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
Duration: 8 min 9 s
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)

Kuş embriyolar IUCAC yönetmelikleri uyarınca omurgalı hayvanlar olarak kabul edilmez. 1. Cerrahi Embriyolar Alınması <ol><li> 80 inkübe - Hamilton ve Hamburger (HH) aşamasında embriyolar elde etmek için yaklaşık 72 saat boyunca 40 ° C&#39;de bir nemlendirilmiş (% 60) rocker inkübatör 90 döllenmiş Bovan tavuk yumurtası (kör uç yukarı) 17. dayalı yumurta sayısını tam olarak belirlemek güç analizi ve embriyoların 17 sağkalım oranı. yeterli hava dolaşımı sağlamak için inkübasyon için plastik yumurta tepsileri kullanın. </li><li> Bu istenilen gelişimsel aşamada elde edildikten sonra, 100 mm x 26 mm Petri kabı içine yaklaşık 20 ml (sodyum bikarbonat (1 g / L) ile desteklenmiş) Sıcak (37 ° C), Tyrode tampon dökün. </li><li> % 70 etanol ile bir yumurta kabuğu sterilize edin. </li><li> Yavaşça neşter sapı ile kabuk çatlak ve dikkatle Tyrode tampon içeren çanak içine içeriğini bırakın. onlar görsel görünür eğer embriyolar (i) atın anormal (ii)Sağ gelişim aşamasında (iii) yanlış sarısı ve / veya (iv) herhangi bir kanama meydana üzerine odaklı değil. Hemen gelişimi uzlaşma şekilde 40 ° C &#39;de ex ovo yerleştirildikten sonra cerrahi tabi herhangi bir embriyolar saklayın. Not: Takma Hamilton ve Hamburger 18 dayanarak yapılır. civciv embriyo anormallik çıplak gözle ve diseksiyon kapsamında belirlenir. Embriyo, uygun katlama ve büküm ve damarsal olduğundan emin olun. </li></ol> 2. OFT Bantlama <ol><li> Tek 11/0 naylon cerrahi dikiş tek tek 1 cm uzunluğunda konuları dışarı tweeze ve gevşek bir düğüm oluşturur. Tüm önceden oluşturulmuş knot sterilize UV. </li><li> Diseksiyon mikroskobu altında, görsel kalp normal hızında (~ 120 atım / dk) yenerek emin olun. Değilse, embriyo atın. </li><li> Bir transfer pipet kullanarak embriyo yüzeyine sıcak (37 ° C) Tyrode tampon 6 ml - Sadece ameliyattan önce, 5 uygulayın. </li><li> , OFT altında önceden düğüm bir serbest ucunu geçirin OFT / ventrikül kavşak (OVJ) (ya da istenilen yere) at dikiş yerleştirin ve böylece kalbi çevreleyen zarların dahil OFT daraltıcı düğüme serbest ucunu geçmektedir. </li><li> Sıcak 6 ml (37 ° C) Tyrode tampon bir transfer pipet kullanarak - Ameliyattan sonra 5 ile sarısının yüzeyini ıslak. </li><li> Kontrol embriyolar bantlı embriyolar olarak ex ovo korumak; Ancak cerrahi onları maruz bırakmayın. </li><li> nemlendirilmiş (% 60) kuluçka makinesi içinde 40 ° C &#39;de, arzu edilen bir zaman miktarı için, embriyolar inkübe edin. </li><li> İstenilen zaman noktası ulaşıldığında, düz makas kullanarak sarısından embriyo tüketim. Ince forseps ile kalbi incelemek ve bu nedeniyle değişmiş hemodinami 17 kardiyak morfoloji değişiklikleri gibi birçok aşağı çalışmalar için kullanın. </li></ol> 3. Yapıştırma Müdahale Hemodinamiğin bir değişikliğe neden olduğunu onaylama Not: bantlama müdahalesiyle kısmen daralma OVJ kan akış hızında bir artışa neden olur. Bu parametrelerden uygun deney istenen bir zaman noktasında gerçekleştirilir 2D ultrason görüntüleme kullanılarak değerlendirilir. <ol><li> yalnızca tek bir embriyo, bir zaman görüntülenebilir için, 40 ° C kuluçka makinesi içinde ultrason görüntüleme için belirlenmiş diğer embriyolar korur. </li><li> embriyolu petri 40 ° C&#39;ye ayarlanmış bir ısıtma yastığı görüntülenmek üzere yerleştirin. </li><li> Sıcak (37 ° C) Tyrode tampon ile, ağzına kadar, çanak doldurun. sağlam yumurta sarısı tutmak için çok yavaş çanak içine tampon çözelti dökün. Bununla birlikte, yumurta sarısı bütünlüğü, bu adım sırasında tehlikeye, embriyo atın. </li><li> embriyonun merkezi ekseni ayarlanabilir bir standdan süspansiyon haline ultrason probu dik olacak Orient embriyo gibi. </li><li> ultrason makinesi opera ileting B-modunda, ekranda kalpte bir 2D görüntü elde ve OFT, ventrikül ve OVJ açıkça görülebilir şekilde aşama (ısıtma yastığı) hareket ettirin. </li><li> İstenilen darbe tekrarlama frekansı (örneğin, 20 kHz) de, darbeli dalga (PW) moduna geçmek ve OVJ tam olarak hız verileri elde. kalpte bir B-mod görüntü ekranda elde edilir. <ol><li> OFT, ventrikül ve OVJ görmek için sahne taşıyın. ultrason makinesi yazılımı kullanarak, tam OVJ de hız ölçümleri elde edin. </li></ol></li></ol> Not: embriyonun kalp hızı görüntüleme sırasında azalırsa, bu şekilde elde edilen hız veri analizi için kullanılmamalıdır. Hız ölçümleri için kullanılan tüm embriyolar, tercihen ultrason görüntüleme ile başka deneyler için kullanılmamalıdır.

