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.

クラシック胎生学者は伝統的に胚発生を制御するメカニズムを調べるために物理的技術を採用します。 1960年代と1970年代に研究者らは、胚形成の間、誘導信号の重要性を実証するために、発生中の胚の組織の間に挿入された不透過性の障壁を使用する技術を開発しました。私たちの関心は肢芽の伸長の前にどのようなイベント脊椎動物の四肢の開発に、特にあります。例えば、バリアの挿入は、対、無操作側に正常に進行させながら、ニワトリ胚の片側に発生肢芽の成長を防止することができます。これらの障壁が変化するために使用される材料。例えば、マイカ板1とタンタル箔2。これらの障壁は、効果的に、近軸中胚葉から形成された体節から、から肢芽の形態、側板中胚葉（LPM）を分離します。外科的移植する技術は、その密接な関係のウィットを実証します肢芽の開始はLPM 3 4で発生することがある場合は時間体節が不可欠です。 80年代と90年代に発達生物学者は、発生過程の制御に不可欠である拡散性シグナル伝達分子を発見しました。これらのシグナル伝達分子に浸したビーズは、胚の特定の領域に移植し、胚発生に変化を生み出すことができます。ニワトリ胚に注入し、数字5の鏡像複製を生成することができる場合、例えば、イオン交換ビーズによって取り込まレチノイン酸（RA）は、24時間にわたって放出されます。 FGFタンパク質を含有するヘパリンアクリルビーズは、肢のLPM 6から肢芽の伸長を開始することができます。 さらに最近では、マウスや魚の遺伝学は、（7件）新たな時代に、脊椎動物の四肢開始の研究をとっています。肢芽の開始に先立って体節でRAの存在は、LPによって必要不可欠拡散信号であることを十分な証拠がありますMは、四肢の出芽を開始します。私たちは、肢芽開始の頃LPMにおける遺伝子発現に対する障壁注入の効果を分析するためにニワトリ胚のアクセシビリティと実験的な堅牢性を悪用したかったです。本手法の新規適応はバリアによってブロックされた外因的に供給候補内因性因子に対する障壁にバリアをビーズの配置、横方向と軸方向の組織からの遮断信号を組み合わせることです。次のプロトコルは、肢芽の誘導および開始のメカニズムを引き出すために開発されたものです。 肢芽の開始を研究することは、早期段階の胚を使用することを意味します。最初の皮下注射針で気嚢を通してアルブミンの一部を除去することにより、多くの研究者ウィンドウの鶏の卵。卵黄の上に位置する胚は、その後、卵の下位に位置します。これは、エレクトロポレーションのようないくつかの技術のために有利であるが、胚の外科的処置が所望される場合に不利であることができます。我々は可能な限り卵の開口部に近いが利点であるとして、胚を持つ見つけます。 質問は、脊椎動物の肢芽の誘導と開始を制御する信号についてのままであり、以下のプロトコルは、それらに対処するのに適しています。私たちは、早い段階12 8より段階でニワトリ肢芽の伸長を防止するための障壁を挿入するためのプロトコルを開発する最初のものです。これらのプロトコルの修正版もアクセス可能な誘導組織が誘導されている原基に隣接している誘導性手がかりの起源とタイミング上の他の質問に対処するために使用することができました。胚の領域は、関連する発達段階でのアクセスが設けられて、その後、バリアは2つの組織の間に配置することができ、開発における必然的な変化を研究しました。例としては、脳の発達、軸延長や、肝臓や腎臓の誘導のような可能性の臓器の開発に見ることができます。

