This work was funded by MRC grant MC.PC_13052.

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A., Parker, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+a+gradient+of+a+morphogenetic+substance+in+the+developing+limb.">Evidence for a gradient of a morphogenetic substance in the developing limb.</a> Dev Biol. 54 (2), 297-303 (1976).</li><li>Summerbell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zone+of+polarizing+activity:+evidence+for+a+role+in+normal+chick+limb+morphogenesis.">The zone of polarizing activity: evidence for a role in normal chick limb morphogenesis.</a> J Embryol Exp Morphol. 50, 217-233 (1979).</li><li>Wilde, S. M., Wedden, S. 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The authors declare they have no competing financial interests.

We describe the use of impermeable barriers to prevent limb bud formation in the chick embryo. This technique has a number of critical steps. Following preliminary experiments, we found that the hinge shaped barriers used in 9, 10 remain in place in the embryo far better than straight barriers. The distal flat side of the barrier sticks to the vitelline membrane and keeps the barrier in place (Figure 2C&#39;). Careful preparation of hinge shaped barriers from aluminum foil as described in protocol step 1.6 is essential to the success of the operation. A second important step is to ensure that eggs are not left out of the incubator for too long following windowing and staging prior to operating on the embryo. In our experience, embryos survive an operation better if they are at or near incubation temperature when the operation is carried out. Operations should therefore be carried out as quickly as possible and the eggs resealed promptly to ensure good survival rates. Many researchers add a drop of antibiotics to the embryo following microsurgery before returning the eggs to the incubator. The presence of the liquid prevented the barriers from sticking well in position. Addition of antibiotic could also dislodge beads after placement. Therefore not using antibiotics, swift operations and good cleanliness with instruments is recommended to avoid infection. Wiping instruments with 70% ethanol following every stage of an operation is essential to prevent instruments from becoming sticky from contact with the vitelline membrane which can cause both barriers or beads to adhere to forceps (see protocol step 3.1.2.2). Both varieties of beads used could fall out of place following the operation. When the barrier position was checked the next day it was not possible to see through the barrier to ascertain whether the bead remained in place or not. In cases where the bead had fallen out wing bud outgrowth was not rescued (Table 1). Avoiding transferring liquid with the bead when implanting them means that beads stick more easily to the cut face of the LPM (see protocol 2.3.2.3). Finally ensuring that embryos are fixed immediately after harvesting is critical to good subsequent gene expression analysis (see protocol step 4.2).
This protocol modifies previously published experiments that used barriers to prevent limb bud initiation, defines specific optimum widths for barriers and introduces combining barrier and bead placement followed by gene expression analysis. We found using conventional aluminum foil, which is cheap and readily available in all labs, produces outcomes equal to those that previously used tantalum foil. The foil can be left in the embryo when carrying out in situ hybridization (Figure 2C&#39;) but we usually remove it if more than one embryo is being processed to prevent the sharp edges of the foil damaging embryos. Pilot studies demonstrated that the width of leg region barriers is particularly important. Initially the barriers trialed were too short and leg bud outgrowth was not prevented. 1.2-1.3 mm is the optimum width for a barrier to block leg bud outgrowth completely. The narrowest wing barriers that one can place accurately to prevent bud outgrowth give the best survival rates. This can be as narrow as 0.5-0.7 mm. However, barriers that are a little wider are more likely to result in complete blocking of wing bud outgrowth because they allow for a little inaccuracy of placement (we used 0.7-1 mm). Avoidance of blood vessels when making the incision is critical for wing bud level barriers. Semi-permeable barriers have previously been used to bisect the chick limb bud. MacCabe and Parker, 1976 and Summerbell, 1979 both used filters 0.45 &#181;m and 0.8 &#181;m pore size, respectively 16, 17. They reported that diffusible signals could pass through the pores in the filters and that results gained using semi-permeable barriers were midway between a limb bud with no barrier present and one with an impermeable tantalum foil barrier. This protocol could be adapted to use semi-permeable barriers of varying pore size to differentiate candidate signals based on size or to modify the dynamics of signal diffusion.
This protocol has various limitations mainly associated with the model organism itself. Such techniques could be used to probe further questions in limb development and also other questions of vertebrate embryogenesis. A proviso is that the organ/area in question should be developing when the chick embryo is accessible to intervention. The presence of large blood vessels in the area where a barrier needs to be placed makes barrier placement later than stage 13 or 14 technically challenging. If large blood vessels are cut during barrier placement this can cause embryo mortality. Our investigation of when the transcription factors Tbx5 and Tbx4 are first induced in the LPM was limited by the earliest stage at which we could place a barrier that would later end up opposite either the wing or leg bud respectively. It could be illuminating to attempt to block Tbx gene induction at earlier stages, closer to gastrulation. The earliest a barrier could be placed in the presumptive wing region successfully was stage 8 and in the leg region stage 10. This protocol is probably not suitable for the study of gene expression beyond limb bud stages because subsequent rapid growth would displace the barrier and results would relate only to the initial position of the barrier.
Having related the limitations of the chick embryo in this context, its advantages over other vertebrate model organisms are many. The chick embryo is a very suitable animal model for this type of physical intervention in a vertebrate embryo. The embryos are relatively large and are easily accessible through the eggshell. One can return to the embryo for further experimentation (e.g., the removal of a bead 18 or even a barrier) or to check on progress by simply removing the tape window. There is possible scope to include live imaging or time lapse imaging. We describe a method of windowing chicken eggs that is particularly suited to operating on early stage embryos. In the case of limb studies it is very important and helpful that there is a contralateral control limb for every experiment that serves as an ideal internal control for each biological replicate.
The combination of using impermeable barriers to prevent limb outgrowth and beads soaked in signaling molecules has not been reported before. Our analysis of mRNA expression following such interventions is also novel. These techniques enabled us to dissect the difficult problem of when limb specific transcription factors Tbx5 and Tbx4 are induced in the LPM and also to show that later, prior to limb bud initiation, the presence of these Tbx genes in the LPM is not sufficient for the activation of Fgf10 transcription and the start of limb bud outgrowth. Figures 2G, 2D and 2E illustrate these findings well. The absence of Tbx4 expression following the insertion of a barrier at stage 10 in Figure 2G is in stark contrast to its presence in 2D and 2E following barrier insertion at stage 15, after the induction of Tbx4. The implantation of RA soaked beads facilitated the rescue of limb bud initiation following barrier insertion and so points to RA from the somites being essential in the LPM before limb bud outgrowth can begin. Such techniques could be used to solve further questions in limb development but also other questions of vertebrate embryogenesis. Areas of the brain, eye and trunk are likely to be suitable.

