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

伦理声明:所有这些实验遵循动物护理和诺丁汉大学的道德准则。 1.准备肝素珠的嫁接<ol><li>在使用前在PBS彻底冲洗肝素珠。注意:珠可以被存储在4℃下作为在PBS浆料。 <ol><li>从股票与20微升吸管入1毫升滴的PBS删除它们选择珠嫁接。然后用微量设置为2微升转移珠串成的PBS在3cm的培养皿20微升下降。选择基于尺寸珠和，使用立体解剖显微镜，具有P2枪头传送所选珠;周围的人直径100微米的理想选择。 </li></ol></li><li>从珠粒中取出PBS中。除去大部分的液体用20微升吸管和通过毛细管作用与细制表商镊子残余液体。注意:以除去尽可能多的这样的生长事实上是重要当它被添加到珠粒r被不稀释。 </li><li>直接吸取生长因子到珠上。对于FGF18添加0.5微升的重组FGF18在PBS中0.1％BSA的复原，在0.5毫克/毫升的。放置数滴水绕盘的边缘，以防止液体蒸发。 </li><li>孵育珠1小时，在室温。从用巴氏吸管的菜卸下水滴置于冰上准备嫁接。 </li><li>如果需要的话（透明珠）转让4-5珠2％酚红嫁接帮助可视化之前。转移到胚胎之前漂洗于PBS中。 </li></ol> 2.制备AG 1-X2珠的嫁接<ol><li>在使用之前，衍生珠粒用0.2N甲酸。 <ol><li>要做到这一点用刮刀珠转移到1.5 ml离心管。添加为0.2N甲酸1毫升和洗了一个惊天的平台上1小时。 </li><li>然后取出甲酸与P1000的移液管，并更换1毫升水中。洗1小时，并重复六次，以除去剩余的甲酸。 </li><li>用的p1000移液管除去剩余的液体，并存储所述珠在4℃下。注意:不必在这个阶段除去所有残留的水。 </li></ol></li><li>除去小颗粒用小刮勺从库存约5-10微升体积珠成3厘米培养皿中，并添加1毫升DMSO中（或任何溶剂的药物溶解在）。注:这应该提供几百珠从中选择那些合适的尺寸。 </li><li>然后用移液管P2设定为2微升约100微米直径转移珠的溶剂中另3厘米培养皿有20微升下降。 </li><li>除去溶剂以20微升吸管和用2微升吸管任何残余的溶剂。替换为要应用20微升的药物。 注:对于一致性溶解该药物在为100μM的DMSO浓度（或曾经使用溶剂），分装成20微升，并储存在-80℃的一些小分子抑制剂是不稳定的，最好是避免反复冻融。如有必要，浸泡珠粒之前然后稀释药物到所需的浓度。有效浓度可能需要凭经验每种药物来决定。 </li><li>孵育1小时。避光保存尽可能多的这些分子是光敏感。 </li><li>前接枝转印4-5珠与p2的吸移管设置为2微升到20微升的2％酚红溶解在水中。执行使用立体声解剖显微镜可视化珠粒此步骤。取出用细镊子制表大师单珠或金属环，并转移到胚胎前冲洗PBS中。使用前不要离开2％酚红珠子超过15分钟，因为这可能会导致失去活性。 </li></ol> 3.准备钨针<ol><li>切细钨丝成3-4厘米的长度。插入一块线到年底巴斯德玻璃吸管和融化在本生灯位置来解决。准备最多10个针，以确保有备用如果它们在使用过程中损坏。 </li><li>放置电线的一端插入喷灯的火焰并保持不动，直至钨发光白色，针尖锋利（约2-3分钟）。注意:在使用期间，针可以清洗，并根据需要重新削尖在喷灯。 注:这些针也可以被用于制造循环用于传送珠。轻轻触碰针板凳，它会弯曲，形成一个循环。 </li></ol>对于珠移植4.准备胚胎<ol><li>孵化鸡蛋的钝端，直到他们达到预期的阶段（3-5天大部分肢芽操作）。使用上的钝端钝钳水龙头破壳，然后使用镊子取下外壳和暴露胚胎。确保蛋壳膜也将被删除。 注:1-2毫升的PBS可以用巴氏吸管，以协助加这如果膜附着在蛋黄。轻轻吸管PBS到蛋黄的表面，把握膜与镊子，注意不要损坏蛋黄，然后轻轻地将其从鸡蛋。 </li><li>从卵用10毫升注射器和一19克针去除高达至5ml蛋白的。根据需要，以确保胚胎不与用于密封蛋作为去除较大的体积可以减少胚胎存活率磁带接触采取只之多。 </li><li>为了形象胚胎加5-6滴的艺术家墨汁到15毫升的PBS含100单位青霉素和0.1毫克链霉素/毫升。注入0.5-1毫升直属胚胎用1ml注射器和一个地下25针。 </li><li>加2-3毫升的PBS，用1％胎牛血清和100U青霉素和0.1mg链霉素/ ml的入卵以确保胚胎不脱水。用削尖的制表大师钳去除卵黄膜并打开羊膜在发展肢芽。确保胚胎不会干出来的如果需要的话，加入更多的PBS / FCS中。 注:肢芽可以清楚地看到作为对胚胎的侧翼配对副产物。在这些阶段的胚胎将转这样的右侧是通常最上部。因此，它通常是更容易移植珠串成的右侧肢体和使用那些留下对侧的控制。 </li><li>用削尖的钨丝做一个切口进入肢芽，其中珠将植入。避免穿过肢芽在可能的情况，虽然在年轻的阶段，这是很难切断所有的方式。 注:在年龄较大的胚​​胎（HH台22及以上）是可能的选择肢芽的特定区域接枝但在更小的阶段，这是更加困难，因为在肢芽本身是小的。同样重要的是要记住，肢芽会成长过去接枝珠如此，尤其是在年轻的阶段，珠粒将保持在肢体近端。 </li><li>拿起无论从生长因子或药物使用任一超等华珠tchmakers镊子或从一个钨针制成的环。如果胎圈已经浸泡在DMSO或酚红，施加到胚胎之前漂洗于PBS中。珠转移到胚胎与钳/钢丝环。然后用锋利的钨丝到胎圈插入到切口。 </li><li>加1-2毫升的PBS / FCS的蛋确保胚胎保持水合，但避免直接施加到胚胎本身，因为这会损坏胚胎和使珠被移位。密封该蛋用胶带，并返回到培养箱中。 </li></ol>

