Name: organotypic culture method to study the development of embryonic chicken tissues
Uploaded: 2018-08-25T12:30:00.000+00:00
Duration: 5 min 12 s
Description: Watch this Scientific Journal Video about Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues at JoVE.com

Os autores gostaria de agradecer Gregory Haller (Mount Saint Vincent University), por seu trabalho preliminar no desenvolvimento do protocolo. Os autores também gostaria de agradecer a Nicholas Jones (Mount Saint Vincent University) por seus conhecimentos técnicos e assistência com a filmagem e produção da parte do áudio/visual do manuscrito. Daniel Andrews foi suportado pelo financiamento do MSVU e as ciências naturais e engenharia pesquisa Conselho de Canadá (NSERC) através de um prêmio de pesquisa de estudante de graduação. Tamara A. Franz-Odendaal é suportada por um fundo de descoberta NSERC.

Nota: Para estágios do embrião, utilizam o Hamburger e Hamilton12 (HH) tabela de preparo.
1. o embrião incubação
<ol><li>Incube os ovos fertilizados de galinha numa incubadora estéril, controlo de temperatura em 37 ° C ± 1 ° C e ~ 40% de umidade.</li>
	<li>Vire os ovos 1 x por dia e permitir que incube ao HH34 (fertilização pós de 8 dias).</li>
</ol>2. preparação e esterilização dos materiais
<ol><li>Para 12 embriões, autoclave 2 L de água destilada, 1 L de solução salina de garota de 0,85%, 12 pipetas de vidro, 1 caixa de tecido de papel, 24 2,5 x 2,5 cm quadrados de papel de filtro semi porosa e praças 24 2,5 x 2,5 cm de fio de aço de malha com as bordas curvadas para baixo. Para esterilizar os materiais, autoclave-los a 121 ° C durante pelo menos 15 minutos. Nota: A esterilização dos materiais pode ser feita com antecedência.</li>
	<li>Esterilize a incubadora com etanol 70% limpando os lados, prateleiras e porta. Coloque 2 recipientes plásticos contendo autoclavado destilaram água dentro da incubadora para manter a umidade. Escaldar a incubadora de cultura a 37 ° C ± 1 ° C e ~ 40% de umidade. Certifique-se da que incubadora está completamente escura; se a porta for transparente, cubra com papel alumínio.</li>
	<li>Escaldar a 100 mL de meio nutritivo e 1 mL de penicilina, colocando-os na incubadora de cultura.</li>
	<li>Cobrir a área de trabalho com uma toalha de papel e esterilizar o banco por pulverização com etanol a 70%. Esterilize 2 pares de pinças, tesouras de dissecação, uma lâmina de barbear e uma colher de plástico com etanol a 70% de forma semelhante.</li>
	<li>Pratos de Petri estéril 100mm lugar 12 e 24 pratos de Petri estéril 35mm no banco para uso. Certifique-se de que pratos permanecem num saco estéril até o uso.</li>
</ol>3. embrião preparação
Nota: A partir deste momento, use uma máscara de protecção contra o pó para evitar a contaminação das culturas. Uma infecção bacteriana ou fúngica vai estragar a cultura... e há um risco de se espalhar rapidamente a todas as culturas na incubadora.
<ol><li>Crack abrir o ovo e transferir o embrião para um prato de Petri de 100mm contém solução salina 0,85% garota. Estágio o embrião de acordo com as características anatômicas descritas no Hamburger e Hamilton encenando orientações12 e confirmar o embrião está no HH34.</li>
	<li>Cortar o pescoço e depois dividem a cabeça do embrião na linha usando uma lâmina de barbear esterilizada. Usando a pinça estéril, remova o cérebro. Deixe o bico intacto.</li>
</ol>4. cultura Setup
<ol><li>2 lugar dos pratos de Petri 35mm dentro de um prato de Petri de 100 mm.</li>
	<li>Coloca uma malha de arame de aço no interior de cada prato de Petri de 35 mm.</li>
	<li>Usando fórceps, transferi 1 das cabeças bisseccionadas para um pedaço de papel de filtro semi porosas de 2,5 x 2,5 cm com o olho virado para cima. Certifique-se o tecido é seguro e não escorregar inclinando-o ligeiramente.</li>
	<li>Usando a pinça, coloque cuidadosamente o tecido do olho e o papel de filtro em cima da malha de arame de aço em 1 dos pratos Petri 35mm, criando um palco Erguido com o tecido do olho em cima.</li>
	<li>Repita as etapas de 4.3 e 4.4, com a outra metade de cabeça.</li>
	<li>Utilizando uma pipeta de vidro estéril, cuidadosamente adicione meio nutritivo diretamente em cada placa de Petri 35mm até atingir o nível do filtro de papel. Não mergulhe a cabeça meia no meio de nutrientes.</li>
	<li>Adicione 50 µ l de 10.000 U/mL de penicilina-estreptomicina no meio de nutrientes em cada placa de Petri.</li>
	<li>Dobre um pedaço de papel de seda em um pequeno quadrado e coloque-a dentro do prato de Petri de 100mm. Umedecê-la com água destilada esterilizada.</li>
	<li>Coloque o prato de cultura dentro da incubadora estéril, escura em 37 ° C ± 1 ° C e ~ 40% de umidade por até 4 dias.</li>
	<li>Repita as etapas 3 e 4 para cada embrião. Nota: O número de embriões necessários dependerá da experiência específica; aqui, descrevemos o protocolo para 12 embriões.</li>
</ol>5. cultura manutenção
<ol><li>Uma vez por dia, encha os recipientes de plástico na incubadora com água destilada esterilizada.</li>
	<li>Verifique diariamente todas as culturas para infecções bacterianas ou fúngicas. Culturas que têm desintegrou-se ou em que o meio mudou de cor estão infectadas. Descarte todas as culturas que estão infectadas.</li>
</ol>6. fixação
<ol><li>Após o cultivo, remover todos os pratos da incubadora.</li>
	<li>Usando um par de pinças, remova suavemente o olho do papel de filtro, tomando cuidado para não rasgar o tecido.</li>
	<li>Reparar o tecido do olho em paraformaldeído 4% em 1x tampão fosfato salino durante a noite a 4 ° C ou em formol a 10% neutro de buffer durante a noite em temperatura ambiente.</li>
	<li>Armazene o tecido a 4 ° C em 1 x fosfato salino.</li>
</ol>

