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.

Declaración de Ética: Todos estos experimentos siguen el cuidado de los animales y las directrices éticas de la Universidad de Nottingham. 1. Preparación de perlas de heparina para Injerto <ol><li> Lávese las perlas de heparina a fondo en PBS antes de su uso. Nota: los granos se pueden almacenar a 4 ° C como una suspensión en PBS. <ol><li> Seleccione los granos para el injerto por sacarlos de la población con una pipeta de 20 l en una gota 1 ml de PBS. A continuación, utilice una micropipeta establece en 2 l para transferir perlas en una gota 20 l de PBS en una placa de Petri 3 cm. Elija cuentas basadas en el tamaño y, usando un estéreo microscopio de disección, la transferencia de granos seleccionados con una punta de pipeta P2; los cerca de 100 m de diámetro son ideales. </li></ol></li><li> Retire la PBS de las perlas. Retire la mayor parte del líquido con una pipeta de 20 l y el líquido residual por capilaridad con relojeros pinzas finas. Nota: Es importante eliminar tanto como sea posible por lo que el crecimiento de factor no se diluye cuando se añade a las perlas. </li><li> Pipetear el factor de crecimiento directamente sobre las perlas. Para FGF18 añadir 0,5 l de FGF18 recombinante reconstituido a 0,5 mg / ml en 0,1% de BSA en PBS. Coloque varias gotas de agua alrededor de los bordes del plato para evitar que el líquido se evapore. </li><li> Se incuban las perlas durante 1 hora a RT. Retire las gotas de agua del plato con una pipeta Pasteur y colocar en hielo listo para el injerto. </li><li> Si se requiere la transferencia (por bolas transparentes) 4-5 perlas al 2% de fenol rojo antes de injertar para ayudar a visualizar ellos. Enjuague en PBS antes de transferir al embrión. </li></ol> 2. Preparación de Cuentas AG 1-X2 para Injerto <ol><li> Antes de su uso derivatizar perlas con 0,2 N de ácido fórmico. <ol><li> Para ello use una espátula para transferir cuentas a un tubo de microcentrífuga de 1,5 ml. Añadir 1 ml de 0,2 N de ácido fórmico y lavado en una plataforma de agitación durante 1 hora. </li><li> A continuación, retire el ácido fórmico con una pipeta p1000 y reemplazar con 1ml de agua. Lave durante 1 hora y repetir seis veces para eliminar el resto de ácido fórmico. </li><li> Retire el líquido residual con una pipeta p1000 y almacenar los granos a 4 ° C. Nota: No es necesario eliminar toda el agua residual en esta etapa. </li></ol></li><li> Retirar una pequeña pellet de perlas de aproximadamente 5-10 l de volumen de las existencias utilizando una pequeña espátula en una placa Petri 3 cm y añadir 1 ml de DMSO (o cualquier disolvente el fármaco se disuelve en). Nota: Esto debería proporcionar varios cientos de cuentas de las que permite seleccionar las del tamaño adecuado. </li><li> A continuación, utilice una pipeta P2 se establece en 2 l de transferir cuentas de aproximadamente 100 m de diámetro a una caída de 20 l de disolvente en otra placa de Petri 3 cm. </li><li> Eliminar el disolvente con una pipeta de 20 l y cualquier disolvente residual con una pipeta de 2 l. Reemplazar con 20 l de la droga que se aplicarán. NOTA: Para obtener la consistencia disolver el fármaco a una concentración de 100 mM en DMSO (o que se utiliza cada vez disolvente),alícuota en 20 l y se almacena a -80 ° C, ya que algunos inhibidores de moléculas pequeñas son inestables y es mejor evitar repetida congelación-descongelación. Si es necesario después diluir la droga a la concentración deseada antes de remojo los granos. Las concentraciones eficaces pueden necesitar ser determinado empíricamente para cada fármaco. </li><li> Incubar durante 1 hora. Proteger de la luz ya que muchas de estas moléculas son sensibles a la luz. </li><li> Antes de injerto de transferencia 4-5 perlas con una pipeta p2 establece en 2 l en 20 l de 2% de fenol rojo disueltos en agua. Realice este paso usando un microscopio de disección estéreo para visualizar las cuentas. Eliminar un solo grano con relojeros pinzas finas o un asa de alambre y enjuague en PBS antes de ser transferido al embrión. No deje perlas en un 2% de rojo fenol durante más de 15 minutos antes de su uso ya que esto puede causar la pérdida de la