<ol>
	<li>Davey, M. G., Tickle, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chicken+as+a+model+for+embryonic+development.">The chicken as a model for embryonic development.</a> Cytogenetic Genome Research. 117, 231-239 (2007).</li><li>Le Douarin, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Nogent+Institute--50+years+of+embryology.">The Nogent Institute--50 years of embryology.</a> International Journal of Developmental Biology. 49, 85-103 (2005).</li><li>Le Douarin, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+avian+embryo+as+a+model+to+study+the+development+of+the+neural+crest:+a+long+and+still+ongoing+story.">The avian embryo as a model to study the development of the neural crest: a long and still ongoing story.</a> Mechanisms of Development. 121, 1089-1102 (2004).</li><li>Bell, E., Brickell, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication-competent+retroviral+vectors+for+expressing+genes+in+avian+cells+in+vitro+and+in+vivo.">Replication-competent retroviral vectors for expressing genes in avian cells in vitro and in vivo.</a> Molecular Biotechnology. 7, 289-298 (1997).</li><li>Abu-Elmagd, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt/Lef1+signaling+acts+via+Pitx2+to+regulate+somite+myogenesis.">Wnt/Lef1 signaling acts via Pitx2 to regulate somite myogenesis.</a> Developmental Biology. 337, 211-219 (2010).</li><li>Munsterberg, A. E., Lassar, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+signals+from+the+neural+tube,+floor+plate+and+notochord+induce+myogenic+bHLH+gene+expression+in+the+somite.">Combinatorial signals from the neural tube, floor plate and notochord induce myogenic bHLH gene expression in the somite.</a> Development. 121, 651-660 (1995).</li><li>McGrew, 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=Efficient+production+of+germline+transgenic+chickens+using+lentiviral+vectors.">Efficient production of germline transgenic chickens using lentiviral vectors.</a> EMBO Reports. 5, 728-733 (2004).</li><li>Valasek, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+molecular+investigations+into+the+development+of+the+pectoral+girdle.">Cellular and molecular investigations into the development of the pectoral girdle.</a> Developmental Biology. 357, 108-116 (2011).</li><li>Tickle, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+contribution+of+chicken+embryology+to+the+understanding+of+vertebrate+limb+development.">The contribution of chicken embryology to the understanding of vertebrate limb development.</a> Mechanisms of Development. 121, 1019-1029 (2004).</li><li>Riddle, R. D., Johnson, R. L., Laufer, E., Tabin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonic+hedgehog+mediates+the+polarizing+activity+of+the+ZPA.">Sonic hedgehog mediates the polarizing activity of the ZPA.</a> Cell. 75, 1401-1416 (1993).</li><li>Tickle, C., Alberts, B., Wolpert, L., Lee, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+application+of+retinoic+acid+to+the+limb+bond+mimics+the+action+of+the+polarizing+region.">Local application of retinoic acid to the limb bond mimics the action of the polarizing region.</a> Nature. 296, 564-566 (1982).</li><li>Rosello-Diez, A., Torres, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulative+patterning+in+limb+bud+transplants+is+induced+by+distalizing+activity+of+apical+ectodermal+ridge+signals+on+host+limb+cells.">Regulative patterning in limb bud transplants is induced by distalizing activity of apical ectodermal ridge signals on host limb cells.</a> Developmental Dynamics. 240, 1203-1211 (2011).</li><li>Anakwe, 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=Wnt+signalling+regulates+myogenic+differentiation+in+the+developing+avian+wing.">Wnt signalling regulates myogenic differentiation in the developing avian wing.</a> Development. 130, 3503-3514 (2003).</li><li>Amthor, H., Christ, B., Weil, M., Patel, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+importance+of+timing+differentiation+during+limb+muscle+development.">The importance of timing differentiation during limb muscle development.</a> Current Biology. 8, 642-652 (1998).</li><li>Brand-Saberi, B., Muller, T. S., Wilting, J., Christ, B., Birchmeier, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scatter+factor/hepatocyte+growth+factor+(SF/HGF)+induces+emigration+of+myogenic+cells+at+interlimb+level+in+vivo.">Scatter factor/hepatocyte growth factor (SF/HGF) induces emigration of myogenic cells at interlimb level in vivo.</a> Developmental Biology. 179, 303-308 (1996).