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Os autores não têm nada para divulgar.

Este protocolo faz uso de tecido estabelecido técnicas de cultivo para o crescimento de um olho galinha embrionário dia 8 (HH34) em vitro por 4 dias. Este método de cultivo a grade foi originalmente descrito por Trowell15. Nós aperfeiçoamos um protocolo de Pinto e do Hall 1991 estudo16 utilizando uma membrana semi porosa com a grade para estudar sinais indutivos entre camadas de tecido separada da galinha embrionários olho15. Usando es...

A galinha embrionária (Gallus gallus) é um organismo de excelente modelo comumente usado no campo da biologia. Seu período de incubação é de aproximadamente 21 dias e muitos ovos podem ser incubados simultaneamente, tornando a experimentação rápida e eficiente. Talvez mais importante, o embrião é também facilmente manipulado, permitindo o estudo extensivo de processos-chave do desenvolvimento e dos genes e proteínas que impulsionam estes processos.
O olho embrionárias de galinha é um órgão complexo que se desenvolve através da interação de um número de diferentes tecidos semelhantes a muitos outros sistemas do corpo. Esse método permite que o estudo do desenvolvimento desses tecidos, particularmente em estágios avançados de desenvolvimento. Por exemplo, a retina multi-camadas pode ser de particular interesse para os que estudam o desenvolvimento do sistema nervoso. Métodos alternativos que permitem o estudo de outros tecidos oculares, tais como a córnea, o corpo de vitreal, a lente, a esclera e as pálpebras são de benefício para os investigadores. O olho embrionárias de galinha também contém uma série de ossos chatos, os ossículos scleral, que podem ser usados como um modelo para o estudo da indução óssea intramembranosa e ossificação em vertebrados1.
Atualmente, existem vários métodos utilizados para estudar o desenvolvimento embrionário. Microinjeções de inibitórios anticorpos ou outras moléculas inibidoras2,3, cirurgicamente microbeads embebido em inibidor4e eletroporação5 são todos os métodos que podem ser usados para downregulate genes ou proteínas de interesse em um embrião. Métodos similares são usados para upregulate proteínas. Esses métodos não são sem suas limitações. Por exemplo, ao usar produtos químicos para alterar o desenvolvimento embrionário, os efeitos letais sobre o embrião devem ser avaliados, e isso limita o uso dos métodos acima mencionados para sites localizados de aplicação em doses baixas o suficiente para garantir a sobrevivência da embrião.
In vitro , tecido cultivo tem sido utilizado em uma ampla gama de organismos, para estudar o desenvolvimento e pode ser usado para contornar algumas das limitações acima mencionadas. Por exemplo, os fêmures6, pena botões7,8e membros9 do frango todos foram estudadas usando tecido cultivo métodos, como têm os testículos de rato10 e as raízes e caules de plantas11. Esses métodos concedem cientistas um alto grau de controle sobre o desenvolvimento de tecidos, tais como a capacidade de variar a temperatura e alterar a disponibilidade de nutrientes. O isolamento do tecido do embrião inteiro também torna muito menos suscetíveis aos efeitos letais de produtos químicos, permitindo estudos de manipulação em escala global em altas concentrações. Outra vantagem notável em vitro cultivo é a preservação do ambiente celular do tecido; o arranjo dos tecidos permanece relativamente inalterado, tornando possível estudar as interações entre diferentes tecidos tipos9. Assim, em vitro abre portas adicionais experimental de cultivo se aproxima não disponível nos modelos no vivo ou no ovo .
