Los autores desean agradecer Gregory Haller (Universidad de Mount Saint Vincent) para sus trabajos preliminares en el desarrollo del protocolo. Los autores también gustaría agradecer Nicholas Jones (Mount Saint Vincent University) por su experiencia técnica y asistencia con el rodaje y producción de la parte audiovisual del manuscrito. Daniel Andrews fue apoyado por fondos de MSVU y las ciencias naturales e Ingeniería investigación Consejo de Canadá (NSERC) a través de un premio de investigación del estudiante de pregrado. Tamara A. Franz-Odendaal es apoyada por una subvención de descubrimiento de NSERC.

Nota: Para las etapas de embrión, utilizar el Hamburger y Hamilton12 (HH) escenificando la mesa.
1. embrión incubación
<ol><li>Incubar huevos de gallina fecundados en una incubadora estéril, control de temperatura a 37 ° C ± 1 ° C y 40% humedad.</li>
	<li>Gire los huevos 1 x por día y que puedan incubar a HH34 (8 días tras la fertilización).</li>
</ol>2. preparación y esterilización de los materiales
<ol><li>12 embriones, autoclave 2 L de agua destilada, 1 L de solución salina al 0,85% pollo, 12 pipetas de vidrio, 1 caja de papel de fumar, 24 2,5 x 2,5 cm cuadrados semi porosa del papel de filtro y plazas 24 2,5 x 2,5 cm de alambre de acero del acoplamiento con los bordes curvados hacia abajo. Para esterilizar los materiales, autoclave a 121 ° C durante al menos 15 minutos. Nota: El autoclave de los materiales puede hacerse por adelantado.</li>
	<li>Esterilizar la incubadora con etanol al 70% por limpiar los lados estantes y puerta. Coloque 2 recipientes de plástico que contienen autoclave destilación agua dentro de la incubadora para mantener la humedad. Precaliente la incubadora de cultivo a 37 ° C ± 1 ° C y 40% humedad. La incubadora esté completamente a oscuras; Si la puerta es transparente, cubrirlo con papel de aluminio.</li>
	<li>Precaliente 100 mL de medio nutritivo y 1 mL de la penicilina por colocarlos en la incubadora de la cultura.</li>
	<li>Cubrir el espacio de trabajo con una toalla de papel y esterilizar el Banco rociando con etanol al 70%. Esterilizar 2 pares de pinzas, tijeras de disección, una hoja de afeitar y una cuchara de plástico con etanol al 70% de manera similar.</li>
	<li>Lugar 12 placas de Petri estériles de 100 mm y 24 placas de Petri estériles de 35 mm en el Banco para el uso. Mantener los platos en una bolsa estéril hasta su utilización.</li>
</ol>3. embrión preparación
Nota: De aquí en adelante, usar una máscara protectora de polvo para evitar la contaminación de las culturas. Una infección bacteriana o micótica arruinará la cultura y existe un riesgo de separarse rápidamente a todas las culturas en la incubadora.
<ol><li>Crack abre el huevo y transferir el embrión a un plato de Petri de 100 mm que contiene solución salina al 0,85% chick. Etapa del embrión según las características anatómicas descritas en la Hamburger y Hamilton escenificando las pautas12 y confirmar el embrión está en HH34.</li>
	<li>Cortar el cuello y luego atraviesan la cabeza del embrión en la línea media usando una navaja esterilizada. Utilizando pinzas estériles, quite el cerebro. Deje intacto el pico.</li>
</ol>4. cultura configuración
<ol><li>2 lugar de los platos de Petri de 35 mm dentro de un plato de Petri de 100 mm.</li>
	<li>Colocar una malla de alambre de acero dentro de cada plato de Petri de 35 mm.</li>
	<li>Con unas pinzas, transferencia 1 de las cabezas bisecadas una pieza de 2,5 x 2,5 cm semi poroso papel de filtro con el ojo mirando hacia arriba. Asegúrese de que el tejido es seguro y no se deslice fuera inclinándola ligeramente.</li>
	<li>Utilizando pinzas, coloque con cuidado el tejido ocular y el papel de filtro sobre la malla de alambre de acero en 1 de los platos de Petri de 35 mm, creando un escenario levantado con el tejido del ojo en la parte superior.</li>
	<li>Repita los pasos 4.3 y 4.4, con la otra media cabeza.</li>
	<li>Cuidadosamente con una pipeta de vidrio estéril, añadir medio nutritivo directamente en cada plato de Petri de 35 mm hasta alcanzar el nivel del papel de filtro. No sumerja la cabeza medio en el medio nutritivo.</li>
	<li>Añadir 50 μl de 10.000 U/mL de penicilina-estreptomicina en el medio nutritivo en cada placa de Petri.</li>
	<li>Doble un pedazo de papel de tejido en una pequeña plaza y colocar dentro de la caja de Petri de 100 mm. Humedecer con agua destilada esterilizada.</li>
	<li>Coloque la placa de cultivo en la incubadora a 37 ° C ± 1 ° C y 40% humedad estéril, oscurezca durante 4 días.</li>
	<li>Repita los pasos 3 y 4 para cada embrión. Nota: El número de embriones necesarios dependerá de la experiencia específica; aquí, describimos el protocolo de 12 embriones.</li>
</ol>5. cultura mantenimiento
<ol><li>Una vez al día, depositar los envases de plástico en la incubadora con agua destilada esterilizada.</li>
	<li>Diario comprobar todas las culturas para las infecciones bacterianas o por hongos. Infectan a las culturas que se han desintegrado o que el medio ha cambiado de color. Deseche todas las culturas que están infectadas.</li>
</ol>6. fijación
<ol><li>Siguiente cultivo, retirar todos los platos de la incubadora.</li>
	<li>Usando un par de pinzas, eliminar suavemente el ojo desde el papel de filtro, teniendo cuidado de no para rasgar el tejido.</li>
	<li>Fijar el tejido ocular en paraformaldehído al 4% en el 1 x de solución salina con tampón fosfato durante la noche a 4 ° C o en formalina tamponada neutra 10% durante la noche a temperatura ambiente.</li>
	<li>Guarde el tejido a 4 ° C en 1 x solución salina con tampón fosfato.</li>
</ol>