</li><li>Mok, G. F., Cardenas, R., Anderton, H., Campbell, K. H. S., Sweetman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+FGF18+and+retinoic+acid+regulate+differentiation+of+chick+embryo+limb+myoblasts.">Interactions between FGF18 and retinoic acid regulate differentiation of chick embryo limb myoblasts.</a> Developmental Biology. 396, 214-223 (2014).</li><li>Abou-Elhamd, A., Cooper, O., M&#252;nsterberg, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Klhl31+is+associated+with+skeletal+myogenesis+and+its+expression+is+regulated+by+myogenic+signals+and+Myf-5.">Klhl31 is associated with skeletal myogenesis and its expression is regulated by myogenic signals and Myf-5.</a> Mechanisms of Development. , 126-852 (2009).</li><li>Schmidt, M., Tanaka, M., Munsterberg, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+(beta)-catenin+in+the+developing+chick+myotome+is+regulated+by+myogenic+signals.">Expression of (beta)-catenin in the developing chick myotome is regulated by myogenic signals.</a> Development. 127, 4105-4113 (2000).</li><li>Stavridis, M. P., Lunn, J. S., Collins, B. J., Storey, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+discrete+period+of+FGF-induced+Erk1/2+signalling+is+required+for+vertebrate+neural+specification.">A discrete period of FGF-induced Erk1/2 signalling is required for vertebrate neural specification.</a> Development. 134, 2889-2894 (2007).</li><li>Martinez-Morales, P. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF+and+retinoic+acid+activity+gradients+control+the+timing+of+neural+crest+cell+emigration+in+the+trunk.">FGF and retinoic acid activity gradients control the timing of neural crest cell emigration in the trunk.</a> Journal of Cell Biology. 194, 489-503 (2011).</li><li>Olivera-Martinez, I., Storey, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+signals+provide+a+timing+mechanism+for+the+FGF-retinoid+differentiation+switch+during+vertebrate+body+axis+extension.">Wnt signals provide a timing mechanism for the FGF-retinoid differentiation switch during vertebrate body axis extension.</a> Development. 134, 2125-2135 (2007).</li><li>Logan, M., Simon, H. G., Tabin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+regulation+of+T-box+and+homeobox+transcription+factors+suggests+roles+in+controlling+chick+limb-type+identity.">Differential regulation of T-box and homeobox transcription factors suggests roles in controlling chick limb-type identity.</a> Development. 125, 2825-2835 (1998).</li><li>Borycki, A. G., Mendham, L., Emerson, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+somite+patterning+by+Sonic+hedgehog+and+its+downstream+signal+response+genes.">Control of somite patterning by Sonic hedgehog and its downstream signal response genes.</a> Development. 125, 777-790 (1998).</li><li>Sweetman, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF-4+signaling+is+involved+in+mir-206+expression+in+developing+somites+of+chicken+embryos.">FGF-4 signaling is involved in mir-206 expression in developing somites of chicken embryos.</a> Developmental Dynamics. 235, 2185-2191 (2006).</li><li>Chapman, S. C., Collignon, J., Schoenwolf, G. C., Lumsden, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+method+for+chick+whole-embryo+culture+using+a+filter+paper+carrier.">Improved method for chick whole-embryo culture using a filter paper carrier.</a> Developmental Dynamics. 220, 284-289 (2001).</li><li>Sweetman, D., Wagstaff, L., Cooper, O., Weijer, C., M&#252;nsterberg, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+migration+of+paraxial+and+lateral+plate+mesoderm+cells+emerging+from+the+late+primitive+streak+is+controlled+by+different+Wnt+signals.">The migration of paraxial and lateral plate mesoderm cells emerging from the late primitive streak is controlled by different Wnt signals.</a> BMC Developmental Biology. 8, 63 (2008).</li><li>Scott, B. B., Lois, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+tissue-specific+transgenic+birds+with+lentiviral+vectors.">Generation of tissue-specific transgenic birds with lentiviral vectors.</a> Proceedings of the National Academy of Sciences of the U.S.A. 102, 16443-16447 (2005).</li><li>Seidl, A. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+quail+as+a+model+for+research+in+the+avian+nervous+system:+a+comparative+study+of+the+auditory+brainstem.">Transgenic quail as a model for research in the avian nervous system: a comparative study of the auditory brainstem.</a> Journal of Comparative Neurology. 521, 5-23 (2013).</li></ol>