Classical embryologists traditionally employed physical techniques to interrogate the mechanisms controlling embryonic development. In the 1960s and 1970s investigators developed techniques that used impermeable barriers inserted between tissues of the developing embryo to demonstrate the importance of inductive signals during embryogenesis. Our interest is in vertebrate limb development and in particular what events precede limb bud outgrowth. For example, insertion of a barrier can prevent limb bud outgrowth from occurring on one side of the chick embryo while allowing it to proceed normally on the contralateral, unoperated side. Materials used to make these barriers varied; for example, mica plates 1 and tantalum foil 2. These barriers effectively separate the lateral plate mesoderm (LPM), from which the limb bud forms, from the somites formed from the paraxial mesoderm. Surgical grafting techniques demonstrate that close association with the somites is essential if limb bud initiation is to occur in the LPM 34. In the 80s and 90s developmental biologists discovered diffusible signaling molecules that are essential in controlling developmental processes. Beads soaked with these signaling molecules can be implanted in specific regions of the embryo and produce alterations to embryonic development. For example, retinoic acid (RA) taken up by ion exchange beads is released over a 24-hour period when implanted into a chick embryo and can produce mirror-image duplication of the digits 5. Heparin acrylic beads containing FGF protein can initiate limb bud outgrowth from the interlimb LPM 6.
More recently mouse and fish genetics have taken the study of vertebrate limb initiation into a new era (reviewed in 7). There is good evidence that RA present in the somites prior to limb bud initiation is the essential diffusible signal required by the LPM to initiate limb budding. We wanted to exploit the accessibility and experimental robustness of the chick embryo to dissect the effect of barrier implantation on gene expression in the LPM around the time of limb bud initiation. A novel adaptation of our method is to combine a barrier that blocks signals from axial tissues with bead placement lateral to the barrier to exogenously supply candidate endogenous factors that have been blocked by the barrier. The following protocols are those developed to tease out the mechanisms of limb bud induction and initiation.
Studying limb bud initiation means using early stage embryos. Many researchers window chicken eggs by first removing some of the albumin through the air sac with a hypodermic needle. The embryo, which lies on top of the yolk, will then lie lower in the egg. This is an advantage for some techniques, such as electroporation, but can be a disadvantage if surgical manipulation of the embryo is desired. We find having the embryo as close to the opening in the egg as possible is an advantage.
Questions remain regarding the signals that control vertebrate limb bud induction and initiation and the following protocols are suitable to address them. We are the first to develop a protocol for the insertion of barriers to prevent chick limb bud outgrowth at a stage earlier than stage 12 8. Modified versions of these protocols could also be used to address other questions on the origin and timing of inductive cues where an accessible inducing tissue lies adjacent to the primordium being induced. Provided the region of the embryo is accessible at the relevant developmental stage, then a barrier could be placed between the two tissues and consequent changes in development studied. Examples may be found in brain development, axis extension and possibly organ development such as liver or kidney induction.