Atualmente, estudar o desenvolvimento do olho embrionárias de galinha usando produtos químicos é particularmente desafiadora. Um número de membranas extraembryonic cobre o embrião, tornando difícil aplicar microbeads ou produtos químicos; o embrião também é muito ativo dentro do ovo, como ele fica mais velho, complicando ainda mais um método já difícil. Este protocolo permite acesso fácil para os olhos e seus tecidos circundantes, eliminando as barreiras, proporcionando também novas oportunidades para examinar os processos de desenvolvimento dentro do olho. Este protocolo foi criado para estudar a indução de scleral ossículos dentro do olho embrionário.

<table><tbody><tr><td>35 mm cell culture petri dishes</td><td>Corning</td><td>353001</td><td>easy grip tissue culture dish, polystyrene, non-pyrogenic</td></tr><tr><td>100 mm cell culture petri dishes</td><td>Corning</td><td>353003</td><td>tissue culture dish, polystyrene, non-pyrogenic</td></tr><tr><td>paper tissue</td><td>Kimtech</td><td>34155</td><td>Kimtech Science Brand Task Wipers, 280 per carton</td></tr><tr><td>wire mesh</td><td>n/a</td><td>n/a</td><td>stainless steel wire mesh (grid size 0.7 mm)</td></tr><tr><td>disposable glass pipettes</td><td>VWR</td><td>14673-010</td><td>Borosilicate glass disposable 5 3/4&quot;</td></tr><tr><td>nutrient medium</td><td>Gibco</td><td>12591-038</td><td>Fitton-Jackson Modification, [+] L-glutamine with phenol red (BGJB)</td></tr><tr><td>penicillin-streptomycin</td><td>Sigma-Aldrich</td><td>P4458</td><td>10000 units/mL penicillin streptomycin solution stabilised</td></tr><tr><td>filter paper</td><td>Whatman</td><td>1454 090</td><td>semi-porous filter paper 90mm</td></tr><tr><td>fertilized chicken eggs</td><td>Dalhousie University Agricultural College</td><td>n/a</td><td>can be obtained from local farms</td></tr><tr><td>sodium chloride (NaCl)</td><td>EMD</td><td>SX0420-3</td><td>sodium chloride crystals, reagent grade</td></tr><tr><td>1 L glass bottle</td><td>VWR</td><td>89000-240</td><td>1 L pyrex autoclavable glass bottle</td></tr><tr><td>ethanol</td><td>Fisher Scientific</td><td>BP82011</td><td>70% molecular biology grade</td></tr><tr><td>tupperware containers</td><td>n/a</td><td>n/a</td><td>store-bought and sterilized with EtOH</td></tr><tr><td>disposable razor blades</td><td>VWR</td><td>55411-050</td><td>single edge industrial razor blades (surgical carbon steel)</td></tr><tr><td>plastic spoons</td><td>n/a</td><td>n/a</td><td>store-bought and sterilized with EtOH</td></tr><tr><td>dust mask</td><td>3M</td><td>n/a</td><td>3M 8500 Comfort Mask</td></tr><tr><td>paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td>paraformaldehyde, reagent grade, crystalline</td></tr><tr><td>neutral-buffered formalin</td><td>Fisher Scientific</td><td>72210</td><td>10% neutral buffered formalin</td></tr><tr><td>phosphate buffered saline (PBS)</td><td>n/a</td><td>n/a</td><td>10X phosphate buffered saline pH 7.4 (137mM NaCl, 2.5mM KCl, 4.3mM Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 1.4mM KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td></tr><tr><td>15 ml falcon tubes</td><td>VWR</td><td>21008-216</td><td>presterilized centrifuge tubes</td></tr><tr><td>forceps</td><td>FST</td><td>n/a</td><td>fine forceps</td></tr><tr><td>chick saline&amp;nbsp;</td><td>n/a</td><td>n/a&amp;nbsp;</td><td>0.85% NaCl</td></tr><tr><td>tinfoil</td><td>n/a</td><td>n/a</td><td>store-bought</td></tr><tr><td>paper towel</td><td>n/a</td><td>n/a</td><td>store-bought</td></tr></tbody></table>

