Name: a novel ex ovo banding technique to alter intracardiac hemodynamics in an embryonic chicken system
Uploaded: 2016-05-13T13:00:00.000+00:00
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Description: Watch this Scientific Journal Video about A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System at JoVE.com

The authors would like to acknowledge Dr. Robert Price and the staff of the Instrumentation Resource Facility at the University of South Carolina School of Medicine. This work was partially supported by a SPARC Graduate Research Grant from the Office of the Vice President for Research at the University of South Carolina (JDP/VM). In addition this work was supported by Cook Biotech research agreement (JDP) and by FirstString Research Inc (JDP) and NIH 2 P20-RR016434-06 (JDP). In addition, NIH IDeA Networks of Biomedical Research Excellence (INBRE) grant for South Carolina P20GM103499 (JE)

embrioni aviaria non sono considerati animali vertebrati ai sensi della normativa IUCAC. 1. Ottenere embrioni per la chirurgia <ol><li> Incubare 80 - 90 uova di gallina Bovan fecondate (smussato finire) in un umidificata (60%) bilanciere incubatore a 40 ° C per circa 72 ore per ottenere embrioni a Hamilton e Hamburger (HH) fase 17. determinare il numero esatto di uova sulla base di analisi della potenza e il tasso di sopravvivenza degli embrioni 17. Utilizzare i vassoi di uova di plastica per l&#39;incubazione per consentire un&#39;adeguata circolazione dell&#39;aria. </li><li> Dopo questa fase di sviluppo desiderato è raggiunto, versare circa 20 ml di acqua calda (37 ° C) di tampone di Tyrode (integrato con bicarbonato di sodio (1 g / L)) in un x 26 mm piastra di Petri a 100 mm. </li><li> Sterilizzare un guscio d&#39;uovo con il 70% di etanolo. </li><li> rompere delicatamente il guscio con un manico bisturi e rilasciare con cautela il contenuto nel piatto contenente tampone di Tyrode. Scartare gli embrioni se (i) in cui appaiono visivamente anomala (ii)non sono in fase di sviluppo a destra (iii) sono impropriamente orientati sulla tuorlo e / o (iv) si verifica un sanguinamento. Conservare eventuali embrioni non soggetti a un intervento chirurgico subito dopo essere state poste ex ovo a 40 ° C in modo da non compromettere lo sviluppo. Nota: Staging è effettuata sulla base di Hamilton e Hamburger 18. Anomalie di embrioni di pollo è determinata ad occhio nudo e nell&#39;ambito di applicazione dissezione. Assicurarsi che l&#39;embrione ha piegatura appropriata e flessione e vascolare. </li></ol> 2. OFT Banding <ol><li> Tweeze fuori singoli thread lunghi 1 cm da un unico 11/0 nylon sutura chirurgica e formare un nodo sciolto. UV sterilizzare tutti i nodi preformati. </li><li> Sotto un microscopio dissezione, assicurarsi visivamente che il cuore batte a una velocità normale (~ 120 battiti / min). In caso contrario, eliminare l&#39;embrione. </li><li> Appena prima dell&#39;intervento chirurgico, applicare 5 - 6 ml di acqua calda (37 ° C) di tampone di Tyrode alla superficie embrione utilizzando una pipetta di trasferimento. </li><li> Far