<table><tbody><tr><td>Dumont No. 5 watchmakers forceps, x2</td><td>Fine Science Tools&amp;nbsp; www.finescience.de</td><td>11251-20</td><td></td></tr><tr><td>Dumont No. 4 watchmakers forceps, x2</td><td>Fine Science Tools&amp;nbsp; www.finescience.de</td><td>11241-30</td><td></td></tr><tr><td>Dumont strong forceps, type AA</td><td>Fine Science Tools&amp;nbsp; www.finescience.de</td><td>11210-10</td><td></td></tr><tr><td>Curved scissors with blades 2.5 cm long</td><td>Fine Science Tools&amp;nbsp; www.finescience.de</td><td>14061-11</td><td></td></tr><tr><td>Blunt ended curved forceps, ends approx 0.5&amp;nbsp;cm long</td><td>Fine Science Tools www.finescience.de</td><td>11065-07</td><td></td></tr><tr><td>Steel pin, 0.2 mm wide</td><td>Fine Science Tools&amp;nbsp; www.finescience.de</td><td>26002-20</td><td></td></tr><tr><td>Sharpening stone, square</td><td>Fine Science Tools&amp;nbsp; www.finescience.de</td><td>29008-01</td><td></td></tr><tr><td>Mineral oil</td><td></td><td></td><td></td></tr><tr><td>Needle holder</td><td>Fine Science Tools&amp;nbsp; www.finescience.de</td><td>26016-12</td><td></td></tr><tr><td>Clear tape, 50 mm x 66&amp;nbsp;m</td><td>www.5staroffice.com</td><td>295985</td><td></td></tr><tr><td>FGF4 protein</td><td></td><td></td><td>A gift from Cliff Tabin.</td></tr><tr><td>all-trans-retinoic acid</td><td>Sigma www.sigmaaldrich.com</td><td>R2625-50MG</td><td>Remember to protect from light.</td></tr><tr><td>BMS 493</td><td>Sigma www.sigmaaldrich.com</td><td>B6688-5MG</td><td></td></tr><tr><td>Scalpel blade No. 15</td><td>www.swann-morton.com</td><td>cat no. 0105</td><td></td></tr><tr><td>Affi-Gel Blue, affinity chromatography gel</td><td>Bio-Rad www.bio-rad.com</td><td>153-7301</td><td>Beads for delivering protein.</td></tr><tr><td>AG1-X2 Ion exchange resin</td><td>Bio-Rad www.bio-rad.com</td><td>140-1251</td><td>Beads for delivering RA or BMS 493.</td></tr><tr><td>Incubator for eggs</td><td>Memmert www.hce-uk.com</td><td>LAB128</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma www.sigmaaldrich.com</td><td>P6148-1KG</td><td>Harmful, irritant, corrosive.&amp;nbsp; Handle powder in fumehood with full protective clothing.</td></tr><tr><td>Ethanol</td><td>Sigma www.sigmaaldrich.com</td><td>32221-2.5L</td><td></td></tr><tr><td>DMEM, high glucose</td><td>Gibco www.thermofisher.com</td><td>41965-039</td><td>500 ml</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Fisher Scientific webshop.fishersci.com</td><td>D/4121/PB08</td><td>500 ml</td></tr><tr><td>Binocular Microscope&amp;nbsp;</td><td>Leica www.leica-microsystems.com</td><td>MZ6</td><td>Replaced by M60</td></tr><tr><td>Chicken eggs</td><td>Henry Stewart &amp;amp; Co Ltd sales@medeggs.com</td><td></td><td>Brown Eggs</td></tr></tbody></table>

application of impermeable barriers combined with candidate factor soaked beads to study inductive signals in the chick

The chick embryo provides a superb vertebrate model that can be used to dissect developmental questions in a direct way. Its accessibility and robustness following surgical intervention are key experimental strengths. Mica plates were the first barriers used to prevent chick limb bud initiation1. Protocols that use aluminum foil as an impermeable barrier to wing bud or leg bud induction and or initiation are described. We combine this technique with bead placement lateral to the barrier to exogenously supply candidate endogenous factors that have been blocked by the barrier. The results are analyzed using in situ hybridization of subsequent gene expression. Our main focus is on the role of retinoic acid signaling in the induction and later initiation of the chick embryo fore and hindlimb. We use BMS 493 (an inverse agonist of retinoic acid receptors (RAR)) soaked beads implanted in the lateral plate mesoderm (LPM) to mimic the effect of a barrier placed between the somites (a source of retinoic acid (RA)) and the LPM from which limb buds grow. Modified versions of these protocols could also be used to address other questions on the origin and timing of inductive cues. Provided the region of the chick embryo is accessible at the relevant developmental stage, a barrier could be placed between the two tissues and consequent changes in development studied. Examples may be found in the developing brain, axis extension and in organ development, such as liver or kidney induction.

