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>

<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 border="0" cellpadding="0" cellspacing="0" style="width:1081px;" width="1082">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">35 mm cell culture petri dishes</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">353001</td>
			<td style="width:676px;">easy grip tissue culture dish, polystyrene, non-pyrogenic</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">100 mm cell culture petri dishes</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">353003</td>
			<td>tissue culture dish, polystyrene, non-pyrogenic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">paper tissue</td>
			<td style="width:71px;">Kimtech</td>
			<td style="width:119px;">34155</td>
			<td>Kimtech Science Brand Task Wipers, 280 per carton</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">wire mesh</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>stainless steel wire mesh (grid size 0.7 mm)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">disposable glass pipettes</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">14673-010</td>
			<td>Borosilicate glass disposable 5 3/4&#34;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">nutrient medium</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">12591-038</td>
			<td>Fitton-Jackson Modification, [+] L-glutamine with phenol red (BGJB)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">penicillin-streptomycin</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">P4458</td>
			<td>10000 units/mL penicillin streptomycin solution stabilised</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">filter paper</td>
			<td style="width:71px;">Whatman</td>
			<td style="width:119px;">1454 090</td>
			<td>semi-porous filter paper 90mm</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:216px;">fertilized chicken eggs</td>
			<td style="width:71px;">Dalhousie University Agricultural College</td>
			<td style="width:119px;">n/a</td>
			<td>can be obtained from local farms</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">sodium chloride (NaCl)</td>
			<td style="width:71px;">EMD</td>
			<td style="width:119px;">SX0420-3</td>
			<td>sodium chloride crystals, reagent grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1 L glass bottle</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">89000-240</td>
			<td>1 L pyrex autoclavable glass bottle</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ethanol</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>BP82011</td>
			<td>70% molecular biology grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">tupperware containers</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>store-bought and sterilized with EtOH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">disposable razor blades</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">55411-050</td>
			<td>single edge industrial razor blades (surgical carbon steel)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">plastic spoons</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>store-bought and sterilized with EtOH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dust mask</td>
			<td style="width:71px;">3M</td>
			<td style="width:119px;">n/a</td>
			<td>3M 8500 Comfort Mask</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">paraformaldehyde</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">P6148</td>
			<td>paraformaldehyde, reagent grade, crystalline</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">neutral-buffered formalin</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">72210</td>
			<td>10% neutral buffered formalin</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">phosphate buffered saline (PBS)</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>10X phosphate buffered saline pH 7.4 (137mM NaCl, 2.5mM KCl, 4.3mM Na2HPO4, 1.4mM KH2PO4)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">15 ml falcon tubes</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">21008-216</td>
			<td>presterilized centrifuge tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">forceps</td>
			<td style="width:71px;">FST</td>
			<td style="width:119px;">n/a</td>
			<td>fine forceps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">chick saline&#160;</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a&#160;</td>
			<td>0.85% NaCl</td>
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			<td height="21" style="height:21px;width:216px;">tinfoil</td>
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			<td>store-bought</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">paper towel</td>
			<td style="width:71px;">n/a</td>
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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 ...

organotypic-culture-method-to-study-development-embryonic-chicken

Research

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

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

Cultura da embrionárias mouse cocleares explantes e Gene Transferência de Eletroporação

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

Biologia do Desenvolvimento

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

Manipulação e<em&gt; In Vitro</em&gt; Maturação<em&gt; Xenopus laevis</em&gt; Oócitos, seguido de injeção intracitoplasmática de espermatozóide, para estudar o desenvolvimento embrionário

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA.

Transcriptoma Profiling de<em&gt; In-Vivo</em&gt; embriões produzidos pré-implantação dos bovinos por meio de duas cores Plataforma Microarray

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo ...

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

Regulação epigenética da Cardiac diferenciação de células estaminais embrionárias e Tecidos

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

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.

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).

