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.

Etica Dichiarazione: Tutti questi esperimenti seguono la cura degli animali e le linee guida etiche del l&#39;Università di Nottingham. 1. Preparazione di Beads eparina per innesto <ol><li> Lavare accuratamente perle di eparina in PBS prima dell&#39;uso. Nota: Beads possono essere conservati a 4 ° C come una sospensione in PBS. <ol><li> Selezionare perline per innesto rimuovendoli dal magazzino con una pipetta 20 microlitri in una goccia 1 ml di PBS. Quindi utilizzare una micropipetta set a 2 ml di trasferire perle in un calo del 20 microlitri di PBS in una capsula di Petri 3 centimetri. Scegli perline in base alle dimensioni e, utilizzando un impianto stereo dissezione microscopio, trasferire perline selezionati con la punta della pipetta P2; quelli intorno a 100 micron di diametro sono l&#39;ideale. </li></ol></li><li> Rimuovere la PBS dalle perline. Rimuovere la maggior parte del liquido con una pipetta 20 microlitri e il liquido residuo per azione capillare con orologeria pinza sottile. Nota: È importante rimuovere il più possibile così la crescita factor non è diluito quando viene aggiunto ai talloni. </li><li> Pipettare il fattore di crescita direttamente sulle perline. Per FGF18 aggiungere 0,5 ml di FGF18 ricombinante ricostituito a 0,5 mg / ml in 0,1% di BSA in PBS. Mettere alcune gocce di acqua intorno ai bordi del piatto per evitare che il liquido da evaporare. </li><li> Incubare le perline per 1 ora a RT. Rimuovere le gocce d&#39;acqua dal piatto con una pipetta Pasteur e disporli sul ghiaccio pronta per l&#39;innesto. </li><li> In caso di necessità (per perle trasparenti) trasferimento 4-5 perle al 2% rosso fenolo prima di innesto per aiutarli a visualizzare. Risciacquare in PBS prima del trasferimento per l&#39;embrione. </li></ol> 2. Preparazione di AG Beads 1-X2 per innesto <ol><li> Prima dell&#39;uso derivatize perline con 0,2 N di acido formico. <ol><li> Per fare questo uso una spatola per trasferire perline in una provetta da 1,5 ml microcentrifuga. Aggiungere 1 ml di 0,2 N di acido formico e lavare su una piattaforma di agitazione per 1 ora. </li><li> Quindi rimuovere l&#39;acido formico con una pipetta p1000 e sostituirlo con 1ml di acqua. Lavare per 1 ora e ripetere sei volte per rimuovere residuo acido formico. </li><li> Rimuovere il liquido residuo con una pipetta p1000 e conservare le perline a 4 ° C. Nota: Non è necessario rimuovere tutta l&#39;acqua residua in questa fase. </li></ol></li><li> Rimuovere una piccola pallina di perle di circa il 5-10 volumi microlitri dal magazzino con una piccola spatola in una capsula di Petri 3 centimetri e aggiungere 1 ml di DMSO (o qualsiasi solvente il farmaco è dissolto in). Nota: Questo dovrebbe fornire diverse centinaia di perline da cui selezionare quelli della giusta misura. </li><li> Quindi utilizzare una pipetta P2 impostata su 2 microlitri di trasferire perle di circa 100 micron di diametro per un calo del 20 ml di solvente in un altro piatto di Petri 3 centimetri. </li><li> Rimuovere il solvente con una pipetta 20 microlitri e qualsiasi solvente residuo con una pipetta 2 microlitri. Sostituire con 20 ml di farmaco da applicare. NOTA: Per coerenza sciogliere il farmaco ad una concentrazione di 100 mM in DMSO (o che mai è utilizzato solvente),un&#39;aliquota in 20 microlitri e conservare a -80 ° C, come alcuni piccoli inibitori molecolari sono instabili ed è meglio evitare ripetuti di congelamento-scongelamento. Se necessario diluire il farmaco alla concentrazione desiderata prima di ammollo perline. Concentrazioni efficaci possono avere bisogno di essere determinati empiricamente per ogni farmaco. </li><li> Incubare per 1 ora. Proteggere dalla luce come molti di queste molecole sono sensibile alla luce. </li><li> Prima innesto trasferimento 4-5 perle con una pipetta p2 impostato 2 microlitri in 20 ml di 2% di rosso fenolo disciolti in acqua. Eseguire questo passaggio usando un microscopio da dissezione stereo per visualizzare le perline. Rimuovere una singola goccia di orologiai pinza sottile o un cappio e risciacquare in PBS prima del trasferimento per l&#39;embrione. Non lasciare perline in 2% rosso fenolo per più di 15 minuti prima dell&#39;uso come questo può causare la perdita