passare una estremità libera del nodo preformato sotto l&#39;OFT, posizionare la sutura al OFT / ventricolo giunzione (OVJ) (o posizione desiderata), e passare l&#39;estremità libera nel nodo costrizione in tal modo l&#39;OFT comprese le membrane che circondano il cuore. </li><li> Dopo l&#39;intervento chirurgico, bagnare la superficie del tuorlo con 5 - 6 ml di calda (37 ° C) di tampone di Tyrode utilizzando una pipetta di trasferimento. </li><li> Mantenere gli embrioni di controllo ex ovo come gli embrioni a bande; tuttavia non li soggetti alla procedura chirurgica. </li><li> Incubare gli embrioni, per la quantità di tempo desiderato, a 40 ° C in un (60%) incubatrice umidificata. </li><li> Una volta che il punto di tempo desiderato viene raggiunto, asportare l&#39;embrione dal tuorlo con le forbici diritte. Sezionare il cuore con una pinza sottile e utilizzare per diversi studi a valle, come i cambiamenti nella morfologia cardiaca a causa di alterata emodinamica 17. </li></ol> 3. A conferma che l&#39;intervento bendaggio causa un cambiamento nella Emodinamica Nota: La costrizione parziale dovuto all&#39;intervento bande porta ad un aumento della velocità del flusso sanguigno al OVJ. Questo parametro emodinamico è convenientemente valutata utilizzando immagini ecografiche 2D, che viene eseguita nel punto desiderato tempo dell&#39;esperimento. <ol><li> Poiché solo un singolo embrione può essere ripreso alla volta, mantenere tutti gli altri embrioni designati per l&#39;imaging ad ultrasuoni in un incubatore a 40 °. </li><li> Porre la capsula di Petri contenente l&#39;embrione di essere ripreso su una piastra elettrica fissata a 40 ° C. </li><li> Riempire il piatto, fino all&#39;orlo, con acqua calda (37 ° C) del buffer di Tyrode. Versare lentamente la soluzione tampone nel piatto in modo da mantenere intatto il tuorlo. Se, tuttavia, l&#39;integrità del tuorlo è compromessa durante questa fase, scartare l&#39;embrione. </li><li> Orient dell&#39;embrione tale che l&#39;asse centrale dell&#39;embrione è perpendicolare alla sonda ad ultrasuoni che è sospeso da un supporto regolabile. </li><li> Con l&#39;opera macchina ad ultrasuoniTing in B-mode, ottenere un&#39;immagine 2D del cuore pulsante sullo schermo e spostare il palco (piastra elettrica) in modo tale che l&#39;OFT, ventricolo e OVJ sono chiaramente visibili. </li><li> Passare alla modalità pulsato onda (PW), alla frequenza di ripetizione degli impulsi desiderata (ad esempio, 20 kHz) e di ottenere esattamente dati di velocità al OVJ. Una immagine B-mode del cuore pulsante si ottiene sullo schermo. <ol><li> Spostare lo stadio per vedere l&#39;OFT, ventricolo e la OVJ. Utilizzando il software macchina ad ultrasuoni, ottenere misure di velocità esattamente al OVJ. </li></ol></li></ol> Nota: Se la frequenza cardiaca di un embrione diminuisce durante l&#39;imaging, i dati di velocità così ottenuti non devono essere utilizzati per l&#39;analisi. Tutti embrioni utilizzati per misure di velocità non dovrebbero preferibilmente essere utilizzati per altri esperimenti dopo ecografica.

