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>

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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?...

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&amp;deg;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&amp;deg;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&amp;deg;C. Protect from light.</td></tr><tr><td>Black Indian ink</td><td>Windsor and Newton</td><td>5012572003384 (30 ml)</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, 0.1 mm 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?...

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

Universidad de Cartagena UDEC

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

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).

优化<em&gt;前OVO</em&gt;鸡胚培养，以发展的高级阶段

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

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, ...

Microbead Implantation in the Zebrafish Embryo

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic conservation, as well as similarities in cellular, molecular, and physiological processes, with other vertebrates including humans. During early ontogeny, zebrafish embryos are optically transparent, allowing researchers to visualize the dynamics of organogenesis using a simple stereomicroscope. Microbead implantation is a method that enables tissue manipulation through the alteration of factors in local environments. This allows researchers to assay the effects of any number of signaling molecules of interest, such as secreted peptides, at specific spatial and temporal points within the developing embryo. Here, we detail a protocol for how to manipulate and implant beads during early zebrafish development.

The zebrafish is an excellent model system for genetic and developmental studies. Bead implantation is a valuable tissue manipulation technique that can be used to interrogate developmental mechanisms by introducing alterations in local cellular environments. This protocol describes how to perform microbead implantation in the zebrafish embryo.

微珠植入斑马鱼胚胎

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic ...

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.

配售早期鸡胚生长因子包被的磁珠

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

In Ovo Electroporations of HH Stage 10 Chicken Embryos

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of molecular biological techniques, the chick has become a useful system in which to study gene regulation and function.  By electroporating DNA or RNA constructs into the developing chicken embryo, genes can be expressed or knocked down in order to analyze in vivo gene function.  Similarly, reporter constructs can be used for fate mapping or to examine putative gene regulatory elements.  Compared to similar experiments in mouse, chick electroporation has the advantages of being quick, easy and inexpensive.  This video demonstrates first how to make a window in the eggshell to manipulate the embryo.  Next, the embryo is visualized with a dilute solution of India ink injected below the embryo.  A glass needle and pipette are used to inject DNA and Fast Green dye into the developing neural tube, then platinum electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator.  Finally, the egg is sealed with tape and placed back into an incubator for further development.  Additionally, the video shows proper egg storage and handling and discusses possible causes of embryo loss following electroporation.

Chick in ovo electroporation is a technique which allows genetic manipulation of the avian embryo. Common applications of this technique include functional analysis of genes and putative enhancer elements. This video demonstrates neural tube electroporation in HH 10 chick embryos. Injection technique and proper egg handling are discussed.

在OVO的HH阶段10个鸡胚Electroporations

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of ...

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.

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.

在小鸡学习感应信号与候选因子浸泡珠结合防渗屏障中的应用

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

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.

在鸡胚神经诱导的含量为

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

Single Cell Transfection in Chick Embryos

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a thorough analysis of the fates of single cells under normal and experimental conditions. To this end, transfection methods that target single progenitors are a prerequisite. We describe here a technically straightforward method for transfecting single cells in chicken tissues in-ovo, allowing reliable lineage tracing as well as genetic manipulation. Specific tissue domains are targeted within the somite or neural tube, and DNA is injected directly into the epithelium of interest, resulting in sporadic transfection of single cells. Using reporters, clonal populations may consequently be traced for up to three days, and behavior of genetically manipulated clonal populations can be compared with that of controls. This method takes advantage of the accessibility of the chick embryo along with emerging tools for genetic manipulation. We compare and discuss its advantages over the widely-used electroporation method, and possible applications and use in additional in-vivo models are also suggested. We advocate the use of this method as a significant addition and complement for existing lineage tracing and genetic interference tools.

Using fine tip micropipettes we inject plasmid DNA into subdomains of chicken somites or neural tubes. The concentration of the plasmid is adjusted to generate single transfected cells. We then allow the cells to develop into clonal populations.

单细胞转染鸡胚

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a ...

Chicken Recombinant Limbs Assay to Understand Morphogenesis, Patterning, and Early Steps in Cell Differentiation

Cell differentiation is the fine-tuned process of cell commitment leading to the formation of different specialized cell types during the establishment of developing tissues and organs. This process is actively maintained in adulthood. Cell differentiation is an ongoing process during the development and homeostasis of organs. Understanding the early steps of cell differentiation is essential to know other complex processes such as morphogenesis. Thus, recombinant chicken limbs are an experimental model that allows the study of cell differentiation and pattern generation under embryonic patterning signals. This experimental model imitates an in vivo environment; it assembles reaggregated cells into an ectodermal cover obtained from an early limb bud. Later, ectoderms are transferred and implanted in a chick embryo receptor to allow its development. This assay was mainly used to evaluate mesodermal limb bud cells; however, it can be applied to other stem or progenitor cells from other organisms.

Recombinant limbs are a powerful experimental model that allows for studying the process of cell differentiation and the generation of patterns under the influence of embryonic signals. This protocol presents a detailed method for generating recombinant limbs with chicken limb-mesodermal cells, adaptable to other cell types obtained from different organisms.

鸡重组肢体测定，以了解细胞分化的形态发生，图案化和早期步骤

Cell differentiation is the fine-tuned process of cell commitment leading to the formation of different specialized cell types during the establishment of ...

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.

使用浸泡珠测定分析鸡胚胎发生过程中的细胞分化，形态发生和图案化

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