organotypic culture method to study the development of embryonic chicken tissues

A galinha embrionária é comumente usada como um organismo modelo confiável para o desenvolvimento de vertebrados. Sua acessibilidade e incubação curta período é ideal para a experimentação. Atualmente, o estudo destas vias do desenvolvimento do embrião de galinha é realizado através da aplicação de inibidores e drogas em locais localizados e em baixas concentrações, usando uma variedade de métodos. Em vitro tecido cultivo é uma técnica que permite o estudo dos tecidos separados do organismo hospedeiro, enquanto ao mesmo tempo ignorando muitas das limitações físicas presentes ao trabalhar com embriões inteiro, tais como a susceptibilidade de embriões para altas doses de substâncias químicas potencialmente letais. Aqui, apresentamos um organotypic cultivo protocolo para cultivo a galinha embrionários meia cabeça em vitro, que apresenta novas oportunidades para o exame de processos de desenvolvimento, além dos métodos actualmente estabelecidos.

Usando esse método, olho embrionárias de galinha pode ser cultivado desde o dia 8 do seu desenvolvimento (HH34) em vitro por 4 dias. Quatro dias de desenvolvimento no ovo corresponde ao HH38.
Este método de cultivo apoia o desenvolvimento de brotos de pena em torno do olho e nas pálpebras (figura 1B). Estes botões de penas não estão presentes no ovo em HH34 antes do...

Watch this Scientific Journal Video about Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues at JoVE.com

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

Cultura de organotypic método para estudar o desenvolvimento de tecidos embrionários de galinha

Aqui, apresentamos um organotypic cultivo protocolo para crescer órgãos embrionárias de galinha in vitro. Usando esse método, o desenvolvimento de tecidos embrionários de galinha pode ser estudado, mantendo um alto grau de controle sobre o ambiente de cultura.

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

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JoVE Journal

Developmental Biology

5.9K visualizações.  Mount Saint Vincent University. Aqui, apresentamos um organotypic cultivo protocolo para crescer órgãos embrionárias de galinha in vitro. Usando esse método, o desenvolvimento de tecidos embrionários de galinha pode ser estudado, mantendo um alto grau de controle sobre o ambiente de cultura.

Vídeo: Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

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).

Otimizado<em&gt; Ex-ovo</em&gt; A cultura de Chick embriões para estágio avançado de desenvolvimento

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

Biologia do Desenvolvimento

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of morphogenetic events, such as gastrulation2, neurulation3-5, somitogenesis6, heart bending7,8, and brain formation9-13, during early embryogenesis. Key to understanding morphogenetic processes is to follow them dynamically by time-lapse imaging. The acquisition of time-lapse movies of chick embryogenesis ex ovo has been limited either to short time windows or to the need for an incubator to control temperature and humidity around the embryo14. Here, we present a new technique to culture chick embryos ex ovo for high-resolution time-lapse imaging using transmitted light microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique (EC-culture)1 and allows for the culturing of chick embryos without the need for a climate chamber. The embryo is sandwiched between two identical filter paper carriers and is kept fully submerged in a simple, temperature-controlled medium covered by a layer of light mineral oil. Starting from the primitive streak stage (Hamburger-Hamilton stage 5, HH5)15 up to at least the 28-somite stage (HH16)15, embryos can be cultured with either their ventral or dorsal side up. This allows the acquisition of time-lapse movies covering about 30 hr of embryonic development. Representative time-lapse frames and movies are shown. Embryos are compared morphologically to an embryo cultured in the standard EC-culture.
The submerged filter paper sandwich provides a stable environment to study early dorsal and ventral morphogenetic processes. It also allows for live fluorescence imaging and micromanipulations, such as microsurgery, bead implantation, microinjection, gene silencing, and electroporation, and has a strong potential to be combined with immersion objectives for laser-based imaging (including light-sheet microscopy).