The authors have nothing to disclose.

使用直接应用到发育组织卵珠移植物是一个功能强大的工具的开发给予无双控制在它们所应用的发育阶段和暴露的持续时间期间对解剖的生长因子信号的作用。 珠为每个类型的分子的选择是重要的。小的疏水性分子，如这里所描述的抑制剂和视黄酸，通常很好地结合衍生AG 1-X2珠虽然它需要测试的有效性的各缓蚀剂对这些珠子凭经验。类似地，对于生长因子，可能有必要以测试不同类型的珠。一些珠是透明的，所以可能很难在操作，但很短的治疗酚红随后在PBS冲洗想象会标记它们足够长的时间来进行移植。 当制备珠，特别是那些已被浸泡抑制剂S，以保护它们免受光尽可能是重要的。在其最初的治疗和操作过程中，他们应该被覆盖并且仅当珠被转移除去。最好是使用刚解冻的等分试样作为一些这些试剂是不稳定的，并且可以给出误导负的结果，如果没有存储正常。同样重要的是不要让它们浸泡在酚红太久接枝，因为这可减少胎圈的有效性之前。 使用这两种生长因子和药物的这些珠移植物，浓度，通常非常高; FGF18珠粒浸泡在0.5毫克/毫升和珠为小分子抑制剂的浓度通常是浸泡在100μM的浓度。尽管这比通常用于治疗细胞溶液，这是必要的，因为生长因子和药物的珠吸收，然后释放的量的浓度高得多很难直接测量，但据推测属德水平低于该体内。其结果是浓度范围必须为用于确定的有效剂量每分子进行试验。 这种技术也适用于范围广泛的其他组织中，如体节24，在开发过程中，可用于对胚胎卵内或在培养物，如乳油培养25。另外，也可以生长培养细胞分泌特异性生长因子如丸粒然后可以移植到发育中的胚胎中以同样的方式26。 无障碍的组合，易于操作且成本低的使鸡胚发展的很好的模型。随着转基因技术被越来越多地应用到鸟类和鸡两种转基因线7和鹌鹑27可用经典胚胎移植与这些修改后的鸟技术的组合已经提供了新的见解德velopment 8,28。