<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

ニワトリ胚は、直接的な方法で発達質問を分析するために使用することができる優れた脊椎動物のモデルを提供します。外科的介入後のアクセシビリティと堅牢性が重要な実験的な強みです。マイカ板は、ニワトリ肢芽開始1を防止するために使用される最初の障壁でした。翼の芽や脚の芽の誘導およびまたは開始に不透過性障壁としてアルミ箔を使用するプロトコルが記載されています。我々は、バリアによってブロックされた外因的に供給候補内因性因子への障壁にビーズ配置横でこの技術を兼ね備えています。結果は、その後の遺伝子発現のインサイチュハイブリダイゼーションを用いて分析します。私たちの主な焦点は、誘導にレチノイン酸シグナル伝達およびニワトリ胚の前部と後肢の後に開始の役割にあります。私たちは、BMS 493（レチノイン酸受容体（RAR）の逆アゴニスト）は、バーの効果を模倣するために側板中胚葉（LPM）に移植されたビーズを浸し使用しますRIERは体節（レチノイン酸（RA）のソース）と肢芽が成長するからLPMの間に配置されました。これらのプロトコルの修正版は、誘導手がかりの起源とタイミング上の他の質問に対処するために使用することができました。ニワトリ胚の領域は、関連する発達段階でのアクセスが提供される、バリアは2つの組織の間に配置することができ、開発における必然的な変化を研究しました。例としては、肝臓や腎臓の誘導として、脳の発達、軸延長にと臓器の開発に見ることができます。

