Name: grafting of beads into developing chicken embryo limbs to identify signal transduction pathways affecting gene expression
Uploaded: 2016-01-17T14:00:00
Description: 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

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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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. 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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 ...

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