鸡胚胎已经很多年1提供了有力的发展工具的研究。他们的一个最有用特征是，它们相对容易操纵。外部发展使得能够打开蛋访问胚胎并执行各种微操作包括的例子，如用于研究细胞命运2,3经典鹌鹑小鸡嵌合体系统，逆转录病毒为过度表达在特定组织的注射发展4,5-期间和外植体培养，以确定发育信号源6。最近嵌合体与表达GFP的转基因鸡线移植未标记的主机之间产生表明，传统的嫁接，转基因的组合可以提供重要的见解发展7,8。 与禽流胚胎可以被操纵的方便性使它成为一个很好的模型来研究肢体发展9。具体生长因子开发四肢体内的应用一直在查明改变肢体图案10,11，并继续提供深入了解这个过程中12个因素 。这种方法也被用于研究该调节肌肉发展的因素，并已发现对于许多信号，诸如Wnts 13，骨形态发生蛋白14和HGF 15的作用。 最近这种技术被用于研究在肢芽控制生肌基因表达的信号，并表明，FGF18和视黄酸之间的相互作用可以控制的MyoD表达式16的定时。使用了可加载到珠，然后直接移植到在限定的发育阶段特定的组织的生长因子和小分子的组合提供机会开发期间进行干预，在几乎任何时间和区域。这已被用来INVEstigate许多过程，包括体节图案17,18，神经特19，神经嵴迁移20和轴延伸21。 在这里，我们描述了嫁接珠浸泡在两种生长因子或抑制剂来发展鸡四肢的方法。这已被使用的肌肉特异性基因表达分析用原位杂交技术来确定肌形成这些信号的影响。我们描述了使用肝素浸渍珠，其中用于生长因子，或为小的疏水性分子AG 1-X2珠如视黄酸或特定信号传导途径的小分子抑制剂的移植物。然而，其他的珠子也可已被用于递送二者的FGFs 22和Shh 23。

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

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

使用鸡胚能够直接测试要么生长因子或信号对基因表达和活化的信号转导途径途径的特异性抑制剂的效果。这种技术允许信号分子在特定的时间精确定义的发育阶段的交付。在此之后的胚胎可以收获和基因表达研究，例如，通过原位杂交 ，或在激活与免疫染色观察到的信号转导途径。 在浸泡在FGF18这个视频肝素珠或AG 1-X2珠浸泡在U0126，MEK抑制剂，被嫁接到蛋内的肢芽。这表明，FGF18诱导的MyoD和ERK磷酸化和内源性和FGF18诱导MyoD的表达的表达是通过U0126抑制。珠浸泡在一视黄酸拮抗剂可通过FGF18增强过早MyoD的诱导。 这种方法可以是我们编与各种不同的生长因子和抑制剂和很容易适应在发育中的胚胎等组织。