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The authors have nothing to disclose.

Questa tecnica è relativamente semplice e facile da eseguire, tuttavia alcuni punti chiave devono essere tenuti in considerazione in modo da ottenere risultati accurati valle. Gli embrioni devono essere mantenuti ex ovo in una capsula di Petri che contiene tampone di Tyrode di fornire adeguata reidratazione. E &#39;anche importante per idratare il tuorlo post-chirurgia con tampone di Tyrode e per assicurarsi che la camera di incubazione è adeguatamente idratato. Interventi chirurgici non devono essere eseguite su un embrione eventualmente sanguinamento è visibile o il tuorlo è anche un po &#39;rotto. Questo influenza la sopravvivenza degli embrioni, soprattutto per esperimenti a lungo termine. Un&#39;altra causa potenziale di letalità di embrioni, è se la fascia intorno alla OFT è troppo stretto quando applicato prima, in tal modo compromettere in modo significativo la funzione cardiaca. Allo stato attuale, si usa ultrasuoni come strumento di conferma per determinare la variazione di velocità del flusso sanguigno su OFT banding. In futuro, ci sarà confermare con micro-tomografia computerizzata (CT). Abbiamo anche determinare visivamente la tenuta fascia intorno alla OFT in immagini 2D B-mode. È importante sottolineare che non si osservano differenze nella frequenza cardiaca e del flusso volumetrico (calcolata utilizzando CFD) tra embrioni nei banded e gruppo di controllo 17. Questi indicano che la fisiologia del sistema cardiovascolare non è compromesso negli embrioni bande, così convalidando ulteriormente l&#39;intervento strisce. Al punto di tempo desiderato, dopo strisce, diverse analisi possono essere eseguite per capire l&#39;effetto di emodinamica alterati in processi di sviluppo delle valvole critica. Questi includono, ma non sono limitati a, determinando cambiamenti nei livelli di trascrizione di geni regolatori chiave coinvolti nello sviluppo della valvola, determinando la localizzazione dei componenti di ECM e la determinazione della ultrastruttura della OFT. Infine, questa tecnica migliora l&#39;attuale metodo di banding l&#39;OFT nell&#39;embrione all&#39;interno dell&#39;uovo finestra 19. Tale metodo è gravemente ostacolato dalla incapacità di accumulare embrioni necessari per fare analisi molecolari a valle. Questa nuova tecnica può essere utilizzata in esperimenti di soccorso pure. Questa applicazione permette di bande OFT di essere salvati ed embrione permesso di recuperare l&#39;insulto. Nessun metodo corrente può fare questo ed essere ancora in grado di eseguire analisi a valle statisticamente significative. Abbiamo impiegato con successo questa tecnica per cambiare gli stimoli emodinamici nel cuore di embrioni di pollo in una fase molto precoce dello sviluppo (HH 16 - 17). Il protocollo bendaggio potrebbe presentare una sfida quando si esegue l&#39;intervento nelle fasi successive di sviluppo a causa del rischio di intaccare qualsiasi dei vasi sanguigni extraembrionali. Inoltre, negli stadi di sviluppo, è spesso difficile mantenere il sacco vitellino intatti quando il contenuto delle uova vengono trasferiti nella capsula di Petri.

Abnormally formed outflow valves are the most common type of congenital heart defects 1. However, defective cardiac valve structure and function, even though present at birth, may become symptomatic only in adulthood. In fact, several adult valve diseases can be attributed to a congenital origin. Treatment of such patients often involves replacing defective valves, and, importantly, replaced aortic valves have been shown to have congenital anomalies 2. Given the fact that critical processes involved in valve development begin early during embryogenesis, the importance of better understanding the mechanisms that regulate these events is highlighted. 
The primitive heart tube, which is the first functioning organ in an embryo, exhibits two distinct layers - an endothelial endocardium surrounded by myocardium - separated by extracellular matrix (cardiac jelly) which is mostly produced and secreted by the myocardium 3-5. As development continues, valve primordia (endocardial cushions) are formed, after rightward looping of the embryonic heart, by local expansion of the cardiac jelly at the atrioventricular (AV) canal and the outflow tract (OFT) 4,6. This expansion is mediated by the highly regulated process of epithelial-mesenchymal transition (EMT), during which the cardiac jelly becomes populated by endocardially-derived mesenchymal cells 6. In addition to the mesenchymal population derived through EMT, neural crest cells are also involved in valvulogenesis of the OFT 3. 
Hemodynamic stimuli, such as shear stress, are important epigenetic factors that regulate heart development in the embryo 7,8. Using a 3D in vitro system, we have previously shown shear stress to be a factor influencing the expression and deposition of fibrous extracellular matrix (ECM) proteins in AV and OFT cushions 9,10. Moreover, studies carried out by several researchers have demonstrated that altered blood flow leads to improper valves and septa formation 11-16. Recently, using the novel banding procedure presented here, we have shown that changing hemodynamics in the embryonic chick heart affects the early processes involved in OFT valve formation 17.
The technique described here provides a novel model for altering hemodynamics in the developing chick heart by partially constricting the OFT ex ovo. This reproducible procedure is relatively quick and allows researchers to obtain a sufficient number of embryos/whole hearts/OFT tissue, etc. for downstream analyses including gene expression studies. Moreover, this new model can be used to study 'chronic' effects of altered hemodynamics on OFT valve development.