上記の詳細なプロトコールは、西本らに用いられる方法を説明します。 、2015年論文は、ニワトリ胚の前部と後肢の両方で肢芽の誘導と開始を研究しています。前肢芽の伸長を防ぐために、不浸透性の障壁を挿入し、次の結果は、以前に1、2、9しかし脚芽開始1を防ぐ障壁を記述する唯一の研究がなされている公開されています。西本らから後肢のデータ。 2015年は、バリア挿入、次のmRNA発現の研究​​の代表的な結果として、ここで提示されています。 LPMでTbx4の発現は、後肢伸長を開始するのに十分なされていません。 図2BおよびC（ 表2）は、ステージ15（ 図2A）でそのバリア挿入が（プロトコル3.2を参照）の両方FGF10につながる表示しますそして、FGF8の発現が運営右側に（括弧で表記）脚芽領域に不在です。無操作左側と比較してください。しかし、Tbx4発現が操作側（ 図2DおよびE）にステージ19とステージ23の双方で観測されます。我々は、単独でTbx4発現がLPMから後肢伸長を開始するのに十分ではないと結論付けます。ニワトリにおける他の研究は、TBX遺伝子発現が前肢芽形成12,13を開始するのに十分であることを示唆しています。 図2Cは、「バリアが胚ポスト固定の脚部の領域に埋め込まれたどのように見えるかを説明するために含まれています。例障壁の大部分では前in situハイブリダイゼーション（プロトコル4.3） 中に除去しました。 体節からRAはFGF10誘導肢芽O前にLPMに不可欠ですutgrowth： 図3Aは、ステージ15（プロトコル3.2.1.3）に挿入障壁にRA浸漬ビーズ遠位の添加を示す概略図です。 （アスタリスクで示される）この操作肢芽の伸長、彼らがコントロール左側にあるように芽で表現されている操作右側とTbx4、FGF10とFGF8上で観察されるに続いて（ 図3B、CおよびD、 表2） 。したがって、LPMにRAの添加は障壁と肢芽開始進行の影響を克服します。私たちは、肢芽の伸長を開始する前に、体節からの正常な発達のRAにLPMに不可欠であると結論付けています。その結果後肢芽は、制御側のものよりも一般的に小さくなっています。これは、野生型の状況と同等でないというLPMにRAの用量に起因する可能性があります。 RAの用量が高すぎるとアピカル外胚葉リッジ（AER）を短縮することができ、小さな芽を生じます14、15。 LPMでのRA（体節から）は、通常の肢芽の開始のためにRAR、BMS 493の逆アゴニストを必要としていることを確認するために、使用されました。ビーズは、ステージ15で脚領域LPM（プロトコル3.2.1.4を参照）（ 図3E）に注入されます。 FGF10の発現が制御側（; 表3、 図3F）に比べて小さい後肢芽で、その結果、LPMでBMS 493ビーズの次のアプリケーションをダウンレギュレートされます。制御DMSOビーズは、これらの欠陥（; 表3、 図3Gおよび3H）を引き起こしません。 BMS 493適用後に観察された欠陥はバリア操作によって誘導されるものよりも穏やかです。 すなわち、FGF10 は依然として発現され、肢芽が形成されています。 BMS 493の効果は、ビーズの周りに局所的に制限され、AXIによって生成された全てのRAをアンタゴナイズすることができないかもしれないので、これは可能性がありますアル組織。 初期の軸方向の信号は後でTbx4エクスプレス LPMセルを指定します 。 LPMは将来の脚形成領域にTbx4を表現する能力を取得したとき私たちは、テストしました。バリアがその後脚芽の伸長を阻止することを挿入することができる最も早い段階では、我々はステージよりも前の段階で近軸中胚葉と推定される脚部との間に挿入12.障壁を障壁を挿入するためのプロトコルを開発する最初のものであるステージ10でありますステージ10でのLPMステージ10における軸方向の組織からの信号は以降の発現に必要であることを示唆し、脚形成LPM（ 図2F及び2G、 表2）のブロック脚芽形成とTbx4の発現（プロトコル3.4を参照してください）後肢LPMでTbx4。後でバリア挿入がTbx4の発現を可能にする図2DおよびEでこの結果を比較確立されます。さらに、我々は、この軸方向の信号はRARの逆アゴニストはTbx4の発現を減少させるかどうかを観察することによってRAかもしれないかどうかを試験しました。ステージ10（プロトコル3.4.1.3を参照）で後肢LPMに配置されたBMS 493ビーズをDMSOに浸したコントロールビーズがTbx4式（ 図2Jおよび2K）には影響しません一方、Tbx4式 （ 図2Hおよび2I）をダウンレギュレートします。一緒に、これらの結果は、軸組織からのRA信号を積極後肢LPM 7にTbx4を調節することにより、四肢誘導を調節するモデルをサポートしています。 術後死： 表1、表2、表 3は、術後、収穫前に死亡したニワトリ胚の数を含む実験結果の詳細を与えます。一部の死亡は、この種のマイクロサージェリーの後に予想されます。瀕死の数字は保つことができます<html>楽器が汚れていないことを確認することによって最小限に、卵の穴は、胚は、この最後の点の操作に連動して、最小限の時間のための粘着テープシールの保護なし左とされていることを、最小サイズ必要です可能な限り迅速に行われます。これらの操作はいずれも、非常に時間がかかりませんが、ビーズや障壁に関係する操作は、もう少し時間がかかります。これらの措置にもかかわらず、操作は最も初期の段階で行われ、翼の地域のものは死をもたらす可能性が高いです。死による失血につながる可能性四肢形成領域及びこれらの血管の偶発的な破裂の周りに大きな血管があります。我々卵がステージ8または9で開かれたことがわかり、その胚に操作されていない、多くの場合、翌日までに死んでしまいます。したがって、これらの問題に対する唯一の解決策は、実験の多数を行うことです。 D / 54618 / 54618fig2.jpg &quot;/&gt; 推定的レッグレベルでのバリアまたはビーズの挿入を次の 図2. マーカー遺伝子発現の変化。 （A）ステージ15胚における推定脚レベル（体節26-32）で示す概略図バリア位置（緑の線）。 （B - EおよびG）操作胚上WISH分析。脚部領域は（カッコ）に概説されています。 （B）FGF10発現を操作LPMには存在しないが、強い発現が左側芽において検出されます。 （C）FGF8発現にはAERがありません示唆し、運営側には存在しません。 （C &#39;）の障壁が取り除かれた前に、同じ胚。 （D）Tbx4は、制御芽と同じ吻側-尾側レベルでLPMで表現されています。 （E）Tbx4発現は肢の成長の欠如にもかかわらず、右脚領域に維持されます。 （F）段階の模式図<html>推定脚レベルでのバリア位置（緑の線）を示す10ニワトリ胚。 （G）Tbx4式が運営右LPM（ブラケット）には存在しません。 （H）推定脚レベルでBMS 493ビーズの位置（紫円）を示すステージ10ニワトリ胚の模式図。 （I）Tbx4式は、BMS 493（J）コントロールDMSOビーズ位置（灰色の円）を示すステージ10ニワトリ胚の模式図で処理した後の操作右側にダウンレギュレートされます。 （K）Tbx4はDMSOのみで処理した後の制御左側のと同様のレベルで操作右側に表現されています。この図7から変更されています。 <a href="http://ecsource.jove.com/files/ftp_upload/54618/54618fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> D / 54618 / 54618fig3.jpg &quot;/&gt; 図3. RA救助肢芽の不在は、バリア挿入によるレッグバド伸長する前にLPMに必須です。 （A）推定脚レベル（体節26-32）とRA-浸したビーズ（赤い円）でバリア位置（緑の線）を示す概略図。 （B - D）（アスタリスクで示す）RA救出脚芽伸長。 （B）Tbx4式は、非手術側に類似救出脚芽に存在しています。 （C）FGF10を救出戦で発現している非手術側に似て出芽します。 （D）FGF8は救出右脚芽のAERで表現されています。 BMS 493ビーズ（紫円）はステージ15の胚の推定脚LPMに入れたところ（E）の回路図を示します。 （F）FGF10の発現が運営右側にダウンレギュレートし、足の芽は、BMS 49以下の小さいです3治療。ビーズは、アスタリスクを付しています。 （E）から（G）同様の概略図、制御DMSOビーズ（灰色の円）の位置を示します。 （アスタリスクで示す）（H）DMSOビーズは、FGF10の発現や脚の芽の大きさに影響を与えませんでした。この図7から変更されています。 <a href="http://ecsource.jove.com/files/ftp_upload/54618/54618fig3large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> ステージバリア置か（+ビーズ）* </td><td> 番号操作 </td><td> 行方不明ウィング** </td><td> 現在ウィング** </td><td> 数の死者 </td></tr><tr><td>ステージ12-14 </td><td> 40 </td><td> 24 </td><td> 1 </td><td> 15 </td></tr><tr><td>ステージ8/9 </td><td> 65 </td><td> 23 </td><td> 0 </td><td> 42 </td></tr><tr><td>ステージ12-13（FGF4ビーズ） </td><td> 41 </td><td> 7（ビーズ不足しています） </td><td> 17 </td><td> 17 </td></tr><tr><td>ステージ12-13（RAビーズ） </td><td> 75 </td><td> 9（ビーズ不足しています） </td><td> 32 </td><td> 34 </td></tr><tr><td>ステージ13（コントロールビーズ） </td><td> 5 </td><td> 3 </td><td> 0 </td><td> 2 </td></tr><tr><td colspan="5">体節15-20と側板中胚葉との間に配置された* 0.7〜1ミリメートル箔障壁。 0.35 mg / mlでFGF4ビーズ。 0.05〜0.1 mg / mlでのRAビーズ。 </td></tr><tr><td colspan="5"> DMSOコントロールビーズ。 </td></tr><tr><td colspan="5"> **ニワトリ胚の固定台21-23 </td></tr></tbody></table> 表1.ウィングレベルのバリアとビーズ実験の成果。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> ステージバリア置か（+ビーズ）* </TD&gt; <td> 番号操作 </td><td> 行方不明の脚** </td><td> 脚存在** </td><td> 数の死者 </td></tr><tr><td>ステージ12-15 </td><td> 43 </td><td> 39 </td><td> 0 </td><td> 4 </td></tr><tr><td>ステージ10月11日</td><td> 23 </td><td> 16 </td><td> 0 </td><td> 7 </td></tr><tr><td>ステージ15（FGF4ビーズ） </td><td> 8 </td><td> 0 </td><td> 5 </td><td> 3 </td></tr><tr><td>ステージ15（RAビーズ） </td><td> 18 </td><td> 0 </td><td> 18 </td><td> 0 </td></tr><tr><td colspan="5">体節26-32と側板中胚葉との間に配置された* 0.7〜1.3ミリメートル箔障壁。 0.35 mg / mlでFGF4ビーズ。 0.05〜0.1 mg / mlでのRAビーズ。 </td></tr><tr><td colspan="5"> **ニワトリ胚の固定台18-24 </td></tr></tbody></table> 表2脚レベル障壁とビーズ実験の成果。 <table borデア= &quot;1&quot;のfo：キープtogether.withinページ= &quot;1&quot;のfo：キープで-next.within-ページ= &quot;常に&quot;&gt; <tbody><tr><td> 番号操作 </td><td> 脚は小さな芽 </td><td> 脚は少し小さな芽 </td><td> 脚芽通常のサイズ </td><td> 数の死者 </td></tr><tr><td> BMS 493ビーズ* </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ビーズ** </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ビーズを5mgに浸し/ミリリットルBMS 493は、脚のレベルで右のステージ14-15 LPMに入れました。 </td></tr><tr><td colspan="5"> DMSOに浸し** 2-3ビーズだけでは足のレベルで右ステージ15 LPMに入れました。 </td></tr><tr><td colspan="5">ニワトリ胚は段階で固定17-19。 </td></tr></tbody></table> 表3脚レベルBMS 493ビーズ実験の成果。