在HH阶段21 MyoD的不表达在显影肢成肌细胞虽然染色所用的显影体节（图1A）的肌节中可以看出。 图1B显示了 原位杂交 MyoD的6小时以下的FGF18珠移植物。的MyoD被诱导成肌细胞的附近到胎圈而没有在对侧肢体无表达。共接枝浸泡在U0126珠，MEK，块FGF18诱导MyoD的（ 图1C）的特异性抑制剂。同样嫁接U0126珠在HH阶段21，寻找MyoD基因表达后的24小时减少MyoD的内源性表达与模型对侧肢体（ 图1D，E）进行比较。 在肢芽在HH级19 FGF18不会诱导的MyoD（ 图1F）。然而共接枝珠浸泡在FGF18和维甲酸拮抗剂BMS493可以克服这一点，并在检测异位的MyoD（Figu重新1G） 为了证实FGF18通过MEK胚胎作用收获后1小时珠移植和免疫染色磷酸化ERK，MEK 图1H的目标显示嫁接后的FGF18珠1小时的明图像，而图1I显示，FGF18诱导快速磷酸嫁接珠周围ERK的。 图1J显示了这些图像的叠加。控制珠粒浸泡在0.1％BSA的不诱导ERK磷酸化（图1K，L，M） <img alt="图1" src="/files/ftp_upload/53342/53342fig1.jpg" /> 四肢 图1 MyoD的表达是通过ERK的磷酸化调控由FGF18和视黄酸，未经处理的控制四肢HH阶段21不表达MyoD基因（A），但过早的表达被检测到6小时嫁接的FGF18珠（后B）。联合移植FGF18和U0126珠块这种诱导（C）。内源性的MyoD的表达被认为在HH级21（D），但它已被接枝珠浸泡在U0126（E）。在早期的肢芽过早MyoD的表达不受FGF18（F）引起的，但是是诱发浸泡在FGF18和维甲酸受体拮抗剂BMS493（G）共嫁接珠。移植到HH21四肢FGF18珠诱导ERK磷酸化1小时后:明（H），荧光（I）和合并的HH21肢免疫染色抗phosphoERK的（J）的图像。浸泡在BSA对照珠不诱导ERK磷酸化:（K），荧光（L）和合并的HH21肢免疫染色抗phosphoERK（M）的图像。这个数字已经被修改莫等人，2014年16。黑色星号:肝素珠;白ASTE风险:AG 1-X2珠;箭头显示的肢芽MyoD的异位表达。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53342/53342fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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

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

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

珠嫁接到发展鸡胚四肢识别信号转导途径影响基因表达

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

grafting-beads-into-developing-chicken-embryo-limbs-to-identify

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

8.8K 次观看.  University of Nottingham. 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.

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

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution.

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here the robust and efficient expression of fluorescent proteins after mRNA injection into unfertilized oocytes of Branchiostoma lanceolatum. The development of the microinjection technique in this basal chordate will pave the way for far-reaching technical innovations in this emerging model system, including in vivo imaging and gene-specific manipulations.

荧光蛋白的表达<em&gt;披文昌鱼</em&gt;通过基因注射到未受精的卵母细胞

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the ...

科研

发育生物学

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

All developing organs need to be connected to both the nervous system (for sensory and motor control) as well as the vascular system (for gas exchange, fluid and nutrient supply). Consequently both the nervous and vascular systems develop alongside each other and share striking similarities in their branching architecture. Here we report embryonic manipulations that allow us to study the simultaneous development of neural crest-derived nervous tissue (in this case the enteric nervous system), and the vascular system. This is achieved by generating chicken chimeras via transplantation of discrete segments of the neural tube, and associated neural crest, combined with vascular DiI injection in the same embryo. Our method uses transgenic chickGFP embryos for intraspecies grafting, making the transplant technique more powerful than the classical quail-chick interspecies grafting protocol used with great effect since the 1970s. ChickGFP-chick intraspecies grafting facilitates imaging of transplanted cells and their projections in intact tissues, and eliminates any potential bias in cell development linked to species differences. This method takes full advantage of the ease of access of the avian embryo (compared with other vertebrate embryos) to study the co-development of the enteric nervous system and the vascular system.

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

Here we report dual labeling of neural crest cells and blood vessels using chickGFP neural tube intraspecies grafting combined with intra-vascular DiI injection. This experimental technique allows us to simultaneously visualize and study development of the NCC-derived (enteric) nervous system and the vascular system, during organogenesis.

使用小鸡GFP 神经管移植和碳水化合物染料 DiI 注射，在鸡胚胎内对神经囊细胞和血管进行双重标记

All developing organs need to be connected to both the nervous system (for sensory and motor control) as well as the vascular system (for gas exchange, ...