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The authors have nothing to disclose.

Bu teknik nispeten hızlı ve kolay uygulanabilir olması, ancak bazı önemli noktaları doğru aşağı sonuçlar elde edecek şekilde akılda tutulması gerekir. Embriyolar, yeterli rehidrasyon sağlamak Tyrode tamponu ihtiva eden bir petri tabağına ovo ex muhafaza edilmelidir. Tyrode tamponu ile yumurta sarısı sonrası ameliyat hidrat ve inkübasyon odası yeterince hidrate emin olmak için de önemlidir. Herhangi bir kanama görülebilir veya sarısı hatta biraz kırık ise ameliyatları bir embriyo üzerinde yapılmamalıdır. Bu, özellikle uzun vadeli deneyler için, embriyo canlılığı etkiler. embriyolar letalitesinin başka bir potansiyel nedeni, ilk böylece önemli ölçüde kalp fonksiyonlarını ödün, uygulandığında OFT etrafında bant çok sıkı olup olmadığıdır. Şu anda, biz OFT bantlama üzerine kan akım hızı değişimi belirlemek için bir doğrulayıcı bir araç olarak ultrason kullanmayın. Gelecekte, (mikro-bilgisayarlı tomografi kullanılarak teyit edecekBT). Biz de görsel olarak B-mod 2D görüntülerde OFT etrafında bant gerginliği belirler. Önemlisi, kalp atım hızı ve hacim akışı (CFD kullanılarak hesaplanan) arasında fark banded ve kontrol grubu 17 embriyoları arasında görülmektedir. Bunlar kalp-damar sistemi fizyolojisi, böylece daha fazla bantlama müdahalesi doğrulayarak, bantlı embriyolar bozulmamış olduğunu göstermektedir. İstenilen zaman noktasında, bantlama sonra, birkaç analizler kritik vana gelişim süreçleri üzerinde değişmiş hemodinami etkisini anlamak için yapılabilir. Bunlar arasında, ancak ECM bileşenleri lokalizasyonunu belirlemek ve sıkça ultra yapısını belirleyen valf gelişiminde rol oynayan temel düzenleyici gen transkript seviyeleri değişiklikleri belirlemek, bunlarla sınırlı değildir. Son olarak, bu teknik pencereli yumurta 19 içinde embriyo olarak oft bantlama mevcut yönteme geliştirir. Bu yöntem, şiddetlily aşağı moleküler analiz yapmak için gerekli embriyolar birikir yetersizlik engel. Bu yeni teknik, hem kurtarma deneylerinde kullanılabilir. OFT bantlı kurtarıldı ve embriyo hakaret kurtarmak için izin verilmesi için Bu uygulama sağlar. Hiçbir geçerli yöntem bunu hala istatistiksel olarak anlamlı aşağı analizleri yapmak mümkün olabilir. Biz başarılı bir şekilde çok erken gelişim aşamasında (- 17 HH 16) de civciv embriyo merkezinde hemodinamik uyaranlara değiştirmek için bu tekniği kullanmışlardır. geliştirme sonraki aşamalarında ameliyat yaparken bantlama protokolü nedeniyle extraembryonic kan damarlarının herhangi nicklemek riski bir sorun mevcut olabilir. Ayrıca, gelişim geç evrelerinde, yumurta içeriği petri aktarılır zaman bozulmadan kesesi sarısı tutmak oldukça zordur.