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Here, we present a new technique to culture chick embryos ex ovo for time-lapse imaging using transmitted light and fluorescence microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique1 that allows for the culturing of chick embryos fully-submerged in a liquid culture medium.

Um Sanduíche papel de filtro submerso por longo prazo<em&gt; Ex Ovo</em&gt; Imagem Time-lapse de galinha precoce embriões

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of ...

Method for Culture of Early Chick Embryos ex vivo (New Culture)

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 patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

Método para a Cultura de Embriões início pintainho ex vivo (Nova Cultura)

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

Biologia

Two-step Approach to Explore Early- and Late-stages of Organ Formation in the Avian Model: The Thymus and Parathyroid Glands Organogenesis Paradigm

The avian embryo, as an experimental model, has been of utmost importance for seminal discoveries in developmental biology. Among several approaches, the formation of quail-chicken chimeras and the use of the chorioallantoic membrane (CAM) to sustain the development of ectopic tissues date back to the last century. Nowadays, the combination of these classical techniques with recent in vitro methodologies offers novel prospects to further explore organ formation.
Here we describe a two-step approach to study early- and late-stages of organogenesis. Briefly, the embryonic region containing the presumptive territory of the organ is isolated from quail embryos and grown in vitro in an organotypic system (up to 48 h). Cultured tissues are subsequently grafted onto the CAM of a chicken embryo. After 10 days of in ovo development, fully formed organs are obtained from grafted tissues. This method also allows the modulation of signaling pathways by the regular administration of pharmacological agents and tissue genetic manipulation throughout in vitro and in ovo developmental steps. Additionally, developing tissues can be collected at any time-window to analyze their gene-expression profile (using quantitative PCR (qPCR), microarrays, etc.) and morphology (assessed with conventional histology and immunochemistry).
The described experimental procedure can be used as a tool to follow organ formation outside the avian embryo, from the early stages of organogenesis to fully formed and functional organs.

This article provides an updated approach to the classical quail-chicken chimera system to study organ formation, by combining novel in vitro and in ovo experimental procedures.

Abordagem em duas fases para explorar e tarde-estágios iniciais de formação do órgão no modelo aviária: O Timo e glândulas paratireoides organogênese paradigma

The avian embryo, as an experimental model, has been of utmost importance for seminal discoveries in developmental biology. Among several approaches, the ...

Chicken Embryo Spinal Cord Slice Culture Protocol

Slice cultures can facilitate the manipulation of embryo development both pharmacologically and through gene manipulations. In this reduced system, potential lethal side effects due to systemic drug applications can be overcome. However, culture conditions must ensure that normal development proceeds within the reduced environment of the slice. We have focused on the development of the spinal cord, particularly that of spinal motor neurons. We systematically varied culture conditions of chicken embryo slices from the point at which most spinal motor neurons had been born. We assayed the number and type of motor neurons that survived during the culture period and the position of those motor neurons compared to that in vivo. We found that serum type and neurotrophic factors were required during the culture period and were able to keep motor neurons alive for at least 24 hr and allow those motor neurons to migrate to appropriate positions in the spinal cord. We present these culture conditions and the methodology of preparing the embryo slice cultures using eviscerated chicken embryos embedded in agarose and sliced using a vibratome.

Slice cultures facilitate the manipulation of embryo development by gene and pharmacological perturbations. However, culture conditions must ensure that normal development can proceed within the reduced environment of the slice. We illustrate a protocol that facilitates normal spinal cord development to proceed for at least 24 hr.

Embrião de galinha Spinal Cord Protocolo Cultura Slice

Slice cultures can facilitate the manipulation of  embryo development both pharmacologically and through gene manipulations. In  this reduced system, ...