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

技术目标显微注射到显影<em&gt;爪</em&gt;肾

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. The inner ear auditory sensory organ is critical for our ability to hear. It houses a highly specialized sensory epithelium that consists of mechano-transducing hair cells (HCs) and surrounding glial-like supporting cells (SCs). Despite structural differences in the auditory organs, molecular mechanisms regulating the development of the auditory organ are evolutionarily conserved between mammals and aves. In ovo electroporation is largely limited to early stages at E1 - E3. Due to the relative late development of the auditory organ at E5, manipulations of the auditory organ by in ovo electroporation past E3 are difficult due to the advanced development of the chicken embryo at later stages. The method presented here is a transient gene transfer method for targeting genes of interest at stage E4 - E4.5 in the developing chicken auditory sensory organ via in ovo micro-electroporation. This method is applicable for gain- and loss-of-functions with conventional plasmid DNA-based expression vectors and can be combined with in ovo cell proliferation assay by adding EdU (5-ethynyl-2&#180;-deoxyuridine) to the whole embryo at the time of electroporation. The use of green or red fluorescent protein (GFP or RFP) expression plasmids allows the experimenter to quickly determine whether the electroporation successfully targeted the auditory portion of the developing inner ear. In this method paper, representative examples of GFP electroporated specimens are illustrated; embryos were harvested 18 - 96 hr&#160;after electroporation and targeting of GFP to the pro-sensory area of the auditory organ was confirmed by RNA in situ hybridization. The method paper also provides an optimized protocol for the use of the thymidine analog EdU to analyze cell proliferation; an example of an EdU based cell proliferation assay that combines immuno-labeling and click EdU chemistry is provided.

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

The auditory organ mediates hearing. Here we present a modified in ovo micro-electroporation method optimized for studying auditory progenitor cell proliferation and differentiation in the developing chicken auditory organ.

基因转移到鸡听觉器官通过<em&gt;在卵</em&gt;微电

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. ...

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments.
In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

Live tracking of individual WT retinal progenitors in distinct genetic backgrounds allows for the assessment of the contribution of cell non-autonomous signaling during neurogenesis. Here, a combination of gene knockdown, chimera generation via embryo transplantation and in vivo time-lapse confocal imaging was utilized for this purpose.

在嵌合斑马鱼胚胎的个别视网膜祖细胞转基因表达的体内成像来研究细胞非自治的影响

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

Tracing Gene Expression Through Detection of &#946;-galactosidase Activity in Whole Mouse Embryos

The Escherichia coli LacZ gene, encoding &#946;-galactosidase, is largely used as a reporter for gene expression and as a tracer in cell lineage studies. The classical histochemical reaction is based on the hydrolysis of the substrate X-gal in combination with ferric and ferrous ions, which produces an insoluble blue precipitate that is easy to visualize. Therefore, &#946;-galactosidase activity serves as a marker for the expression pattern of the gene of interest as the development proceeds. Here we describe the standard protocol for the detection of &#946;-galactosidase activity in early whole mouse embryos and the subsequent method for paraffin sectioning and counterstaining. Additionally, a procedure for clarifying whole embryos is provided to better visualize X-gal staining in deeper regions of the embryo. Consistent results are obtained by performing this procedure, although optimization of reaction conditions is needed to minimize background activity. Limitations in the assay should be also considered, particularly regarding the size of the embryo in whole mount staining. Our protocol provides a sensitive and a reliable method for &#946;-galactosidase detection during the mouse development that can be further applied to the cryostat sections as well as whole organs. Thus, the dynamic gene expression patterns throughout development can be easily analyzed by using this protocol in whole embryos, but also detailed expression at the cellular level can be assessed after paraffin sectioning.

Here we describe the standard protocol for the detection of &#946;-galactosidase activity in early whole mouse embryos and the method for paraffin sectioning and counterstaining. This is an easy and quick procedure to monitor gene expression during development that can also be applied to tissue sections, organs or cultured cells.

通过检测全小鼠胚胎β乳糖酶活性追踪基因表达

The Escherichia coli LacZ gene, encoding &#946;-galactosidase, is largely used as a reporter for gene expression and as a tracer in cell lineage studies. ...