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.

Şekil 1&#39;de gösterildiği OFT bantlama için gerekli araçları tavsiye edilir. Kapakla kapatıldı embriyo yok etmeyecek şekilde embriyo eski ihtiva eden petri (Şekil 1A) ovo yeterince derin olması gerekmektedir. Derin Petri kutuları (Şekil 1C), aynı zamanda Tyrode tamponu yeterli miktarda sarısı üstüne döküldü izin vermek için, ultrason görüntüleme için kullanılmalıdır. Tek bir 1 cm iplik (Şekil 2A) 11/0 naylon sütür (Şekil 1A) dışarı tweezed ve gevşek bir düğüm (Şekil 1B) içine bağlıdır. Tüm önceden oluşturulmuş knot UV sterilize edilir. Şekil 3A döllenmiş tavuk yumurtası, istenen aşamasına kadar inkübe edilir nemlendirilmiş bir rocker odasını göstermektedir. Şekil 3B bantlı ve kontrol embriyolar ovo ex tutulur bir embriyo kuluçka gösterir </ezaman istenilen miktarda m&gt;. Hem inkübatör türleri 40 ° C&#39;de muhafaza edilir. Şekil 4A&#39;da gösterilen sarısı ex ovo. Şekil 4B&#39;de üstünde HH17 embriyo HH17 embriyo OVJ de dikiş (bant) konumunu gösteren şematik bir temsilidir olmasıdır. Şekil 4C görüntülü bantlı embriyo Örnek resimdir ameliyat sonrası 48 saat. daralma sitesi aracılığıyla kan akım hızına bant müdahalenin etkisi 2B ultrason görüntüleme ile ölçülür. Beklendiği gibi, OVJ hızın kontrol (Şekil 5) göre bantlı embriyo daha yüksektir. Biz son zamanlarda OFT kan akım hızı ve duvar kayma gerilmesi 17 bantlama etkilerini gösteren 24 saat zaman noktasında bantlı ve kontrol kalpleri üzerinde hesaplamalı akışkanlar dinamiği (CFD) analizi gerçekleştirdik. &lt;keep-together.within sayfa = &quot;1&quot;&gt;: fo p class = &quot;jove_content&quot; <img alt="Şekil 1" src="/files/ftp_upload/53955/53955fig1.jpg" /> Şekil 1. Cerrahi Aletler. (A) 11/0 naylon sütür. (B) ön şekillendirilmiş knot içeren Petri tabağı. (C) ex ovo embriyo kültürü ve ultrason görüntüleme için derin petri. (D) Transfer pipet. (E) neşter yumurta kabuğu kırmak için anlaştım. (F) ameliyat sonrası çalışmalar için embriyo eksizyon için düz makas. (G) Güzel forseps <a href="https://www.jove.com/files/ftp_upload/53955/53955fig1large.jpg" target="_blank">bu rakamın daha büyük bir versiyonunu görmek için lütfen buraya tıklayınız.</a> <img alt="şekil 2" src="/files/ftp_upload/53955/53955fig2.jpg" /> OFT Bandlama 2. Sütür rakam. (A) Tek 1 cm parçacığı bir sütür dışarı tweezed. <strong&gt; (B) Önceden oluşturulmuş düğüm. <a href="https://www.jove.com/files/ftp_upload/53955/53955fig2large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="Şekil 3," src="/files/ftp_upload/53955/53955fig3.jpg" /> 3. Kuluçka Chambers rakam. (A) döllenmiş yumurta Nemlendirilmiş rocker inkübatör. (B) bantlı ve kontrol embriyoların kültürü ovo ex Nemlendirilmiş odası. <a href="https://www.jove.com/files/ftp_upload/53955/53955fig3large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="Şekil 4," src="/files/ftp_upload/53955/53955fig4.jpg" /> 4. OFT bantlama Şekil. (A) Tüm HH17 civciv embriyo daralma gösteren, ölçek çubuğu = 10 mm (B) şematik gösterimi ex ovo muhafazaSite (kırmızı). (C) Bantlı embriyo OFT bantlama sonra 48 saat görüntülü, ölçek çubuğu = 0.5 mm. <a href="https://www.jove.com/files/ftp_upload/53955/53955fig4large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="Şekil 5," src="/files/ftp_upload/53955/53955fig5.jpg" /> Şekil 5. Hız Ölçümleri Ultrason kaynaklı. (A) kontrol ve (B) Temsilcisi hız profilleri 1 saat zaman noktasında elde edilen embriyolar bantlı. <a href="https://www.jove.com/files/ftp_upload/53955/53955fig5large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</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).