di attività. </li></ol> 3. Preparazione tungsteno aghi <ol><li> Tagliare multa filo di tungsteno in 3-4 cm di lunghezza. Inserire un pezzo di filo al fine diuna pipetta Pasteur di vetro e si fondono in un becco Bunsen per fissare in posizione. Preparare fino a 10 aghi per assicurare che non vi siano parti di ricambio se danneggiati durante l&#39;uso. </li><li> Posizionare l&#39;estremità del filo in una fiamma ossidrica e tenerla lì fino a quando il tungsteno diventa bianco e la punta è affilata (circa 2-3 minuti). Nota: Durante l&#39;uso l&#39;ago può essere pulito e ri-affilata nella fiamma ossidrica, se necessario. NOTA: Questi aghi possono essere utilizzati anche per creare dei cicli per il trasferimento di perline. Toccare delicatamente l&#39;ago per la panchina e si piega a formare un anello. </li></ol> 4. Gli embrioni Preparazione per Bead innesti <ol><li> Incubare le uova con l&#39;estremità smussata fino a quando non raggiungono lo stadio desiderato (3-5 giorni per la maggior parte le manipolazioni gemma arto). Utilizzando pinze smussato toccare la punta smussata per rompere il guscio e quindi utilizzare le pinze per rimuovere il guscio ed esporre l&#39;embrione. Assicurarsi che la membrana guscio d&#39;uovo viene rimosso anche. NOTA: 1-2 ml di soluzione salina può essere aggiunto con una pipetta Pasteur per assistere conquesto se la membrana aderisce al tuorlo. Pipetta delicatamente la PBS sulla superficie del tuorlo, afferrare la membrana con le pinze, facendo attenzione a non danneggiare il tuorlo, e tirare delicatamente via dall&#39;uovo. </li><li> Rimuovere fino a 5 ml di albume dall&#39;uovo con una siringa da 10 ml e un ago 19 G. Prendere solo quanto necessario per garantire l&#39;embrione non entrare in contatto con il nastro utilizzato per sigillare l&#39;uovo come rimozione di volumi più grandi in grado di ridurre i tassi di sopravvivenza degli embrioni. </li><li> Per visualizzare l&#39;embrione aggiungere un 5-6 gocce di artisti china a 15 ml di PBS contenente 100 U penicillina e 0,1 mg di streptomicina / ml. Iniettare 0,5-1 ml direttamente sotto l&#39;embrione con una siringa da 1 ml e un ago 25 G. </li><li> Aggiungere 2-3 ml di PBS 1% con siero fetale bovino e 100 U di penicillina e 0,1 mg di streptomicina / ml nell&#39;uovo di garantire l&#39;embrione non disidratarsi. Utilizzando affilate orologiai pinze rimuovere la membrana vitellina e aprire il sacco amniotico sulle gemme degli arti in via di sviluppo. Assicurarsi che l&#39;embrione non si secchi e, Se necessario, aggiungere altro PBS / FCS. NOTA: Le gemme degli arti può essere visto chiaramente come escrescenze appaiati sul fianco dell &#39;embrione. A queste fasi dell&#39;embrione girerà come il lato destro è normalmente superiore. Il risultato è normalmente più facile innestare perle nelle arti di destra e utilizzare quelli lasciati come controlli controlaterale. </li><li> Utilizzare un filo di tungsteno affilato per fare un&#39;incisione nella gemma arto dove sarà impiantato il tallone. Evitare di tagliare tutto il percorso attraverso la gemma arto, se possibile, anche se a fasi più giovani questo è difficile. NOTA: in embrioni più anziani (HH stadio 22 e sopra) è possibile selezionare specifiche regioni del bocciolo arto per l&#39;innesto, ma nelle fasi più giovani questo è più difficile come l&#39;arto gemma in sé è piccolo. E &#39;anche importante ricordare che la gemma arto crescerà oltre il tallone innestato così, soprattutto nelle fasi più giovani, il tallone rimarrà nell&#39;arto prossimale. </li><li> Prendete una perlina sia dal fattore di crescita o di droga utilizzando wa extra finetchmakers forcipe o un ciclo fatto da un ago di tungsteno. Se il cordone è stato immerso in uno DMSO o rosso fenolo, lavare in PBS prima di applicare per l&#39;embrione. Trasferire il tallone per l&#39;embrione con la pinza / cappio. Quindi utilizzare il filo di tungsteno affilato per inserire il tallone nell&#39;incisione. </li><li> Aggiungere 1-2 ml di PBS / FCS all&#39;uovo assicurare che l&#39;embrione è mantenuta idratata ma evitare di applicare direttamente sulla dell&#39;embrione stesso come questo può danneggiare gli embrioni e causare il tallone da sloggiato. Sigillare l&#39;uovo con nastro e tornare alla incubatrice. </li></ol>