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Los autores no tienen nada que revelar.

Este protocolo hace uso de técnicas de cultivo del tejido establecido para lograr el crecimiento de un ojo de pollo del día embrionario 8 (HH34) en vitro por 4 días. Este método de cultivo de rejilla fue descrito originalmente por Trowell15. Hemos optimizado un protocolo de Pinto y Hall 1991 estudio16 utilizando una membrana semi porosa con la red para el estudio de señales inductivas entre capas de tejido separadas del embrionario pollo ojo1...

El embrionario pollo (Gallus gallus) es un organismo de excelente modelo comúnmente utilizado en el campo de la biología. Su período de incubación es de aproximadamente 21 días y muchos huevos pueden incubarse simultáneamente, haciendo experimentación rápida y eficiente. Tal vez lo más importante, el embrión es también fácilmente manipulable, permite el estudio exhaustivo de procesos clave del desarrollo y de los genes y las proteínas que impulsan estos procesos.
El ojo de embrión de pollo es un órgano complejo que se desarrolla a través de la interacción de un número de diferentes tejidos similares a muchos otros sistemas del cuerpo. Este método permite el estudio del desarrollo de estos tejidos, especialmente en etapas avanzadas de desarrollo. Por ejemplo, la retina varias capas puede ser de particular interés para quienes estudian el desarrollo del sistema nervioso. Métodos alternativos que permiten el estudio de otros tejidos oculares como la córnea, el cuerpo de vitreal, la lente, la esclera y los párpados son de beneficio para los investigadores. El ojo de embrión de pollo también contiene una serie de huesos planos, los osículos esclerales, que pueden ser utilizados como un modelo para el estudio de inducción ósea intramembranosa y osificación en vertebrados1.
Actualmente, hay una serie de métodos utilizados para estudiar el desarrollo embrionario. Microinyecciones de anticuerpos inhibitorios u otras moléculas inhibidoras2,3, implantaron quirúrgicamente microbeads en inhibidor de la4, y electroporación5 son todos los métodos que pueden utilizarse para regular a la baja los genes o proteínas de interés en un embrión. Métodos similares se utilizan a las proteínas del alza. Estos métodos no están sin sus limitaciones. Por ejemplo, al utilizar productos químicos para alterar el desarrollo embrionario, los efectos letales sobre los embriones deben ser evaluados, y esto limita el uso de los métodos mencionados para sitios localizados de la aplicación en dosis lo suficientemente bajas para asegurar la supervivencia de la embrión.
In vitro cultivo de tejidos se ha utilizado en una amplia gama de organismos para el estudio de desarrollo y se puede utilizar para saltarse algunas de las limitaciones mencionadas. Por ejemplo, los fémures6pluma yemas7,8y miembros9 del pollo se todos han estudiado usando métodos de cultivo de tejidos como los testículos del ratón10 y las raíces y tallos de las plantas11. Estos métodos otorgan a los científicos un alto grado de control sobre el desarrollo de tejido, tales como la capacidad de fluctuar la temperatura y alterar la disponibilidad de nutrientes. El aislamiento de los tejidos del embrión entero también hace mucho menos susceptible a los efectos letales de sustancias químicas, lo que permite estudios de manipulación a escala global en concentraciones más altas. Otra ventaja notable de cultivo en vitro es la preservación del medio ambiente celular del tejido; el arreglo de los tejidos permanece relativamente sin cambios, lo que es posible estudiar las interacciones entre tipos de tejido diferentes9. Así, en vitro cultivo abre puertas a más experimental acercamientos no disponible en modelos en vivo o en ovo .
Actualmente, estudiando el desarrollo del ojo de pollo embrionario utilizando productos químicos es particularmente desafiante. Cubierta de una serie de membranas extraembrionarias del embrión, lo que hace difícil aplicar microesferas o productos químicos. el embrión también es muy activo dentro del huevo como se pone más viejo, complicando aún más un método ya difícil. Este protocolo permite el acceso fácil para el ojo y los tejidos circundantes, eliminando esas barreras, mientras que también proporciona nuevas oportunidades para examinar los procesos del desarrollo dentro del ojo. Este protocolo fue establecido para estudiar la inducción de los osículos esclerales en el ojo embrionario.