Bir roman<em&gt; Ex Ovo</em&gt; Yapıştırma Tekniği bir Embriyonik tavuk Sistemde İntrakardiyak hemodinamiği Alter

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

Research

JoVE Journal

Developmental Biology

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

6.5K Görüntüleme.  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.

Primer Hücre Kültürü Araştırmaları Chick retina Verimli Gen Transferi: Bir<em&gt; Eski-ovo</em&gt; Elektroporasyon Yaklaşımı

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

Gelişim Biyolojisi

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).

Optimize<em&gt; Ex-ovo</em&gt; Kalkınma Gelişmiş Aşamaları Chick Embriyolar ekimi

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.

ChickGFP Nöral Tüp Greftleme ve Karbosiyanine Boya DiI Enjeksiyonu Kullanılarak Tavuk Embriyoları İçinde Nöral Kret Hücrelerinin ve Kan Damarlarının Çift Etiketlenimi

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.

Embriyonik tavuk doku gelişimi çalışmaya Organotypic kültür yöntemi

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.

Embriyonik Cardiomyocytes kuş embriyo implantasyonu Vivo için Mikroenjeksiyon tabanlı sistem

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.

Gelişimsel Kardiyotoksikliklerin Değerlendirilmesinde Tavuk Embriyosunun Güçlü Bir Araç Olarak Kullanılması

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.

Bir Eski-ovo</emGörüntüleme ve Mikrocerrahi Uygulamaları için uygundur> Tavuk Embriyo Kültürü Sistemi

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

Biyoloji

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.

Burada: Ovo Tavuk Embriyolarında İntravasküler Enjeksiyon

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

HLHS ve Kardiyovasküler Araştırmalar için Avian Embryonic Sol Atriyal Ligasyon Modeli

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.

Yazar Spotlight: Sol Atriyal Ligasyonun Kuş Embriyonik Kalpleri Üzerindeki Etkisi ve HLHS Etkileri 

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.

Erken Vasküler Gelişim Sırasında Değişen Hemodinamik Yükleme için Bir Model Olarak Kuş Embriyosunda Sol Atriyal Ligasyon

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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