actividad. </li></ol> 3. Preparación de tungsteno Agujas <ol><li> Cortar alambre de tungsteno fino en 3-4 cm de longitud. Inserte un trozo de alambre en el extremo deuna pipeta Pasteur de vidrio y se funden en un mechero Bunsen para fijar en su posición. Prepara hasta 10 agujas para asegurarse de que hay piezas de repuesto si están dañados durante el uso. </li><li> Coloque el extremo del cable en una llama de soplete y mantenerlo allí hasta que el tungsteno se ilumina en blanco y la punta es aguda (unos 2-3 minutos). Nota: Durante el uso de la aguja se puede limpiar y volver a afilarse en el soplete, según sea necesario. NOTA: Estas agujas también pueden ser utilizados para hacer bucles para la transferencia de perlas. Toque suavemente la aguja para el banco y que se dobla para formar un bucle. </li></ol> 4. Los embriones Preparación de los injertos del grano <ol><li> Incubar huevos con el extremo romo hacia arriba hasta que alcanzan la etapa deseada (3-5 días para la mayoría de las manipulaciones extremidad Bud). Con unas pinzas contundente golpe en el extremo romo de romper la cáscara y luego usar las pinzas para quitar la cáscara y exponer el embrión. Asegúrese de que la membrana de la cáscara de huevo también se elimina. NOTA: 1-2 ml de PBS se puede agregar con una pipeta Pasteur para ayudar conesto si la membrana se adhiere a la yema. Pipeta suavemente la PBS sobre la superficie de la yema, sujete la membrana con las pinzas, teniendo cuidado de no dañar la yema, y ​​tire de él lejos del huevo. </li><li> Remover hasta 5 ml de albumen del huevo con una jeringa de 10 ml y una aguja de 19 G. Tome sólo tanto como sea necesario para asegurar que el embrión no tiene contacto con la cinta utilizada para sellar el huevo como la eliminación de grandes cantidades puede reducir las tasas de supervivencia de embriones. </li><li> Para visualizar el embrión añadir unas 5-6 gotas de artistas tinta china 15 ml de PBS que contenía 100 U de penicilina y estreptomicina 0,1 mg / ml. Inyectar 0,5 a 1 ml directamente bajo el embrión con una jeringa de 1 ml y una aguja de 25 G. </li><li> Añadir 2-3 ml de PBS con 1% de suero de ternera fetal y 100 U de penicilina y estreptomicina 0,1 mg / ml en el huevo para asegurar el embrión no deshidratar. Usando afiladas pinzas de relojero quitar la membrana vitelina y abrir el amnios en los esbozos de los miembros en desarrollo. Asegúrese de que el embrión no se seque y, Si es necesario, agregue más PBS / FCS. NOTA: Las yemas de las extremidades se pueden ver claramente como excrecencias emparejados en el flanco del embrión. En estas etapas el embrión se convertirá el lado este derecho es normalmente más alta. Como resultado normalmente es más fácil para injertar cuentas en las extremidades derechas y utilizar las que quedan como controles contralaterales. </li><li> Use un alambre de tungsteno afilado hacer una incisión en la yema de la extremidad en la que se implanta la perla. Evite cortar todo el camino a través de la yema de la extremidad cuando sea posible, aunque en etapas más jóvenes esto es difícil. NOTA: En los embriones de más edad (estadio HH 22 y más) es posible seleccionar regiones específicas de la yema de la extremidad para injertar, pero en etapas más jóvenes esto es más difícil, ya que la extremidad del brote en sí es pequeña. También es importante recordar que el primordio de la extremidad crecerá más allá del talón injertado así, especialmente en las etapas más jóvenes, el cordón se mantendrá en la extremidad proximal. </li><li> Recoge un grano, ya sea del factor de crecimiento o de drogas utilizando wa extra finotchmakers fórceps o un lazo hecho de una aguja de tungsteno. Si el cordón se ha empapado en DMSO o rojo de fenol, enjuague en PBS antes de aplicar para el embrión. Transferir el talón al embrión con el fórceps / asa de alambre. A continuación, utilice el alambre de tungsteno afilada para insertar el cordón en la incisión. </li><li> Añadir 1-2 ml de PBS / FCS al huevo asegurar que el embrión se mantiene hidratado pero evite aplicar directamente sobre el propio embrión ya que esto puede dañar los embriones y causar el talón sea desalojado. Sellar el huevo con cinta y volver a la incubadora. </li></ol>

