This work was funded by MRC grant MC.PC_13052.

<ol>
	<li>Murillo-Ferrol, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Causal+study+of+the+earliest+differentiation+of+the+morphological+rudiments+of+the+extremities.+Experimental+analysis+on+bird+embryos.">Causal study of the earliest differentiation of the morphological rudiments of the extremities. Experimental analysis on bird embryos.</a> Acta Anat (Basel). 62 (1), 80-103 (1965).</li><li>Sweeney, R. M., Watterson, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rib+development+in+chick+embryos+analyzed+by+means+of+tantalum+foil+blocks).">Rib development in chick embryos analyzed by means of tantalum foil blocks).</a> Am J Anat. 126 (2), 127-149 (1969).</li><li>Kieny, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+relations+between+somatic+and+somatopleural+mesoderm+before+and+during+primary+induction+of+chick+embryo+limbs.">On the relations between somatic and somatopleural mesoderm before and during primary induction of chick embryo limbs.</a> C R Acad Sci Hebd Seances Acad Sci D. 268 (26), 3183-3186 (1969).</li><li>Pinot, 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+role+of+the+somitic+mesoderm+in+the+early+morphogenesis+of+the+limbs+in+the+fowl+embryo.">The role of the somitic mesoderm in the early morphogenesis of the limbs in the fowl embryo.</a> J Embryol Exp Morphol. 23 (1), 109-151 (1970).</li><li>Eichele, G., Tickle, C., Alberts, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcontrolled+release+of+biologically+active+compounds+in+chick+embryos:+beads+of+200-microns+diameter+for+the+local+release+of+retinoids.">Microcontrolled release of biologically active compounds in chick embryos: beads of 200-microns diameter for the local release of retinoids.</a> Anal Biochem. 142 (2), 542-555 (1984).</li><li>Cohn, M. 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=Fibroblast+growth+factors+induce+additional+limb+development+from+the+flank+of+chick+embryos.">Fibroblast growth factors induce additional limb development from the flank of chick embryos.</a> Cell. 80 (5), 739-746 (1995).</li><li>Nishimoto, S., 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=RA+Acts+in+a+Coherent+Feed-Forward+Mechanism+with+Tbx5+to+Control+Limb+Bud+Induction+and+Initiation.">RA Acts in a Coherent Feed-Forward Mechanism with Tbx5 to Control Limb Bud Induction and Initiation.</a> Cell Rep. 12 (5), 879-891 (2015).</li><li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.">A series of normal stages in the development of the chick embryo.</a> J Morphol. 88 (1), 49-92 (1951).</li><li>Stephens, T. D., McNulty, T. R. <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+metameric+pattern+in+the+development+of+the+chick+humerus.">Evidence for a metameric pattern in the development of the chick humerus.</a> J Embryol Exp Morphol. 61, 191-205 (1981).</li><li>Strecker, T. R., Stephens, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+nerves+do+not+play+a+trophic+role+in+limb+skeletal+morphogenesis.">Peripheral nerves do not play a trophic role in limb skeletal morphogenesis.</a> Teratology. 27 (2), 159-167 (1983).</li><li>Riddle, R. 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=Sonic+hedgehog+mediates+the+polarizing+activity+of+the+ZPA.">Sonic hedgehog mediates the polarizing activity of the ZPA.</a> Cell. 75 (7), 1401-1416 (1993).</li><li>Ng, J. K., 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=The+limb+identity+gene+Tbx5+promotes+limb+initiation+by+interacting+with+Wnt2b+and+Fgf10.">The limb identity gene Tbx5 promotes limb initiation by interacting with Wnt2b and Fgf10.</a> Devel. 129 (22), 5161-5170 (2002).</li><li>Takeuchi, J. K., 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=Tbx5+and+Tbx4+trigger+limb+initiation+through+activation+of+the+Wnt/Fgf+signaling+cascade.">Tbx5 and Tbx4 trigger limb initiation through activation of the Wnt/Fgf signaling cascade.</a> Devel. 130 (12), 2729-2739 (2003).</li><li>Lee, J., 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=Retinoic+acid+and+pattern+formation+in+the+developing+chick+wing:+SEM+and+quantitative+studies+of+early+effects+on+the+apical+ectodermal+ridge+and+bud+outgrowth.">Retinoic acid and pattern formation in the developing chick wing: SEM and quantitative studies of early effects on the apical ectodermal ridge and bud outgrowth.</a> J Embryol Exp Morphol. 90, 139-169 (1985).