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.

チックに誘導シグナルを研究するために、候補因子浸しビーズと組み合わせることで不浸透性の障壁の応用

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

Research

JoVE Journal

Developmental Biology

9.3K ビュー.  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.

ビデオ: 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).

最適化された<em&gt;元OVO</em&gt;開発の進んだ段階にニワトリ胚の培養

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

発生生物学

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.

遺伝子発現に影響を​​及ぼすシグナル伝達経路を同定するために、ニワトリ胚手足の開発にビーズのグラフト

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.

アーリーステージのニワトリ胚に成長因子コートビーズの配置

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

生物学

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.

長期用水中フィルターペーパーサンドイッチ<em&gt;例OVO</emニワトリ初期胚の&gt;タイムラプスイメージング

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.

鶏胚における神経誘導のためのアッセイ

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.

ニワトリ胚における軸索軌道とシナプスターゲットを追跡する後脳のエレクトロ

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

神経科学

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.

浸漬ビーズアッセイを用いたニワトリ胚発生時の細胞分化、形態形成、パターニングの解析

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.

鳥類の内耳の発達を研究するためのOvoおよびEx Ovoの方法

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.

頭蓋顔面の開発時には外胚葉上皮のシグナリング特性を評価する

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.

ニワトリ孵化子の聴覚脳幹応答の評価

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

チックに誘導シグナルを研究するために、候補因子浸しビーズと組み合わせることで不浸透性の障壁の応用

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遺伝子発現に影響を及ぼすシグナル伝達経路を同定するために、ニワトリ胚手足の開発にビーズのグラフト

アーリーステージのニワトリ胚に成長因子コートビーズの配置

長期用水中フィルターペーパーサンドイッチ

鶏胚における神経誘導のためのアッセイ

ニワトリ胚における軸索軌道とシナプスターゲットを追跡する後脳のエレクトロ

浸漬ビーズアッセイを用いたニワトリ胚発生時の細胞分化、形態形成、パターニングの解析

鳥類の内耳の発達を研究するためのOvoおよびEx Ovoの方法

頭蓋顔面の開発時には外胚葉上皮のシグナリング特性を評価する

ニワトリ孵化子の聴覚脳幹応答の評価

チックに誘導シグナルを研究するために、候補因子浸しビーズと組み合わせることで不浸透性の障壁の応用

要約

さらに動画を探す

この動画の章

最適化された

遺伝子発現に影響を​​及ぼすシグナル伝達経路を同定するために、ニワトリ胚手足の開発にビーズのグラフト

アーリーステージのニワトリ胚に成長因子コートビーズの配置

長期用水中フィルターペーパーサンドイッチ

鶏胚における神経誘導のためのアッセイ

ニワトリ胚における軸索軌道とシナプスターゲットを追跡する後脳のエレクトロ

浸漬ビーズアッセイを用いたニワトリ胚発生時の細胞分化、形態形成、パターニングの解析

鳥類の内耳の発達を研究するためのOvoおよびEx Ovoの方法

頭蓋顔面の開発時には外胚葉上皮のシグナリング特性を評価する

ニワトリ孵化子の聴覚脳幹応答の評価

遺伝子発現に影響を及ぼすシグナル伝達経路を同定するために、ニワトリ胚手足の開発にビーズのグラフト