Neurociência

Isolation of Embryonic Tissues and Formation of Quail-Chicken Chimeric Organs Using The Thymus Example

The capacity to isolate embryonic tissues was an essential step for establishing the quail-chicken chimera system, which in turn has provided undisputed contributions to unveiling key processes in developmental biology.
Herein is described an optimized method to isolate embryonic tissues from quail and chickens by microsurgery and enzymatic digestion while preserving its biological properties. After isolation, tissues from both species are associated in an in vitro organotypic assay for 48 h. Quail and chicken tissues can be discriminated by distinct nuclear features and molecular markers allowing the study of the cellular cross-talk between heterospecific association of tissues. This approach is, therefore, a useful tool for studying complex tissue interactions in developmental processes with highly dynamic spatial modifications, such as those occurring during pharyngeal morphogenesis and the formation of the foregut endoderm-derived organs. This experimental approach was first developed to study the epithelial-mesenchymal interactions during early-stages of thymus formation. In this, the endoderm-derived prospective thymic rudiment and mesoderm-derived mesenchyme, were isolated from quail and chicken embryos, respectively.
The capacity of the associated tissues to generate organs can be further tested by grafting them onto the chorioallantoic membrane (CAM) of a chicken embryo. The CAM provides nutrients and allows gas exchanges to the explanted tissues. After 10 days of in ovo development, the chimeric organs can be analyzed in the harvested explants by conventional morphological methods. This procedure also allows studying tissue-specific contributions during organ formation, from its initial development (in vitro development) to the final stages of organogenesis (in ovo development).
Finally, the improved isolation method also provides three-dimensionally (3D) preserved embryonic tissues, that can also be used for high-resolution topographical analysis of tissue-specific gene-expression patterns.

This article provides a method to isolate pure embryonic tissues from quail and chicken embryos that can be combined to form ex vivo chimeric organs.

Isolamento de tecidos embrionários e formação de órgãos quiméricoes codorna-frango, usando o exemplo do Timo

The capacity to isolate embryonic tissues was an essential step for establishing the quail-chicken chimera system, which in turn has provided undisputed ...

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.

Um Ex-ovo Sistema de Cultura de embriões de frango Indicado para aplicações de imagem e Microcirurgia

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

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.

Análise da diferenciação celular, morfogênese e padronização durante a embriogênese de frango usando o ensaio de contas encharcadas

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

Uma revisão abrangente dos protocolos de cultura de explante de pele embrionária aviária

Using Avian Skin Explants to Study Tissue Patterning and Organogenesis

The developing avian skin during embryogenesis is a unique model that can provide valuable insights into tissue patterning. Here three variations on skin explant cultures to examine different aspects of skin development are described. First, ex vivo organ cultures and manipulations offer researchers opportunities to observe and study the development of feather buds directly. Skin explant culture can grow for 7 days enabling direct analysis of cellular behavior and 4D imaging at intervals during this growth period. This also allows for physical and molecular manipulations of culture conditions to visualize tissue response. For example, growth factor-coated beads can be applied locally to induce changes in feather patterning in a limited area. Alternatively, viral transduction can be delivered globally in the culture media to up or downregulate gene expression. Second, the skin recombination protocol allows researchers to investigate tissue interactions between the epidermis and mesenchyme that are derived from different skin regions, different life stages, or different species. This affords an opportunity to test the time window in which the epithelium is competent to respond to signals and its ability to form different skin appendages in response to signals from different mesenchymal sources. Third, skin reconstitution using dissociated dermal cells overlaid with intact epithelium resets skin development and enables the study of the initial processes of periodic patterning. This approach also enhances our ability to manipulate gene expression among the dissociated cells before creating the reconstituted skin explant. This paper provides the three culture protocols and exemplary experiments to demonstrate their utility.

Autor em destaque: Insight sobre as atuais metodologias experimentais de explante de pele aviária

Here we describe protocols for three types of avian embryonic skin explant cultures that can be used to examine tissue interactions, 4D imaging timelapse movie (3D plus time), global or local perturbation of molecular function, and systems biology characterization.

Usando explantes de pele aviária para estudar padrões de tecidos e organogênese

The developing avian skin during embryogenesis is a unique model that can provide valuable insights into tissue patterning. Here three variations on skin ...

Cultura de organotypic método para estudar o desenvolvimento de tecidos embrionários de galinha

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Usando explantes de pele aviária para estudar padrões de tecidos e organogênese

Usando explantes de pele aviária para estudar padrões de tecidos e organogênese