</li><li>Tickle, C., Crawley, A., Farrar, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+application+to+chick+wing+buds+leads+to+a+dose-dependent+reorganization+of+the+apical+ectodermal+ridge+that+is+mediated+by+the+mesenchyme.">Retinoic acid application to chick wing buds leads to a dose-dependent reorganization of the apical ectodermal ridge that is mediated by the mesenchyme.</a> Devel. 106 (4), 691-705 (1989).</li><li>MacCabe, J. 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. E., 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=Retinoids+reprogramme+pre-bud+mesenchyme+to+give+changes+in+limb+pattern.">Retinoids reprogramme pre-bud mesenchyme to give changes in limb pattern.</a> Devel. 100 (4), 723-733 (1987).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" width="926">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Dumont No.5 watchmakers forceps, x2&#160;</td>
			<td style="width:277px;">Fine Science Tools&#160; www.finescience.de</td>
			<td style="width:149px;">11251-20</td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont No.4 watchmakers forceps, x2&#160;&#160;&#160;</td>
			<td>Fine Science Tools&#160; www.finescience.de</td>
			<td>11241-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont strong forceps, type AA&#160;&#160;</td>
			<td>Fine Science Tools&#160; www.finescience.de</td>
			<td>11210-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Curved scissors with blades 2.5 cm long</td>
			<td>Fine Science Tools&#160; www.finescience.de</td>
			<td>14061-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blunt ended curved forceps,&#160;</td>
			<td>Fine Science Tools&#160; www.finescience.de</td>
			<td>11065-07</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ends approx 0.5 cm long</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Steel pin, 0.2 mm wide</td>
			<td>Fine Science Tools&#160; www.finescience.de</td>
			<td>26002-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sharpening stone, square</td>
			<td>Fine Science Tools&#160; www.finescience.de</td>
			<td>29008-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mineral oil</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Needle holder</td>
			<td>Fine Science Tools&#160; www.finescience.de</td>
			<td>26016-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clear tape, 50 mm x 66 m</td>
			<td>www.5staroffice.com</td>
			<td align="right">295985</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FGF4 protein</td>
			<td></td>
			<td></td>
			<td>A gift from Cliff Tabin.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">all-trans-retinoic acid</td>
			<td>Sigma&#160; www.sigmaaldrich.com</td>
			<td>R2625-50MG</td>
			<td>Remember to protect from light.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BMS 493</td>
			<td>Sigma&#160; www.sigmaaldrich.com</td>
			<td>B6688-5MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpel blade No. 15</td>
			<td>www.swann-morton.com</td>
			<td>cat no. 0105</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Affi-Gel Blue, affinity chromatography gel</td>
			<td>Bio-Rad&#160; www.bio-rad.com</td>
			<td>153-7301</td>
			<td>Beads for delivering protein.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AG1-X2 Ion exchange resin</td>
			<td>Bio-Rad&#160; www.bio-rad.com</td>
			<td>140-1251</td>
			<td>Beads for delivering RA or BMS 493.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator for eggs</td>
			<td>Memmert&#160; www.hce-uk.com</td>
			<td>LAB128</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde (PFA)</td>
			<td>Sigma&#160; www.sigmaaldrich.com</td>
			<td>P6148-1KG</td>
			<td>Harmful, irritant, corrosive.&#160; Handle powder in fumehood with full protective clothing.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol</td>
			<td>Sigma&#160; www.sigmaaldrich.com</td>
			<td>32221-2.5L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM, high glucose</td>
			<td>Gibco&#160; www.thermofisher.com</td>
			<td>41965-039</td>
			<td>500ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dimethyl sulfoxide (DMSO)</td>
			<td>Fisher Scientific&#160; webshop.fishersci.com</td>
			<td>D/4121/PB08</td>
			<td>500ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Binocular Microscope&#160;</td>
			<td>Leica&#160;&#160; www.leica-microsystems.com</td>
			<td>MZ6</td>
			<td>Replaced by M60</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chicken eggs</td>
			<td>Henry Stewart &#38; Co Ltd&#160;&#160; 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="https://cloudflare-jove-com.remotexs.ntu.edu.sg/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="https://cloudflare-jove-com.remotexs.ntu.edu.sg/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 ...