<table><tbody><tr><td>Fertilized Bovan chicken eggs</td><td>Clemson University, Clemson, SC</td><td></td><td></td></tr><tr><td>11 / 0 Nylon suture</td><td>Ashaway</td><td>S30001</td><td>UV sterilize knots before surgery</td></tr><tr><td>100 x 26 mm petri dish</td><td>VWR</td><td>25387-030</td><td></td></tr><tr><td>Transfer pipettes</td><td>Thermo Scientific&amp;nbsp;</td><td>232-20S</td><td></td></tr><tr><td>Scalpel handle #3</td><td>Fine Science Tools</td><td>91003-12</td><td></td></tr><tr><td>Straight scissor</td><td>Roboz</td><td>RS-6702</td><td></td></tr><tr><td>Dumont #5 fine forceps</td><td>Fine Science Tools</td><td>11254-20</td><td></td></tr><tr><td>Tyrodes buffer</td><td>Sigma-Aldrich</td><td>2145-10L</td><td>Filter sterlize before use&amp;nbsp;</td></tr><tr><td>Sodium bicarbonate</td><td>Fisher Scientific</td><td>S233-500</td><td></td></tr><tr><td>Vevo 770 Ultrasound Imaging system</td><td>VisualSonics, Inc.</td><td>VS-11392</td><td></td></tr><tr><td>708 Ultrasound transducer&amp;nbsp;</td><td>VisualSonics, Inc.</td><td>VS-11171</td><td></td></tr></tbody></table>

a novel ex ovo banding technique to alter intracardiac hemodynamics in an embryonic chicken system

The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and molecular level. To alter intracardiac hemodynamics, fertilized chicken eggs are incubated in a humidified chamber to obtain embryos of the desired stage (HH17). Once this developmental stage is achieved, the embryo is maintained ex ovo and hemodynamics in the embryonic heart are altered by partially constricting the outflow tract (OFT) with a surgical suture at the junction of the OFT and ventricle (OVJ). Control embryos are also cultured ex ovo but are not subjected to the surgical intervention. Banded and control embryos are then incubated in a humidified incubator for the desired period of time, after which 2D ultrasound is employed to analyze the change in blood flow velocity at the OVJ as a result of OFT banding. Once embryos are maintained ex ovo, it is important to ensure adequate hydration in the incubation chamber so as to prevent drying and eventually embryo death. Using this new banded model, it is now possible to perform analyses of changes in the expression of key players involved in valve development and to understand the role of hemodynamics on cellular responses in vivo, which could not be achieved previously.