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The authors have nothing to disclose.

El uso de injertos de cuentas aplicados directamente al desarrollo de tejidos in ovo es una herramienta poderosa para diseccionar el papel de la señalización del factor de crecimiento durante el desarrollo dando un control sin precedentes sobre el estado de desarrollo en que se aplican y la duración de la exposición. La elección del talón para cada tipo de molécula es importante. Moléculas hidrofóbicas pequeños, tales como los inhibidores aquí descritos y el ácido retinoico, por ...

Embriones de aves han proporcionado una poderosa herramienta para el estudio del desarrollo durante muchos años 1. Una de sus características más útiles es que son relativamente fáciles de manipular. Desarrollo externo hace que sea posible abrir el huevo para acceder al embrión y realizar diversas micromanipulaciones incluyendo ejemplos tales como el sistema quimera codorniz-chick clásico para estudiar el destino celular 2,3, la inyección de los retrovirus para la sobreexpresión en tejidos específicos durante el desarrollo 4,5 y la cultura explante para identificar las fuentes de señalización de desarrollo 6. Más recientemente quimeras generada entre hosts sin marcar con injertos de una línea de pollo transgénico que expresa GFP ha demostrado que la combinación de injerto clásica y la modificación genética puede proporcionar importantes conocimientos sobre el desarrollo de 7,8. La facilidad con la que el embrión aviar puede ser manipulado ha hecho que sea un modelo excelente para el estudio de las extremidadesdesarrollo 9. La aplicación de factores de crecimiento específicos para los miembros en desarrollo in vivo ha sido fundamental en los factores que alteran los patrones extremidad 10,11 y continúa proporcionando conocimientos sobre este proceso 12 identificación. Este enfoque también se ha utilizado para estudiar los factores que regulan el desarrollo muscular y ha descubierto roles para numerosas señales tales como Wnts 13, BMPs 14 y 15 HGF. Recientemente, esta técnica se ha utilizado para investigar las señales que controlan la expresión génica en myogenic la yema del miembro y ha demostrado que las interacciones entre FGF18 y el ácido retinoico puede controlar el momento de la expresión de MyoD 16. Usando una combinación de factores de crecimiento y moléculas pequeñas que se pueden cargar en perlas y luego injertados directamente en los tejidos específicos en las etapas de desarrollo definidas da la oportunidad de intervenir en casi cualquier momento y la región durante el desarrollo. Esto ha sido utilizado para investigate muchos procesos incluyendo somito patrones 17,18, especificación neural 19, la migración de la cresta neural 20 y la extensión del eje 21. Aquí se describe un método para injertar perlas empapadas en cualquiera de los factores de crecimiento o inhibidores en el desarrollo de las extremidades de pollo. Esto ha sido utilizado para determinar los efectos de estas señales en la miogénesis mediante el análisis de la expresión génica específica de músculo con hibridación in situ. Se describen injertos utilizando heparina empapada perlas, que se utilizan para factores de crecimiento, o perlas de AG 1-X2 para las pequeñas moléculas hidrófobas tales como el ácido retinoico o inhibidores de moléculas pequeñas de las vías de señalización específicas. Sin embargo otras perlas están también disponibles que se han utilizado para ofrecer tanto FGFs 22 y 23 Shh.