The protocol detailed above describes the methods employed in Nishimoto et al., 2015. The paper studies limb bud induction and initiation in both the fore and hindlimb of the chick embryo. Results following the insertion of impermeable barriers to prevent the outgrowth of the forelimb bud have been published previously 1, 2, 9 but there has been only one study describing barriers preventing leg bud initiation 1. Hindlimb data from Nishimoto et al., 2015 are presented here as representative results of the study of mRNA expression following barrier insertion.
Tbx4 Expression in the LPM Is Not Sufficient to Initiate Hindlimb Outgrowth: 
Figures 2B and C (Table 2) show that barrier insertion at stage 15 (Figure 2A) (see protocol 3.2) leads to both Fgf10 and Fgf8 expression being absent in the leg bud region (denoted by bracket) on the operated right side. Compare with the unoperated left side. However, Tbx4 expression is observed at both stage 19 and stage 23 on the operated side (Figure 2D and E). We conclude that Tbx4 expression alone is not sufficient to initiate hindlimb outgrowth from the LPM. Other studies in the chick have suggested that Tbx gene expression is sufficient to initiate forelimb bud formation 12, 13.
Figure 2C&#39; is included to illustrate how a barrier looks imbedded in the leg region of the embryo post fixation. In the majority of cases barriers were removed prior to in situ hybridization (protocol 4.3).
RA from the Somites Is Essential in the LPM before Fgf10 Induces Limb Bud Outgrowth: 
Figure 3A is a schematic representing the addition of an RA soaked bead distal to a barrier inserted at stage 15 (protocol 3.2.1.3). Subsequent to this operation limb bud outgrowth (denoted by an asterisk) is observed on the operated right side and Tbx4, Fgf10 and Fgf8 are expressed in the bud as they are in the control left side (Figure 3B, C and D, Table 2). Thus addition of RA to the LPM overcomes the effect of the barrier and limb bud initiation proceeds. We conclude that in normal development RA from the somites is essential in the LPM before limb bud outgrowth can be initiated. The resulting hindlimb buds are generally smaller than those on the control side. This may be due to the RA dose in the LPM being not equivalent to the wild type situation. If the RA dose is too high the Apical Ectodermal Ridge (AER) can be shortened and smaller buds would result 14, 15.
To confirm that RA (from the somites) in the LPM is required for normal limb bud initiation an inverse agonist of RAR, BMS 493, was used. Beads are implanted into the leg region LPM at stage 15 (see protocol 3.2.1.4) (Figure 3E). Fgf10 expression is downregulated following application of BMS 493 beads in the LPM, resulting in smaller hindlimb buds compared to the control side (Figure 3F; Table 3). Control DMSO beads do not cause these defects (Figures 3G and 3H; Table 3). The defects observed following BMS 493 application are milder than those induced by a barrier operation; i.e., Fgf10 is still expressed and limb buds are formed. This is likely to be because the effects of BMS 493 are restricted locally around the beads and may not be able to antagonize all the RA produced by axial tissues.
Early Axial Signals Specify the LPM Cells that Later Express Tbx4: 
We tested when the LPM acquires its ability to express Tbx4 in the prospective leg-forming region. The earliest stage at which a barrier can be inserted that will subsequently block leg bud outgrowth, is stage 10. We are the first to develop a protocol to insert barriers at stages earlier than stage 12. Barriers inserted between the paraxial mesoderm and the presumptive leg LPM at stage 10 (see protocol 3.4) block leg bud formation and the expression of Tbx4 in the leg-forming LPM (Figures 2F and 2G, Table 2), suggesting that a signal from axial tissues at stage 10 is required for later expression of Tbx4 in hindlimb LPM. Compare this result with Figures 2D and E where later barrier insertion allows Tbx4 expression to be established. Furthermore, we tested whether this axial signal could be RA by observing whether the inverse agonist of RAR reduces Tbx4 expression. A BMS 493 bead placed in the hindlimb LPM at stage 10 (see protocol 3.4.1.3) downregulates Tbx4 expression (Figures 2H and 2I), whereas control beads soaked in DMSO do not affect Tbx4 expression (Figures 2J and 2K). Together, these results support a model that an RA signal from axial tissues regulates limb induction by positively regulating Tbx4 in the hindlimb LPM 7.
Post-operative Death: 
Tables 1, 2 and 3 give details of experimental outcomes including the number of chick embryos that died post operation and before harvesting. Some deaths are to be expected after microsurgery of this nature. The numbers dying can be kept to a minimum by making sure that instruments are clean, the hole in the egg is the minimum size required, that the embryos are left without the protection of the sticky tape seal for a minimum time and, in conjunction with this last point that the operation is carried out as quickly as possible. None of these operations are very time consuming but operations involving beads and barriers take a little more time. Despite these measures, operations carried out at the earliest stages and those in the wing region are more likely to result in death. There are large blood vessels around the limb-forming regions and accidental rupture of these vessels may lead to death due to blood loss. We find that eggs opened at stage 8 or 9, whose embryos have not been operated on, will often die by the next day. Therefore, the only solution to these problems is to carry out a high number of experiments.