Assessment of Ultrastructural Neuroplasticity Parameters After In Utero Transduction of the Developing Mouse Brain and Spinal Cord

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ultrastructural parameters in unambiguously identified topographical structures, affected by a protein of interest that is introduced into the organism via viral transfer. This combined approach allows for a smooth transition from macrostructural to ultrastructural identification by following topographical navigation maps in a tissue atlas. High-resolution electron microscopy of the in-utero-transduced tissue reveals the fine ultrastructure of the neuropil and its plasticity parameters, such as cross-sectioned synaptic bouton areas, the number of synaptic vesicles and mitochondria within a bouton profile, the length of synaptic contacts, cross-sectioned axonal areas, the thickness of myelin sheaths, the number of myelin lamellae, and cross-sectioned areas of mitochondria profiles. The analysis of these parameters reveals essential insights into changes of ultrastructural plasticity in the areas of the nervous system that are affected by the viral transfer of the genetic construct. This combined method can not only be used for studying the direct effect of genetically engineered biomolecules and/or drugs on neuronal plasticity but also opens the possibility to study the in utero rescue of neuronal plasticity (e.g., in the context of neurodegenerative diseases).

The combination of transmission electron microscopy and in utero transduction is a powerful approach for studying morphological changes in the fine ultrastructure of the nervous system during development. This combined method allows deep insights into changes in structural details underlying neuroplasticity with respect to their topographical representation.

小鼠脑脊髓子宫转导后超微结构神经可塑性参数的评价

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ...

Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human HSC emergence in vitro are required to overcome limitations in studying this complex developmental process. Here, we describe a protocol to generate hematopoietic stem and progenitor-like cells from human dermal fibroblasts employing a direct cell reprogramming approach. These cells transit through a hemogenic intermediate cell-type, resembling the endothelial-to-hematopoietic transition (EHT) characteristic of HSC specification. Fibroblasts were reprogrammed to hemogenic cells via transduction with GATA2, GFI1B and FOS transcription factors. This combination of three factors induced morphological changes, expression of hemogenic and hematopoietic markers and dynamic EHT transcriptional programs. Reprogrammed cells generate hematopoietic progeny and repopulate immunodeficient mice for three months. This protocol can be adapted towards the mechanistic dissection of the human EHT process as exemplified here by defining GATA2 targets during the early phases of reprogramming. Thus, human hemogenic reprogramming provides a simple and tractable approach to identify novel markers and regulators of human HSC emergence. In the future, faithful induction of hemogenic fate in fibroblasts may lead to the generation of patient-specific HSCs for transplantation.

This protocol demonstrates the induction of a hemogenic program in human dermal fibroblasts by enforced expression of the transcription factors GATA2, GFI1B and FOS to generate hematopoietic stem and progenitor cells.

通过转录因子的强制表达，人类纤维细胞的造血再编程

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human ...

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.

在Vitro重建空间细胞接触模式与孤立的Caenorhabditis elegans胚胎冲击器和粘性聚苯乙烯珠

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

Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and molecular complexity of skeletal elements increases with development. Tissue heterogeneity, such as at the interface of cartilaginous and osseous elements with each other or with surrounding tissues, is one obstacle to the study of developing cartilage and bone. Our protocol provides a rapid method of tissue processing and isolation of cartilage and bone that yields high quality RNA for gene expression analysis. Fresh frozen tissues of mouse embryos are sectioned and brief cresyl violet staining is used to visualize cartilage and bone with colors distinct from surrounding tissues. Slides are then rapidly dehydrated, and cartilage and bone are isolated subsequently by LCM. The minimization of exposure to aqueous solutions during this process maintains RNA integrity. Mouse Meckel&rsquo;s cartilage and mandibular bone at E16.5 were successfully collected and gene expression analysis showed differential expression of marker genes for osteoblasts, osteocytes, osteoclasts, and chondrocytes. High quality RNA was also isolated from a range of tissues and embryonic ages. This protocol details sample preparation for LCM including cryoembedding, sectioning, staining and dehydrating fresh frozen tissues, and precise isolation of cartilage and bone by LCM resulting in high quality RNA for transcriptomic analysis.

This protocol describes laser capture microdissection for the isolation of cartilage and bone from fresh frozen sections of the mouse embryo. Cartilage and bone can be rapidly visualized by cresyl violet staining and collected precisely to yield high quality RNA for transcriptomic analysis.

用于基因表达分析的小鼠胚胎软骨和骨骼的激光捕获微分

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and ...