Mostrato nella Figura 1 sono strumenti necessari per OFT bande raccomandato. La capsula di Petri (Figura 1A) contenente l&#39;embrione ex ovo deve essere sufficientemente profonda in modo da non distruggere l&#39;embrione quando coperto con il coperchio. Capsule di Petri profondi (Figura 1C) dovrebbero essere utilizzati anche per l&#39;imaging ad ultrasuoni per consentire un adeguato volume di tampone di Tyrode da versare in cima al tuorlo. Una singola 1 centimetro filo (Figura 2A) è tweezed fuori da un 11/0 sutura di nylon (Figura 1A) e legato in un nodo sciolto (Figura 1B). Tutti i nodi preformati sono sterilizzati ai raggi UV. La figura 3A mostra una camera rocker umidificata cui uova di pollo fecondate sono incubate fino alla fase desiderata. La figura 3B mostra un incubatore dell&#39;embrione, dove hanno legato e gli embrioni di controllo sono mantenuti ex ovo </em&gt; per la quantità di tempo desiderato. Entrambi i tipi incubatori sono mantenuti a 40 ° C. Mostrato in figura 4A è un embrione HH17 sopra il tuorlo ex ovo. Figura 4B è una rappresentazione schematica che mostra la posizione della sutura (banda) al OVJ di un embrione HH17. Figura 4C è una immagine rappresentativa di un embrione fasciato ripreso 48 ore dopo l&#39;intervento chirurgico. L&#39;effetto dell&#39;intervento bande sulla velocità del flusso di sangue attraverso il sito costrizione viene misurata mediante ecografia 2D. Come previsto, la velocità alla OVJ è maggiore nell&#39;embrione bande rispetto al controllo (Figura 5). Abbiamo recentemente eseguito fluidodinamica computazionale analisi (CFD) su cuori bande e di controllo in corrispondenza del punto di tempo di 24 ore che mostra gli effetti della OFT strisce sulla velocità del flusso sanguigno e di taglio della parete dello stress 17. &lt;p class = &quot;jove_content&quot; fo: keep-together.within-page = &quot;1&quot;&gt; <img alt="Figura 1" src="/files/ftp_upload/53955/53955fig1.jpg" /> Figura 1. strumenti chirurgici. (A) 11/0 nylon di sutura. (B) piastra di Petri contenente nodi pre-formato. (C) profonda capsula di Petri per la coltura degli embrioni ex ovo e ecografia. (D) Trasferimento pipetta. (E) Scalpel handle per rompere il guscio dell&#39;uovo. (F) forbici dritte per embrione escissione per gli studi post-operatorio. (G) una pinza sottile <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53955/53955fig1large.jpg" target="_blank">clicca qui per vedere una versione più grande di questa figura.</a> <img alt="figura 2" src="/files/ftp_upload/53955/53955fig2.jpg" /> Figura 2. sutura per OFT Banding. (A) Un unico filo 1 centimetro tweezed fuori da una sutura. <strong&gt; (B) nodo pre-formata. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53955/53955fig2large.jpg" target="_blank">Fai clic qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 3" src="/files/ftp_upload/53955/53955fig3.jpg" /> Figura 3. incubazione Chambers. (A) umidificata incubatore bilanciere per le uova fecondate. (B) camera umidificata per la cultura ex ovo di embrioni bande e di controllo. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53955/53955fig3large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 4" src="/files/ftp_upload/53955/53955fig4.jpg" /> Figura 4. OFT Banding. (A) Tutta la HH17 embrione di pollo mantenuto ex ovo, bar scala = 10 mm (B) Rappresentazione schematica raffigurante la costrizionesito (rosso). (C) fasciato embrione ripreso 48 ore dopo l&#39;OFT strisce, bar scala = 0,5 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53955/53955fig4large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 5" src="/files/ftp_upload/53955/53955fig5.jpg" /> Figura 5. Ultrasound-derivato Velocity misurazioni. Profili di velocità rappresentativo di controllo (A) e (B) fasciato embrioni ottenuti in un punto nel tempo di 1 ora. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53955/53955fig5large.jpg" target="_blank">Si prega di cliccare qui per vedere una versione più grande di questa figura.</a>

Watch this Scientific Journal Video about A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System at JoVE.com

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

For the first time we present here a reproducible banding procedure to alter hemodynamics in the developing heart ex ovo. This is achieved by partially constricting the outflow tract (OFT).

Un romanzo<em&gt; Ex Ovo</em&gt; Tecnica Banding alterare intracardiaca Emodinamica in un sistema embrionali di pollo

The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and ...

a-novel-ex-ovo-banding-technique-to-alter-intracardiac-hemodynamics

Nanyang Technological University

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JoVE Journal

Developmental Biology

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

6.5K Views.  School of Medicine, University of South Carolina. For the first time we present here a reproducible banding procedure to alter hemodynamics in the developing heart ex ovo. This is achieved by partially constricting the outflow tract (OFT). 

Video: A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying the biology of retinal neurons, particularly photoreceptors. The applications of this system go beyond basic research, as they can easily be adapted to high throughput technologies for drug development. However, genetic manipulation of retinal photoreceptors in these cultures has proven to be very challenging, posing an important limitation to the usefulness of the system. We have recently developed and validated an ex&#160;ovo plasmid electroporation technique that increases the rate of transfection of retinal cells in these cultures by five-fold compared to other currently available protocols1. In this method embryonic chick eyes are enucleated at stage 27, the RPE is removed, and the retinal cup is placed in a plasmid-containing solution and electroporated using easily constructed custom-made electrodes. The retinas are then dissociated and cultured using standard procedures. This technique can be applied to overexpression studies as well as to the downregulation of gene expression, for example via the use of plasmid-driven RNAi technology, commonly achieving transgene expression in 25% of the photoreceptor population. The video format of the present publication will make this technology easily accessible to researchers in the field, enabling the study of gene function in primary retinal cultures. We have also included detailed explanations of the critical steps of this procedure for a successful outcome and reproducibility.