<img alt="Figure 2" src="/files/ftp_upload/54618/54618fig2.jpg" /> 
Figure 2. Marker Gene Expression Changes following Barrier or Bead Insertion at Presumptive Leg Level. (A) Schematic showing barrier position (green line) at the presumptive leg level (somites 26-32) in a stage 15 embryo. (B-E and G) WISH analysis on operated embryos. The leg region is outlined (brackets). (B) Fgf10 expression is absent in the operated LPM, but robust expression is detected in the left bud. (C) Fgf8 expression is absent in the operated side, suggesting there is no AER. (C&#39;) The same embryo before the barrier was removed. (D) Tbx4 is expressed in the LPM at the same rostro-caudal level as the control bud. (E) Tbx4 expression is maintained in the right leg region despite absence of limb growth. (F) Schematic diagram of a stage 10 chick embryo showing barrier position (green line) at the presumptive leg level. (G) Tbx4 expression is absent in the operated right LPM (bracket). (H) Schematic diagram of a stage 10 chick embryo showing BMS 493 bead position (purple circle) at the presumptive leg level. (I) Tbx4 expression is downregulated on the operated right side following treatment with BMS 493. (J) Schematic diagram of a stage 10 chick embryo showing control DMSO bead position (gray circle). (K) Tbx4 is expressed on the operated right side at a similar level to that of control left side following treatment with DMSO alone. This figure has been modified from 7. <a href="http://cloudflare.jove.com/files/ftp_upload/54618/54618fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54618/54618fig3.jpg" /> 
Figure 3. RA Rescues Limb Bud&#39;s Absence Caused by Barrier Insertion and is Essential in the LPM Prior to Leg Bud Outgrowth. (A) Schematic diagram indicating barrier position (green line) at the presumptive leg level (somites 26-32) and an RA-soaked bead (red circle). (B-D) RA rescues leg bud outgrowth (shown by an asterisk). (B) Tbx4 expression is present in the rescued leg bud similar to the un-operated side. (C) Fgf10 is expressed in the rescued leg bud similar to the un-operated side. (D) Fgf8 is expressed in the AER of the rescued right leg bud. (E) Schematic indicating where BMS 493 beads (purple circles) were placed in the presumptive leg LPM of stage 15 embryos. (F) Fgf10 expression is downregulated on the operated right side and the leg bud is small following BMS 493 treatment. Beads are asterisked. (G) Similar schematic to (E), indicating the position of control DMSO beads (gray circles). (H) DMSO beads (shown by asterisks) did not affect Fgf10 expression or leg bud size. This figure has been modified from 7. <a href="http://cloudflare.jove.com/files/ftp_upload/54618/54618fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Stage barrier placed (+ bead) *</td>
			<td>Number operated</td>
			<td>Wing missing **</td>
			<td>Wing present **</td>
			<td>Number dead</td>
		</tr>
		<tr>
			<td>Stage 12-14&#160;&#160;</td>
			<td>40</td>
			<td>24</td>
			<td>1</td>
			<td>15</td>
		</tr>
		<tr>
			<td>Stage 8/9</td>
			<td>65</td>
			<td>23</td>
			<td>0</td>
			<td>42</td>
		</tr>
		<tr>
			<td>Stage 12-13 (FGF4 bead)</td>
			<td>41</td>
			<td>7 (bead missing)</td>
			<td>17</td>
			<td>17</td>
		</tr>
		<tr>
			<td>Stage 12-13 (RA bead)</td>
			<td>75</td>
			<td>9 (bead missing)</td>
			<td>32</td>
			<td>34</td>
		</tr>
		<tr>
			<td>Stage 13 (control bead)</td>
			<td>5</td>
			<td>3</td>
			<td>0</td>
			<td>2</td>
		</tr>
		<tr>
			<td colspan="5">* 0.7-1 mm foil barriers placed between somites 15-20 and the lateral plate mesoderm. 0.35 mg/ml FGF4 bead. 0.05-0.1 mg/ml RA bead.</td>
		</tr>
		<tr>
			<td colspan="5">DMSO control bead.</td>
		</tr>
		<tr>
			<td colspan="5">** Chick embryos fixed stages 21-23</td>
		</tr>
	</tbody>
</table>
Table 1. Wing Level Barrier and Bead Experiment Outcomes.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Stage barrier placed (+ bead) *</td>
			<td>Number operated</td>
			<td>Leg missing **</td>
			<td>Leg present **</td>
			<td>Number dead</td>
		</tr>
		<tr>
			<td>Stage 12-15</td>
			<td>43</td>
			<td>39</td>
			<td>0</td>
			<td>4</td>
		</tr>
		<tr>
			<td>Stage 10/11</td>
			<td>23</td>
			<td>16</td>
			<td>0</td>
			<td>7</td>
		</tr>
		<tr>
			<td>Stage 15 (FGF4 bead)</td>
			<td>8</td>
			<td>0</td>
			<td>5</td>
			<td>3</td>
		</tr>
		<tr>
			<td>Stage 15 (RA bead)</td>
			<td>18</td>
			<td>0</td>
			<td>18</td>
			<td>0</td>
		</tr>
		<tr>
			<td colspan="5">* 0.7-1.3 mm foil barriers placed between somites 26-32 and the lateral plate mesoderm. 0.35 mg/ml FGF4 bead. 0.05-0.1 mg/ml RA bead.</td>
		</tr>
		<tr>
			<td colspan="5">** Chick embryos fixed stages 18-24</td>
		</tr>
	</tbody>
</table>
Table 2. Leg Level Barrier and Bead Experiment Outcomes.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Number operated</td>
			<td>Leg bud small</td>
			<td>Leg bud slightly small</td>
			<td>Leg bud normal size</td>
			<td>Number dead</td>
		</tr>
		<tr>
			<td>BMS 493 beads *</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15</td>
			<td>12</td>
			<td>3</td>
			<td>0</td>
			<td>0</td>
		</tr>
		<tr>
			<td>DMSO beads **</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>12</td>
			<td>0</td>
			<td>3</td>
			<td>9</td>
			<td>0</td>
		</tr>
		<tr>
			<td colspan="5">* 2-3 beads soaked in 5 mg/ml BMS 493 placed in the right stage 14-15 LPM at leg level.</td>
		</tr>
		<tr>
			<td colspan="5">** 2-3 beads soaked in DMSO alone placed in the right stage 15 LPM at leg level.</td>
		</tr>
		<tr>
			<td colspan="5">Chick embryos fixed at stages 17-19.</td>
		</tr>
	</tbody>
</table>
Table 3. Leg Level BMS 493 Bead Experiment Outcomes.