<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

El uso de embriones de pollo es posible probar directamente los efectos de cualquiera de los factores de crecimiento o inhibidores específicos de las vías de señalización sobre la expresión génica y la activación de vías de transducción de señal. Esta técnica permite la entrega de moléculas de señalización en las etapas de desarrollo definidas con precisión para momentos específicos. Después de esto, los embriones pueden ser cosechadas y examinaron la expresión de genes, por ejemplo mediante hibridación in situ, o activación de las vías de transducción de señales observados con la inmunotinción. En vídeo perlas de heparina empapados en FGF18 o AG perlas 1-X2 empapados en U0126, un inhibidor de MEK, están injertados en el esbozo de la extremidad in ovo. Esto demuestra que FGF18 induce la expresión de MyoD y la fosforilación de ERK y la expresión MyoD tanto endógenas como inducida FGF18 es inhibida por U0126. Cuentas empapados en un antagonista del ácido retinoico pueden potenciar la inducción prematura MyoD por FGF18. Este enfoque puede ser nosotrosed con una amplia gama de diferentes factores de crecimiento e inhibidores y se adapta fácilmente a otros tejidos en el embrión en desarrollo.

En la etapa HH 21 MyoD no se expresa en el desarrollo de las extremidades mioblastos aunque la tinción se puede ver en la miotoma de los somitas en desarrollo (Figura 1A). La Figura 1B muestra la hibridación in situ para MyoD 6 hr después de un injerto de talón FGF18. MyoD se induce en mioblastos cercanos a la perla, mientras que no hay expresión en el miembro contralateral. Co-injerto de un grano empapado en U0126, un inhibidor específico de MEK, bloques FGF18 inducción...

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.

Injerto de Cuentas en desarrollo Miembros de embrión de pollo para identificar señales de transducción Pathways afectan la Expresión Génica

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 vistas.  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.

Video: 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).

Optimizado<em&gt; Ex-ovo</em&gt; El cultivo de embriones del polluelo a etapas avanzadas de desarrollo

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

Investigación

Biología del Desarrollo

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.

Etiquetado Dual De Las Células De La Cresta Neural Y Los Vasos Sanguíneos Dentro De Los Embriones De Pollo Usando ChickGFP Injerto De Tubo Neural Y Carbocianina Tinte DiI Inyección

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.

Microbead Implantación en el embrión de pez cebra

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.

La colocación de crecimiento recubierto Factor-bolas en los embriones tempranos de la etapa de pollo

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

Biología

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.

En Electroporations Ovo de embriones de pollo HH Etapa 10

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.

La aplicación de barreras impermeables combinadas con empapó las bolas Factor Candidato para el Estudio de señales inductivas en el polluelo

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.

Ensayo de inducción neural en el embrión de pollo

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.

Transfección de células individuales en embriones de pollo

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.

Ensayo de extremidades recombinantes de pollo para comprender la morfogénesis, los patrones y los primeros pasos en la diferenciación celular

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.

Análisis de la diferenciación celular, la morfogénesis y el patrón durante la embriogénesis del pollo utilizando el ensayo de cuentas empapadas

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

Injerto de Cuentas en desarrollo Miembros de embrión de pollo para identificar señales de transducción Pathways afectan la Expresión Génica

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Etiquetado Dual De Las Células De La Cresta Neural Y Los Vasos Sanguíneos Dentro De Los Embriones De Pollo Usando Chick^GFPInjerto De Tubo Neural Y Carbocianina Tinte DiI Inyección