Um romance<em&gt; Ex Ovo</em&gt; Técnica Banding para Alter intracardíaca Hemodinâmica em um sistema embrionário de galinha

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

Culture of Murine Embryonic Metatarsals: A Physiological Model of Endochondral Ossification

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or longitudinal bone growth. As such, it is imperative that we further our understanding of the underpinning molecular mechanisms involved in such disorders so as to provide advances towards human and animal patient benefit. The mouse metatarsal organ explant culture is a highly physiological ex vivo model for studying endochondral ossification and bone growth as the growth rate of the bones in culture mimic that observed in vivo. Uniquely, the metatarsal organ culture allows the examination of chondrocytes in different phases of chondrogenesis and maintains cell-cell and cell-matrix interactions, therefore providing conditions closer to the in vivo situation than cells in monolayer or 3D culture. This protocol describes in detail the intricate dissection of embryonic metatarsals from the hind limb of E15 murine embryos and the subsequent analyses that can be performed in order to examine endochondral ossification and longitudinal bone growth.

We present a protocol to dissect and culture embryonic day 15 (E15) murine metatarsal bones. This highly physiological ex vivo model of endochondral ossification provides conditions closer to the in vivo situation than cells in monolayer or 3D culture and is a vital tool for investigating bone growth and development.

Cultura de murino embrionárias Metatarsos: Um modelo fisiológico de Endocondral ossificação

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or ...

Dissection and Culture of Mouse Embryonic Kidney

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney development, the two progenitor tissues, the ureteric bud and the metanephric mesenchyme, communicate and reciprocally induce cellular mechanisms to eventually form the collecting system and the nephrons of the kidney. As mammalian embryos grow intrauterine and therefore are inaccessible to the observer, an organ culture has been developed. With this method, it is possible to study epithelial-mesenchymal interactions and cellular behavior during kidney organogenesis. Furthermore, the origin of congenital kidney and urogenital tract malformations can be investigated. After careful dissection, the metanephric rudiments are transferred onto a filter that floats on culture medium and can be kept in a cell culture incubator for several days. However, one must be aware that the conditions are artificial and could influence the metabolism in the tissue. Also, the penetration of test substances could be limited due to the extracellular matrix and basal membrane present in the explant.
One main advantage of organ culture is that the experimenter can gain direct access to the organ. This technology is cheap, simple, and allows a large number of modifications, such as the addition of biologically active substances, the study of genetic variants, and the application of advanced imaging techniques.

This protocol describes a method for isolating and culturing metanephric rudiments from mouse embryos.

Dissecção e cultura do rim embrionário do rato

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

Análise computacional do Germline Caenorhabditis elegans , para estudar a distribuição de núcleos, as proteínas e o citoesqueleto

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

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 ...

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One challenge is that embryogenesis dynamically unfolds over a period of about 13 h; this half day-long timescale has constrained the scope of experiments by limiting the number of embryos that can be imaged. Here, we describe a semi-high-throughput protocol that allows for the simultaneous 3D time-lapse imaging of development in 80&#8211;100 embryos at moderate time resolution, from up to 14 different conditions, in a single overnight run. The protocol is straightforward and can be implemented by any laboratory with access to a microscope with point visiting capacity. The utility of this protocol is demonstrated by using it to image two custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis. To analyze the data, a custom program that crops individual embryos out of a broader field of view in all channels, z-steps, and timepoints and saves the sequences for each embryo into a separate tiff stack was built. The program, which includes a user-friendly graphical user interface (GUI), streamlines data processing by isolating, pre-processing, and uniformly orienting individual embryos in preparation for visualization or automated analysis. Also supplied is an ImageJ macro that compiles individual embryo data into a multi-panel file that displays maximum intensity fluorescence projection and brightfield images for each embryo at each time point. The protocols and tools described herein were validated by using them to characterize embryonic development following knock-down of 40 previously described developmental genes; this analysis visualized previously annotated developmental phenotypes and revealed new ones. In summary, this work details a semi-high-throughput imaging method coupled with a cropping program and ImageJ visualization tool that, when combined with strains expressing informative fluorescent markers, greatly accelerates experiments to analyze embryonic development.

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80&#8211;100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.

Um método de imagem semi-alta-fonte e um kit de ferramentas de visualização de dados para analisar o desenvolvimento embrionário de C. elegans