application-impermeable-barriers-combined-with-candidate-factor

Nanyang Technological University

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

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice &#8212; A High-throughput Screen in ES Cells

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely limits its potential. We describe here the use of mouse embryonic stem (ES) cells as surrogate cells to screen for a phenotype of interest and subsequently introduce these cells into a host embryo to develop into a living mouse carrying the phenotype. This method provides (1) a cost effective, high-throughput platform for genetic screen in mammalian cells; (2) a rapid way to identify the mutated genes and verify causality; and (3) a short-cut to develop mouse mutants directly from these selected ES cells for whole animal studies. We demonstrated the use of paraquat (PQ) to select resistant mutants and identify mutations that confer oxidative stress resistance. Other stressors or cytotoxic compounds may also be used to screen for resistant mutants to uncover novel genetic determinants of a variety of cellular stress resistance.

Stress resistance is one of the hallmarks for longevity and is known to be genetically governed. Here, we developed an unbiased high-throughput method to screen for mutations that confer stress resistance in ES cells with which to develop mouse models for longevity studies.

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To investigate the underlying mechanisms that bring about various muscle adaptations, a number of exercise protocols such as treadmill, wheel running, and swimming exercise have been used in the animal studies. However, these exercise models require a long period of time to achieve muscle adaptations, which may be also regulated by humoral or neurological factors, thus limiting their applications in studying the muscle-specific contraction-induced adaptations. Indirect low frequency stimulation (10 Hz) to induce chronic contractile activity (CCA) has been used as an alternative model for exercise training, as it can successfully lead to muscle mitochondrial adaptations within 7 days, independent of systemic factors. This paper details the surgical techniques required to apply the treatment of CCA to the skeletal muscle of rats, for widespread application in future studies.

This protocol describes the use of the chronic contractile activity model of exercise to observe stimulation-induced skeletal muscle adaptations in the rat hindlimb.

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the Notch receptor with ligands from neighboring cells induces activation of the signaling pathway (trans-activation), while interaction with ligands from the same cell inhibits signaling (cis-inhibition). Proper balance between trans-activation and cis-inhibition helps establish optimal levels of Notch signaling in some contexts during animal development. Because of the overlapping expression domains of Notch and its ligands in many cell types and the existence of feedback mechanisms, studying the effects of a given post-translational modification on trans- versus cis-interactions of Notch and its ligands in vivo is difficult. Here, we describe a protocol for using Drosophila S2 cells in cell-aggregation assays to assess the effects of knocking down a Notch pathway modifier on the binding of Notch to each ligand in trans and in cis. S2 cells stably or transiently transfected with a Notch-expressing vector are mixed with cells expressing each Notch ligand (S2-Delta or S2-Serrate). Trans-binding between the receptor and ligands results in the formation of heterotypic cell aggregates and is measured in terms of the number of aggregates per mL composed of &#62;6 cells. To examine the inhibitory effect of cis-ligands, S2 cells co-expressing Notch and each ligand are mixed with S2-Delta or S2-Serrate cells and the number of aggregates is quantified as described above. The relative decrease in the number of aggregates due to the presence of cis-ligands provides a measure of cis-ligand-mediated inhibition of trans-binding. These straightforward assays can provide semi-quantitative data on the effects of genetic or pharmacological manipulations on the binding of Notch to its ligands, and can help deciphering the molecular mechanisms underlying the in vivo effects of such manipulations on Notch signaling.

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Complexity of in vivo systems makes it difficult to distinguish between the activation and inhibition of Notch receptor by trans- and cis-ligands, respectively. Here, we present a protocol based on in vitro cell-aggregation assays for qualitative and semi-quantitative evaluation of the binding of Drosophila Notch to trans-ligands vs cis-ligands.

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that makes up much of the parenchyma of the human placenta. The distal villous capillary network is the terminus of the fetal blood supply after several generations of branching of vessels extending out from the umbilical cord. This network has a contiguous cellular sheath, the syncytial trophoblast barrier layer, which prevents mixing of fetal blood and the maternal blood in which it is continuously bathed. Insults to the integrity of the placental capillary network, occurring in disorders such as maternal diabetes, hypertension and obesity, have consequences that present serious health risks for the fetus, infant, and adult. To better define the structural effects of these insults, a protocol was developed for this study that captures capillary network structure on the order of 1 - 2 mm3 wherein one can investigate its topological features in its full complexity. To accomplish this, clusters of terminal villi from placenta are dissected, and the trophoblast layer and the capillary endothelia are immunolabeled. These samples are then clarified with a new tissue clearing process which makes it possible to acquire confocal image stacks to z- depths of ~1 mm. The three-dimensional renderings of these stacks are then processed and analyzed to generate basic capillary network measures such as volume, number of capillary branches, and capillary branch end points, as validation of the suitability of this approach for capillary network characterization.