Watch this Scientific Journal Video about Application of Impermeable Barriers Combined with Candidate Factor Soaked Beads to Study Inductive Signals in the Chick at JoVE.com

Application of Impermeable Barriers Combined with Candidate Factor Soaked Beads to Study Inductive Signals in the Chick

Protocols using impermeable barriers to block induction events between tissues of the main body axis and flank in the chick embryo required for limb formation are described. Beads soaked in candidate inductive signals are used to overcome the effect of barrier placement and analysis of gene expression confirms this.

The chick embryo provides a superb vertebrate model that can be used to dissect developmental questions in a direct way. Its accessibility and robustness ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

9.3K Views.  King's College London, Guy's Campus. Protocols using impermeable barriers to block induction events between tissues of the main body axis and flank in the chick embryo required for limb formation are described. Beads soaked in candidate inductive signals are used to overcome the effect of barrier placement and analysis of gene expression confirms this.

Video: Application of Impermeable Barriers Combined with Candidate Factor Soaked Beads to Study Inductive Signals in the Chick

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos model organisms, such as the chicken, are often used. The chicken is an excellent model organism due to its low cost and minimal maintenance, however they present observational challenges because they are enclosed in an opaque eggshell. In order to properly view the embryo as it develops, the shell must be windowed or removed. Both windowing and ex ovo techniques have been developed to assist researchers in the study of embryonic development. However, each of the methods has limitations and challenges. Here, we present a simple, optimized ex ovo culture technique for chicken embryos that enables the observation of embryonic development from stage HH 19 into late stages of development (HH 40), when many organs have developed. This technique is easy to adopt in both undergraduate classes and more advanced research laboratories where embryo manipulations are conducted.

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Viewing and accessing the chicken embryo during development can be challenging. We have developed an ex ovo method that is simple, cost effective, and can easily be used in a classroom or research setting. This method provides access to the embryo into late stages of embryonic development (HH 40).

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos ...

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.

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 ...

Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos

The neural tube expresses many proteins in specific spatiotemporal patterns during development. These proteins have been shown to be critical for cell fate determination, cell migration, and formation of neural circuits. Neuronal induction and patterning involve bone morphogenetic protein (BMP), sonic hedgehog (SHH), fibroblast growth factor (FGF), among others. In particular, the expression pattern of Fgf8 is in close proximity to regions expressing BMP4 and SHH. This expression pattern is consistent with developmental interactions that facilitate patterning in the telencephalon.

Here we provide a visual demonstration of a method in which an in ovo preparation can be used to test the effects of Fgfs in the formation of the forebrain. Beads are coated with protein and placed in the developing neural tube to provide sustained exposure. Because the procedure uses small, carefully placed beads, it is minimally invasive and allows several beads to be placed within a single neural tube. Moreover, the method allows for continued development so that embryos can be analyzed at a more mature stage to detect changes in anatomy and in neural patterning. This simple but useful protocol allows for real time imaging. It provides a means to make spatially and temporally limited changes to endogenous protein levels.