<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 height="21" style="height:21px;width:216px;">paper towel</td>
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organotypic culture method to study the development of embryonic chicken tissues

El pollo embrionario se utiliza comúnmente como un organismo modelo confiable para el desarrollo de vertebrados. Su accesibilidad e incubación corto período es ideal para la experimentación. En la actualidad, el estudio de estas vías de desarrollo en el embrión de pollo se lleva a cabo mediante la aplicación de inhibidores y las drogas en los sitios localizados y a bajas concentraciones usando una variedad de métodos. In vitro cultivo de tejidos es una técnica que permite el estudio de los tejidos separados del organismo anfitrión, mientras que al mismo tiempo pasando por alto muchas de las limitaciones físicas presentes al trabajar con embriones enteros, como la susceptibilidad de los embriones a altas dosis de químicos potencialmente letales. Aquí, presentamos un organotypic cultivo de protocolo para el cultivo del embrionario pollo media cabeza en vitro, que presenta nuevas oportunidades para el examen de los procesos de desarrollo más allá de los métodos actualmente establecidos.

Usando este método, un ojo de embrión de pollo puede cultivarse del día 8 de su desarrollo (HH34) en vitro durante 4 días. Cuatro días de desarrollo en ovo corresponde a HH38.
Este método de cultivo apoya el desarrollo de brotes de plumas que rodean el ojo y los párpados (figura 1B). Estos brotes de plumas no están presentes en ovo en HH34 antes el cultivo (<strong...

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 Organotypic método para estudiar el desarrollo de tejidos de embrión de pollo

Aquí, presentamos un organotypic cultivo protocolo para crecer órganos embrionarios de pollo en vitro. Usando este método, el desarrollo de tejidos de embrión de pollo puede ser estudiado, manteniendo un alto grado de control sobre el ambiente de cultivo.

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 vistas.  Mount Saint Vincent University. Aquí, presentamos un organotypic cultivo protocolo para crecer órganos embrionarios de pollo en vitro. Usando este método, el desarrollo de tejidos de embrión de pollo puede ser estudiado, manteniendo un alto grado de control sobre el ambiente de cultivo.

Video: 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 de embriones de ratón explantes cocleares y Gene Transfer por electroporación

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

Investigación

Biología del Desarrollo

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.

La manipulación y<em&gt; In Vitro</em&gt; La maduración de<em&gt; Xenopus laevis</em&gt; Los ovocitos, seguido de inyección intracitoplasmática de espermatozoides, para estudiar el desarrollo embrionario

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 de perfiles de<em&gt; In-Vivo</em&gt; Productos fabricados preimplantación de embriones bovinos mediante plataforma de microarrays de dos colores

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.

La regulación epigenética de Cardíaca diferenciación de células madre embrionarias y Tejidos

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).

Una novela<em&gt; Ex Ovo</em&gt; Banda Técnica para alterar intracardiaca Hemodinámica en un sistema embrionario de pollo

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 murinos embrionarias Metatarsianos: Un modelo fisiológico de osificación endocondral

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.

Disección y cultivo de riñón embrionario de ratón

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.

Análisis computacional de lo elegans de Caenorhabditis del Germline para estudiar la distribución de núcleos, proteínas y 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.

Aislamiento de los tejidos embrionarios y formación de órganos quiméricas pollo codorniz utilizando el ejemplo de 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.

Un método de imágenes de semi-alto rendimiento y un kit de herramientas de visualización de datos para analizar el desarrollo embrionario de C. elegans

Cultura Organotypic método para estudiar el desarrollo de tejidos de embrión de pollo

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