This study presents a protocol for the reversible tissue clearing, immunostaining, 3D-rendering and analysis of vascular networks in human placenta villi samples on the order of 1 - 2 mm3.

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that ...

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human developmental processes. Stepwise directed differentiation that recapitulates developmental processes is one of the major ways to generate pancreatic cells including pancreas/duodenum homeobox protein 1+ (PDX1+) pancreatic progenitor cells. Conventional protocols initiate the differentiation with small colonies shortly after the passage. However, in the state of colonies or aggregates, cells are prone to heterogeneities, which might hamper the differentiation to PDX1+ cells. Here, we present a detailed protocol to differentiate hPSCs into PDX1+ cells. The protocol consists of four steps and initiates the differentiation by seeding dissociated single cells. The induction of SOX17+ definitive endoderm cells was followed by the expression of two primitive gut tube markers, HNF1&#946; and HNF4&#945;, and eventual differentiation into PDX1+ cells. The present protocol provides easy handling and may improve and stabilize the differentiation efficiency of some hPSC lines that were previously found to differentiate inefficiently into endodermal lineages or PDX1+ cells.

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Here, we present a detailed protocol to differentiate human pluripotent stem cells (hPSCs) into pancreas/duodenum homeobox protein 1+ (PDX1+) cells for the generation of pancreatic lineages based on the non-colony type monolayer growth of dissociated single cells. This method is suitable for producing homogenous hPSC-derived cells, genetic manipulation and screening.

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human ...

Visualization of Tangential Cell Migration in the Developing Chick Optic Tectum

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture can be recorded under video microscopy. For analyzing cell migration in the developing brain, slice culture is commonly used to observe cell migration parallel to the slice section, such as radial cell migration. However, limited information can be obtained from the slice culture method to analyze cell migration perpendicular to the slice section, such as tangential cell migration. Here, we present the protocols for time-lapse imaging to visualize tangential cell migration in the developing chick optic tectum. A combination of cell labeling by electroporation in ovo and a subsequent flat-mount culture on the cell culture insert enables detection of migrating cell movement in the horizontal plane. Moreover, our method facilitates detection of both individual cell behavior and the collective action of a group of cells in the long term. This method can potentially be applied to detect the sequential change of the fluorescent-labeled micro-structure, including the axonal elongation in the neural tissue or cell displacement in the non-neural tissue.

We describe the methods for fluorescent labeling of tangentially migrating cells by electroporation, and for time-lapse imaging of the labeled cell movement in a flat-mount culture in order to visualize migrating cell behavior in the developing chick optic tectum.

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

In Vitro Reconstitution of Spatial Cell Contact Patterns with Isolated Caenorhabditis elegans Embryo Blastomeres and Adhesive Polystyrene Beads

In multicellular systems, individual cells are surrounded by the various physical and chemical cues coming from neighboring cells and the environment. This tissue complexity confounds the identification of causal link between extrinsic cues and cellular dynamics. A synthetically reconstituted multicellular system overcomes this problem by enabling researchers to test for a specific cue while eliminating others. Here, we present a method to reconstitute cell contact patterns with isolated Caenorhabditis elegans blastomere and adhesive polystyrene beads. The procedures involve eggshell removal, blastomere isolation by disrupting cell-cell adhesion, preparation of adhesive polystyrene beads, and reconstitution of cell-cell or cell-bead contact. Finally, we present the application of this method to investigate the orientation of cellular division axes that contributes to the regulation of spatial cellular patterning and cell fate specification in developing embryos. This robust, reproducible, and versatile in vitro method enables the study of direct relationships between spatial cell contact patterns and cellular responses.

In Vitro Reconstitution of Spatial Cell Contact Patterns with Isolated Caenorhabditis elegans Embryo Blastomeres and Adhesive Polystyrene Beads

Tissue complexities of multicellular systems confound the identification of causal relationship between extracellular cues and individual cellular behaviors. Here, we present a method to study the direct link between contact-dependent cues and division axes using C. elegans embryo blastomeres and adhesive polystyrene beads.

In multicellular systems, individual cells are surrounded by the various physical and chemical cues coming from neighboring cells and the environment. ...