A variety of growth factors and proteins interact to induce cells to take on different cell fates during development. Here we demonstrate the use of an in ovo preparation to address possible interactions between different proteins in development by placing beads on E2.5 chick embryos.

The neural tube expresses many proteins in specific spatiotemporal patterns during development.   These proteins have been shown to be critical for cell ...

Biology

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of morphogenetic events, such as gastrulation2, neurulation3-5, somitogenesis6, heart bending7,8, and brain formation9-13, during early embryogenesis. Key to understanding morphogenetic processes is to follow them dynamically by time-lapse imaging. The acquisition of time-lapse movies of chick embryogenesis ex ovo has been limited either to short time windows or to the need for an incubator to control temperature and humidity around the embryo14. Here, we present a new technique to culture chick embryos ex ovo for high-resolution time-lapse imaging using transmitted light microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique (EC-culture)1 and allows for the culturing of chick embryos without the need for a climate chamber. The embryo is sandwiched between two identical filter paper carriers and is kept fully submerged in a simple, temperature-controlled medium covered by a layer of light mineral oil. Starting from the primitive streak stage (Hamburger-Hamilton stage 5, HH5)15 up to at least the 28-somite stage (HH16)15, embryos can be cultured with either their ventral or dorsal side up. This allows the acquisition of time-lapse movies covering about 30 hr of embryonic development. Representative time-lapse frames and movies are shown. Embryos are compared morphologically to an embryo cultured in the standard EC-culture.
The submerged filter paper sandwich provides a stable environment to study early dorsal and ventral morphogenetic processes. It also allows for live fluorescence imaging and micromanipulations, such as microsurgery, bead implantation, microinjection, gene silencing, and electroporation, and has a strong potential to be combined with immersion objectives for laser-based imaging (including light-sheet microscopy).

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Here, we present a new technique to culture chick embryos ex ovo for time-lapse imaging using transmitted light and fluorescence microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique1 that allows for the culturing of chick embryos fully-submerged in a liquid culture medium.

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of ...

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Electroporation of the chick embryonic neural tube has many advantages such as being quick and efficient for the expression of foreign genes into neuronal cells. In this manuscript we provide a method that demonstrates uniquely how to electroporate DNA into the avian hindbrain at E2.75 in order to specifically label a subset of neuronal progenitors, and how to follow their axonal projections and synaptic targets at much advanced stages of development, up to E14.5. We have utilized novel genetic tools including specific enhancer elements, Cre/Lox - based plasmids and the PiggyBac-mediated DNA transposition system to drive GFP expression in a subtype of hindbrain cells (the dorsal most subgroup of interneurons, dA1). Axonal trajectories and targets of dA1 axons are followed at early and late embryonic stages at various brainstem regions. This strategy contributes advanced techniques for targeting cells of interest in the embryonic hindbrain and for tracing circuit formation at multiple stages of development.

How neuronal networks are established in the embryonic brain is a fundamental question in developmental neurobiology. Here we combined an electroporation technique with novel genetic tools, such as Cre/Lox&#x2013;plasmids and PiggyBac-mediated DNA transposition system in the avian hindbrain to label dorsal interneurons and track their axonal projections and synaptic targets at various developmental stages.

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

Neuroscience

Analysis of Cell Differentiation, Morphogenesis, and Patterning During Chicken Embryogenesis Using the Soaked-Bead Assay

A multitude of genetic programs is activated during embryonic development that orchestrates cell differentiation to generate an astounding diversity of somatic cells, tissues, and organs. The precise activation of these genetic programs is regulated by morphogens, diffusible molecules that direct cell fate at different thresholds. Understanding how genetic activation coordinates morphogenesis requires the study of local interactions triggered by morphogens during development. The use of beads soaked in proteins or drugs implanted into distinct regions of the embryo enables studying the role of specific molecules in the establishment of digits and other developmental processes. This experimental technique provides information on the control of cell induction, cell fate, and pattern formation. Thus, this soaked bead assay is an extremely powerful and valuable experimental tool applicable to other embryonic models.

The soaked bead assay involves targeted delivery of test reagent at any developmental time point to study the regulation of cell differentiation and morphogenesis. A detailed protocol, applicable to any experimental animal model, for preparing three different types of soaked beads and implanting these in the interdigit of a chicken embryo is presented.

A multitude of genetic programs is activated during embryonic development that orchestrates cell differentiation to generate an astounding diversity of ...