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The authors have nothing to disclose.

L&#39;utilizzo di innesti tallone applicati direttamente allo sviluppo di tessuti in ovo è un potente strumento per analizzare il ruolo del fattore di crescita di segnalazione durante lo sviluppo dando il controllo senza precedenti sopra lo stadio di sviluppo in cui vengono applicate e la durata dell&#39;esposizione. La scelta del cordone per ogni tipo di molecola è importante. Piccole molecole idrofobe, come gli inibitori qui descritte ed acido retinoico, di solito si legano bene al deriv...

Embrioni aviaria hanno fornito un potente strumento per lo studio di sviluppo per molti anni 1. Una delle loro caratteristiche più utili è che sono relativamente facili da manipolare. Sviluppo esterno consente di aprire l&#39;uovo per accedere l&#39;embrione ed eseguire varie micromanipolazioni con esempi come il sistema chimera quaglia-chick classico per lo studio il destino della cellula 2,3, l&#39;iniezione di retrovirus per sovraespressione in tessuti specifici durante lo sviluppo 4,5 e la cultura espianto di identificare le fonti di segnalazione di sviluppo 6. Più recentemente chimere generato tra gli host non etichettati con innesti da una linea pollo transgenici che esprimono GFP ha dimostrato che la combinazione di innesto classica e la modificazione genetica in grado di fornire importanti intuizioni sviluppo 7,8. La facilità con cui l&#39;embrione aviaria può essere manipolato ha reso un ottimo modello per lo studio degli artisviluppo 9. L&#39;applicazione di fattori di crescita specifici per gli arti in via di sviluppo in vivo è stato fondamentale per identificare i fattori che alterano arto patterning 10,11 e continua a fornire intuizioni in questo processo 12. Questo approccio è stato utilizzato anche per studiare i fattori che regolano lo sviluppo muscolare e ha scoperto ruoli per numerosi segnali, come Wnts 13, BMP 14 e HGF 15. Recentemente questa tecnica è stata utilizzata per studiare i segnali che controllano l&#39;espressione genica myogenic sul nascere degli arti e ha dimostrato che le interazioni tra FGF18 e acido retinoico può controllare i tempi di espressione MyoD 16. Usando una combinazione di fattori di crescita e piccole molecole che possono essere caricati su perline e poi innestate direttamente nei tessuti specifici a stadi di sviluppo definiti dà la possibilità di intervenire in quasi ogni momento e regione durante lo sviluppo. Questo è stato usato per investigate molti processi tra cui somite patterning 17,18, specificazione neurale 19, neural crest migrazione 20 e l&#39;estensione dell&#39;asse 21. Qui si descrive un metodo per perline imbevute di entrambi i fattori di crescita o inibitori nello sviluppo di arti di pollo innesto. Questo è stato utilizzato per determinare gli effetti di questi segnali su miogenesi analizzando muscolo espressione genica specifica con ibridazione in situ. Descriviamo innesti utilizzando eparina imbevuto perline, che sono utilizzati per fattori di crescita, o perline AG 1-X2 per piccole molecole idrofobe quali acido retinoico o piccole molecole inibitrici di vie di segnalazione specifici. Tuttavia altre perle sono disponibili che sono stati utilizzati per fornire sia FGFs 22 e 23 anche 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