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Embryonic chick retinal cell cultures constitute valuable tools for the study of photoreceptor biology. We have developed an efficient gene transfer technique based on ex&#160;ovo plasmid electroporation of the retinas prior to culture. This technique considerably increases transfection efficiencies over currently available protocols, making genetic manipulation feasible for this system.

Trasferimento efficiente Gene in Chick retine per cellulari primarie Cultura Studies: An<em&gt; Ex-ovo</em&gt; Elettroporazione Approach

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

Ricerca

Biologia dello sviluppo

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 ...

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, ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

Metodo di coltura organotypic per studiare lo sviluppo dei tessuti embrionali di pollo

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a general challenge in developmental biology research. In the embryonic heart, this can be particularly difficult as regional differences in the expression of transcriptional regulators, paracrine/juxtacrine signaling cues, and hemodynamic force are all known to influence cardiomyocyte maturation. A simplified method to alter a developing cardiomyocyte's molecular and biomechanical microenvironment would, therefore, serve as a powerful technique to examine how local conditions influence cell fate and function. To address this, we have optimized a method to physically transplant juvenile cardiomyocytes into ectopic locations in the heart or the surrounding embryonic tissue. This allows us to examine how microenvironmental conditions influence cardiomyocyte fate transitions at single cell resolution within the intact embryo. Here, we describe a protocol in which embryonic myocytes can be isolated from a variety of cardiac sub-domains, dissociated, fluorescently labeled, and microinjected into host embryos with high precision. Cells can then be directly analyzed in situ using a variety of imaging and histological techniques. This protocol is a powerful alternative to traditional grafting experiments that can be prohibitively difficult in a moving tissue such as the heart. The general outline of this method can also be adapted to a variety of donor tissues and host environments, and its ease of use, low cost, and speed make it a potentially useful application for a variety of developmental studies.

In this method, embryonic cardiac tissues are surgically microdissected, dissociated, fluorescently labeled, and implanted into host embryonic tissues. This provides a platform for studying the individual or tissue level developmental organization under ectopic hemodynamic conditions, and/or altered paracrine/juxtacrine environments.

Sistema basato su microiniezione per In Vivo l'impianto di cardiomiociti embrionali nell'embrione aviaria

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a ...

Using Chicken Embryo as a Powerful Tool in Assessment of Developmental Cardiotoxicities

Chicken embryos are a classical model in developmental studies. During the development of chicken embryos, the time window of heart development is well-defined, and it is relatively easy to achieve precise and timely exposure via multiple methods. Moreover, the process of heart development in chicken embryos is similar to mammals, also resulting in a four-chambered heart, making it a valuable alternative model in the assessment of developmental cardiotoxicities. In our lab, the chicken embryo model is routinely used in the assessment of developmental cardiotoxicities following exposure to various environmental pollutants, including per- and polyfluoroalkyl substances (PFAS), particulate matter (PMs), diesel exhaust (DE) and nano materials. The exposure time can be freely selected based on the need, from the beginning of development (embryonic day 0, ED0) all the way to the day prior to hatch. The major exposure methods include air-cell injection, direct microinjection, and air-cell inhalation (originally developed in our lab), and the currently available endpoints include cardiac function (electrocardiography), morphology (histological assessments) and molecular biological assessments (immunohistochemistry, qRT-PCR, western blotting, etc.). Of course, the chicken embryo model has its own limitations, such as limited availability of antibodies. Nevertheless, with more laboratories starting to utilize this model, it can be used to make significant contributions to the study of developmental cardiotoxicities.

Chicken embryos, as a classical developmental model, are used in our lab to assess developmental cardiotoxicities following exposure to various environmental contaminants. Exposure methods and morphological/functional assessment methods established are described in this manuscript.

Utilizzo dell'embrione di pollo come potente strumento nella valutazione delle cardiotossicità dello sviluppo

Chicken embryos are a classical model in developmental studies. During the development of chicken embryos, the time window of heart development is ...

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

Un Ex-ovo Sistema di embrioni di pollo Cultura Adatto per applicazioni di imaging e Microchirurgia

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...