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as the hair cell. Basic research in the inner ear has led to a deep understanding of how the hair cell functions, and how dysregulation can lead to hearing loss and vertigo. For this research, the mouse has been the pre-eminent model system. However, mice, like all mammals, have lost the ability to replace hair cells. Thus, when trying to understand cellular therapies for restoring inner ear function, complementary studies in other vertebrate species could provide further insights. The auditory epithelium of birds, the basilar papilla (BP), is a sheet of epithelium composed of mechanosensory hair cells (HCs) intercalated by supporting cells (SCs). Although the anatomical architecture of the basilar papilla and the mammalian cochlea differ, the molecular mechanisms of inner ear development and hearing are similar. This makes the basilar papilla a useful system for not only comparative studies but also to understand regeneration. Here, we describe dissection and manipulation techniques for the chicken inner ear. The technique shows genetic and small molecule inhibition methods, which offer a potent tool for studying the molecular mechanisms of inner ear development. In this paper, we discuss in ovo electroporation techniques to genetically perturb the basilar papilla using CRIPSR-Cas9 deletions, followed by dissection of the basilar papilla. We also demonstrate the BP organ culture and optimal use of culture matrices, to observe the development of the epithelium and the hair cells.

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The chick is a cost-effective, accessible, and widely available model organism for a variety of studies. Here, a series of protocols is detailed to understand the molecular mechanisms underlying avian inner ear development and regeneration.

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as ...

Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue interactions that regulate patterning and morphogenesis of vertebrates (e.g., 1, 2, 3, 4). Here, we illustrate a method that exploits this accessibility to test the signaling and patterning properties of ectodermal tissues during facial development. In these experiments, we create quail-chick 5 or mouse-chick 6 chimeras by transplanting the surface cephalic ectoderm that covers the upper jaw from quail or mouse onto either the same region or an ectopic region of chick embryos. The use of quail as donor tissue for transplantation into chicks was developed to take advantage of a nucleolar marker present in quail but not chick cells, thus allowing investigators to distinguish host and donor tissues 7. Similarly, a repetitive element is present in the mouse genome and is expressed ubiquitously, which allows us to distinguish host and donor tissues in mouse-chick chimeras 8. The use of mouse ectoderm as donor tissue will greatly extend our understanding of these tissue interactions, because this will allow us to test the signaling properties of ectoderm derived from various mutant embryos.

This article describes a tissue transplantation technique that was designed to test the signaling and patterning properties of surface cephalic ectoderm during craniofacial development.

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue ...

Evaluation of Auditory Brainstem Response in Chicken Hatchlings

The auditory brainstem response (ABR) is an invaluable assay in clinical audiology, non-human animals, and human research. Despite the widespread use of ABRs in measuring auditory neural synchrony and estimating hearing sensitivity in other vertebrate model systems, methods for recording ABRs in the chicken have not been reported in nearly four decades. Chickens provide a robust animal research model because their auditory system is near functional maturation during late embryonic and early hatchling stages. We have demonstrated methods used to elicit one or two-channel ABR recordings using subdermal needle electrode arrays in chicken hatchlings. Regardless of electrode recording configuration (i.e., montage), ABR recordings included 3-4 positive-going peak waveforms within the first 6 ms of a suprathreshold click stimulus. Peak-to-trough waveform amplitudes ranged from 2-11 &#181;V at high-intensity levels, with positive peaks exhibiting expected latency-intensity functions (i.e., increase in latency as a function of decreased intensity). Standardized earphone position was critical for optimal recordings as loose skin can occlude the ear canal, and animal movement can dislodge the stimulus transducer. Peak amplitudes were smaller, and latencies were longer as animal body temperature lowered, supporting the need for maintaining physiological body temperature. For young hatchlings (&#60;3 h post-hatch day 1), thresholds were elevated by ~5 dB, peak latencies increased ~1-2 ms, and peak to trough amplitudes were decreased ~1 &#181;V compared to older hatchlings. This suggests a potential conductive-related issue (i.e., fluid in the middle ear cavity) and should be considered for young hatchlings. Overall, the ABR methods outlined here permit accurate and reproducible recording of in-vivo auditory function in chicken hatchlings that could be applied to different stages of development. Such findings are easily compared to human and mammalian models of hearing loss, aging, or other auditory-related manipulations.

We have used standard auditory brainstem response (ABR) techniques and applied them to hatchling chickens, a precocious avian model for auditory function. The protocol outlines animal preparation and ABR acquisition techniques in detail, with steps that could translate to other avian or rodent models.

The auditory brainstem response (ABR) is an invaluable assay in clinical audiology, non-human animals, and human research. Despite the widespread use of ...

Application of Impermeable Barriers Combined with Candidate Factor Soaked Beads to Study Inductive Signals in the Chick

Summary

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Chapters in this video

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

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

Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Assay for Neural Induction in the Chick Embryo

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Analysis of Cell Differentiation, Morphogenesis, and Patterning During Chicken Embryogenesis Using the Soaked-Bead Assay

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development

Evaluation of Auditory Brainstem Response in Chicken Hatchlings