Usando embrioni di pollo è possibile testare direttamente gli effetti delle due fattori di crescita o inibitori specifici di vie di segnalazione di espressione genica e attivazione di vie di trasduzione del segnale. Questa tecnica permette la consegna di molecole di segnalazione in stadi di sviluppo ben definiti per i tempi specifici. Dopo questo embrioni possono essere raccolte e l&#39;espressione genica esaminati, ad esempio in situ ibridazione, o l&#39;attivazione di vie di trasduzione del segnale osservato con immunocolorazione. In questo video di eparina perline imbevuti di FGF18 o AG perline 1-X2 imbevute di U0126, un inibitore di MEK, si innestano nel bocciolo arto in ovo. Questo dimostra che FGF18 induce l&#39;espressione di MyoD e ERK fosforilazione e espressione MyoD sia endogena e FGF18 indotta è inibito dalla U0126. Perline imbevuto di un antagonista dell&#39;acido retinoico possono potenziare l&#39;induzione prematura MyoD da FGF18. Questo approccio può essere noied una vasta gamma di fattori di crescita e inibitori e si adatta facilmente ad altri tessuti embrioni.

Nella fase HH 21 MyoD non è espresso in mioblasti sviluppo arto sebbene colorazione può essere visto in myotome dei somiti sviluppo (Figura 1A). Figura 1B mostra ibridazione in situ per MyoD 6 ore dopo un innesto FGF18 tallone. MyoD è indotta in mioblasti vicino al tallone, mentre non vi è alcuna espressione nell&#39;arto controlaterale. Co-innestando un branello imbevuto U0126, un inibitore specifico di MEK, induzione blocchi FGF18 di MyoD (Figura 1C). All...

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.

L&#39;innesto di Perline in via di sviluppo di pollo Limbs embrioni per identificare percorsi di trasduzione del segnale che influenzano l&#39;espressione genica

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 Views.  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).

Ottimizzato<em&gt; Ex-ovo</em&gt; Coltura di Chick embrioni a stadi avanzati di sviluppo

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

Ricerca

Biologia dello sviluppo

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.

Doppia etichettatura delle cellule della cresta neurale e dei vasi sanguigni all'interno degli embrioni di pollo utilizzando l'innesto del tubo neuraleGFP del pulcino e l'iniezione di colorante carbocianina

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.

Microperla impianto nel zebrafish

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.

Posizionamento Growth Factor-Coated Perline su embrioni di pollo Stadio

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

Biologia

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.

In Electroporations Ovo della Fase HH 10 embrioni di pollo

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.

L&#39;applicazione di barriere impermeabili combinati con un fattore di Candidato Perle Soaked per studiare i segnali induttivi nel 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 ...

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.

Test di induzione neurale nell&#39;embrione Chick

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.

Cella Transfection singola in embrioni di 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.

Test degli arti ricombinanti di pollo per comprendere la morfogenesi, i modelli e i primi passi nella differenziazione cellulare

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.

Analisi della differenziazione cellulare, della morfogenesi e del pattern durante l'embriogenesi del pollo utilizzando il test Soaked-Bead

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

L'innesto di Perline in via di sviluppo di pollo Limbs embrioni per identificare percorsi di trasduzione del segnale che influenzano l'espressione genica

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Doppia etichettatura delle cellule della cresta neurale e dei vasi sanguigni all'interno degli embrioni di pollo utilizzando l'innesto del tubo neurale^GFPdel pulcino e l'iniezione di colorante carbocianina