual solvent with a two micro-liter pipette.Then, cover the beads with 20 micro-liters of the drug to be applied, dissolved in DMSO. Incubate the beads in the drug solution for one hour. Protect the beads from light, as many drug molecules are light sensitive.To help visualize the beads, transfer four to five beads in two micro-liters of liquid, into 20 micro-liters of two percent phenol red dissolved in water. Remove a single bead with fine watch-maker's forceps and rinse the bead in PBS. Do not leave beads in the phenol red solution for more than 15 minutes before rinsing them, as this can cause lost of activity.Cut fine tungsten wire into three to four centimeter lengths Then, insert one piece of the wire, into the end of a glass Pasteur pipette and melt in a Bunsen burner to fix it in position. Prepare up to 10 needles to ensure that there are spares if they are damaged. Next, place the end of the wire into a blow-torch flame and hold it there until the tungsten glows white and tip is sharp.This takes around two to three minutes. Add five to six drops of artist India ink to 15 milliliters of PBS containing 100 units of Penicillin and 0.1 milligrams of Streptomycin per milliliter. To prepare the chicken embryos, incubate eggs with the blunt end up until they reach the desired stage for most limb bud manipulation, which is typically three to five days.When the desired time point is reached, use blunt forceps to tap on the blunt end of the egg and break the shell. Then, use the forceps to remove the shell and expose the embryo. Ensure the egg cell membrane is also removed.To visualize the embryo, inject PBS supplemented with a few drops of artist India ink, under the embryo with a one milliliter syringe and a 21 gauge needle. The vitelline membrane overlying the embryo, can then be removed with fine forceps. Next, add two to three milliliters of a supplemented PBS with one percent fetal calf serum, 100 units per milliliter of Penicillin, and 0.1 milligrams per milliliter of Streptomycin, into the egg to ensure that embryo does not dehydrate.Using sharpened watch-maker's forceps, open amnion over the developing limb buds. At these stages, the embryo will typically turn, so that the right side is upper most. Ensure the embryo does not dry out by adding more of the supplemented PBS solution when it is needed.The amnion surrounding the embryo can be opened using a fine tungsten needle to reveal the developing limb. Then, use the sharpened tungsten wire to make an incision into the limb bud where the bead will be implanted. Avoid cutting all the way through the limb bud, where possible.Although at younger stages, this is difficult. Pick up a bead, coated with either the growth factor or drug using extra fine, watch-maker's forceps. If the bead has been soaked in either DMSO or phenol red, be sure it gets rinsed with PBS before applying it to the embryo.Transfer the bead to the embryo using the forceps, and then, use the sharpened tungsten wire to insert the bead into the incision location. Add an additional one to two milliliters of the supplemented PBS solution to the egg to ensure that the embryo is kept hydrated. Avoid applying the solution directly onto the embryo itself, as this can damage the embryo and cause the bead to be dislodged.Next, use a ten milliliter syringe, equipped with a 19 gauge needle to remove only as much albumin as is needed to ensure that the embryo does not make contact with the tape used to seal the egg. Ensure that the needle does not damage the yolk, by sliding it down the inside of the shell. Finally, seal the egg with tape and return it to the incubator.In order to confirm that FGF18 is able to act through MEK to phosphorylate ERK, the embryos were harvested one hour after bead grafting and immunostained for the presence of phosphorylated ERK, which can be seen surround the grafted bead in the image on the right. At HH stage 21, MyoD is not expressed in developing limb myoblasts. Although staining can be seen in the myotome of the developing somites.Six hours following an FGF18 bead graft, MyoD can be seen induced in myoblast close to the bead. While there is no expression in the contralateral limb. This expression can be inhibited by co-grafting a bead, coated in a MEK inhibitor such as U0126.Similarly, grafting a U0126 bead at HH Stage 21 reduces the endogenous expression of MyoD after 24 hours, compared with the untreated contralateral limb. In limb buds, at HH stage 19, FGF18 does not induce MyoD. However, co-grafting of beads soaked in FGF18 and a retinoic acid antagonist can overcome this, and ectopic MyoD is detected.Once mastered, this technique can be done in three to four minutes, if it's performed properly. The development of this technique, allowed researchers in the field of development biology to explore growth factors signalling in embryo development. While attempting this technique, it's important to remember not to let the embryo dry out.After watching this video, you should have a good understanding of how to graft beads into developing embryos to determine how growth factors signalling affects development.

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8.7K vistas.  University of Nottingham. The overall goal of this procedure is to show how to determine the effects of growth factors and inhibitors, in the gene expression of developing chicken embryo limbs. This method can help answer key questions in development biology, such as how signalling molecules influence the growth and differentiations of cells in vivo. The main advantage of this technique is that it provides a method to test these factors directly in the embryo.Though this method can provide insight into the limb...