Biologia

In Ovo Intravascular Injection in Chicken Embryos

As a classical model system of embryo biology, the chicken embryo has been used to investigate embryonic development and differentiation. Delivering exogenous materials into chicken embryos has a great advantage for studying gene function, transgenic breeding, and chimera preparation during embryonic development. Here we show the method of in ovo intravascular injection whereby exogenous materials such as plasmid vectors or modified primordial germ cells (PGCs) can be transferred into donor chicken embryos at early developmental stages. The results show that the intravascular injection through the dorsal aorta and head allows injected materials to diffuse into the whole embryo through the blood circulatory system. In the presented protocol, the efficacy of exogenous plasmid and lentiviral vector introduction, and the colonization of injected exogenous PGCs in the recipient gonad, were determined by observing fluorescence in the embryos. This article describes detailed procedures of this method, thereby providing an excellent approach to studying gene function, embryo and developmental biology, and gonad-chimeric chicken production. In conclusion, this article will allow researchers to perform in ovo intravascular injection of exogenous materials into chicken embryos with great success and reproducibility.

In Ovo Intravascular Injection in Chicken Embryos

The overall goal of this paper is to describe how to perform in ovo intracellular injection of exogenous materials into chicken embryos. This approach is very useful to study the developmental biology of chicken embryos.

In zona Ovo Iniezione intravascolare in embrioni di pollo

As a classical model system of embryo biology, the chicken embryo has been used to investigate embryonic development and differentiation. Delivering ...

Modello di legatura dell'atrio sinistro embrionale aviario per HLHS e ricerca cardiovascolare

Left Atrial Ligation in the Avian Embryo as a Model for Altered Hemodynamic Loading During Early Vascular Development

Due to its four-chambered mature ventricular configuration, ease of culture, imaging access, and efficiency, the avian embryo is a preferred vertebrate animal model for studying cardiovascular development. Studies aiming to understand the normal development and congenital heart defect prognosis widely adopt this model. Microscopic surgical techniques are introduced to alter the normal mechanical loading patterns at a specific embryonic time point and track the downstream molecular and genetic cascade. The most common mechanical interventions are left vitelline vein ligation, conotruncal banding, and left atrial ligation (LAL), modulating the intramural vascular pressure and wall shear stress due to blood flow. LAL, particularly if performed in ovo, is the most challenging intervention, with very small sample yields due to the extremely fine sequential microsurgical operations. Despite its high risk, in ovo LAL is very valuable scientifically as it mimics hypoplastic left heart syndrome (HLHS) pathogenesis. HLHS is a clinically relevant, complex congenital heart disease observed in human newborns. A detailed protocol for in ovo LAL is documented in this paper. Briefly, fertilized avian embryos were incubated at 37.5 &#176;C and 60% constant humidity typically until they reached Hamburger-Hamilton (HH) stages 20 to 21. The egg shells were cracked open, and the outer and inner membranes were removed. The embryo was gently rotated to expose the left atrial bulb of the common atrium. Pre-assembled micro-knots from 10-0 nylon sutures were gently positioned and tied around the left atrial bud. Finally, the embryo was returned to its original position, and LAL was completed. Normal and LAL-instrumented ventricles demonstrated statistically significant differences in tissue compaction. An efficient LAL model generation pipeline would contribute to studies focusing on synchronized mechanical and genetic manipulation during the embryonic development of cardiovascular components. Likewise, this model will provide a perturbed cell source for tissue culture research and vascular biology.

Autore in primo piano: Effetto della legatura atriale sinistra sui cuori embrionali aviari e implicazioni HLHS 

Here, we present a detailed visual protocol for executing the left atrial ligation (LAL) model in the avian embryo. The LAL model alters the intracardiac flow, which changes wall shear stress loading, mimicking hypoplastic left heart syndrome. An approach to overcome the challenges of this difficult microsurgery model is presented.

Legatura atriale sinistra nell'embrione aviario come modello per l'alterato carico emodinamico durante lo sviluppo vascolare precoce

Due to its four-chambered mature ventricular configuration, ease of culture, imaging access, and efficiency, the avian embryo is a preferred vertebrate ...

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Trasferimento efficiente Gene in Chick retine per cellulari primarie Cultura Studies: An

Ottimizzato

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

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