Nos gustaría dar las gracias a Paul Poirier, el Productor de Medios, en la Universidad Mount Saint Vincent por su trabajo en la filmación y la edición de la parte de vídeo de este manuscrito. Reconocemos el de Ciencias Naturales e Ingeniería de Investigación de Canadá para su financiación.

Nota: Todos los suministros se enumeran en la Tabla 1. 1. Almacenamiento del pollo embriones <ol><li> Incubar huevos de gallina de la cepa de la especie Gallus gallus horizontalmente a 37 ° C, con aproximadamente 40% de humedad y girar los huevos una vez o dos veces al día. Volviendo huevos es importante para evitar que el embrión se adhiera a la cáscara del huevo. </li><li> No girar el huevo en la 24 horas antes de la creación de la cultura de lo contrario el embrión se encuentra ventral a la masa de yema y puede dañarse al abrir el huevo en el paso 3. Además, mantener los huevos a 4 ° C grados para no más de una semana antes de la incubación a &quot;alto&quot; desarrollo pero esto no es lo ideal. </li></ol> 2. Puesta pollo embriones <ol><li> Etapa los embriones de pollo utilizando la hamburguesa y Hamilton 12 tabla provisional. El escenario ideal para establecer el cultivo ex ovo es HH etapa 19-20 (alrededor de 3 días de incubación) becausa esto es poco después de la cabeza se convierte en 53 HPF. Nota: HH etapa 19-20 se caracteriza por los siguientes rasgos morfológicos: somitas se extienden en la mayoría de la cola, sin embargo, el final de la cola permanece no segmentado, el brote de la cola está curvado, el alantoides es pequeña y ha limitado vasculatura, las piernas brotes son más grandes que los de ala-brotes y los ojos son sin pigmentar o tienen un tono grisáceo. </li></ol> 3. Extracción del Embrión de la Shell <ol><li> Antes de extraer el embrión de la cáscara, rocíe la cáscara con etanol al 70% y deje que se seque. No encienda el huevo rápidamente ya que podría dañar el embrión. </li><li> Con cuidado de no alterar la orientación del huevo, el crack con cuidado el huevo en la parte inferior (es decir, el lado que estaba ventral durante la incubación) y suelte el embrión en una estéril pesan barco (88 x 88 x 23 mm). Limpie sopesar barcos con etanol al 70%. Inspeccionar el embrión para asegurarse de que el saco vitelino no está dañado. Si la yema hcomo quebrado, deseche el embrión, ya que no va a sobrevivir. </li><li> Observar el embrión para asegurarse de que es viable; el corazón está latiendo, vasculatura parece normal, y no hay anomalías obvias están presentes. Asegúrese de que los bordes del recipiente para pesar están secos. </li><li> Con una pipeta, soltar 40 l de penicilina / estreptomicina (5.000 unidades de penicilina, 5 mg de estreptomicina por ml) en la parte superior de la albúmina para ayudar a prevenir la infección. </li></ol> 4. Preparación de la Cámara de humedad <ol><li> Coloque una pequeña pila de Kim-toallitas y / o una almohadilla absorbente hecho de algodón en el fondo de un recipiente de plástico estéril (12 x 12 x 6 cm) limpió con etanol al 70%. </li><li> Añadir, agua destilada estéril para humedecer los Kim-toallitas y / o algodón (aproximadamente 150 ml de agua). </li></ol> 5. Montaje de la Ex Ovo Cultura <ol><li> Coloque el barco pesar que contiene el embrión en la parte superior del relleno húmedo. Luego, coloque la mitad de un plato petri estéril cuadrada (9,5 x 9,5 cm) en la parte superior de la tque pesan barco para formar una tapa suelta. </li><li> Cubra el recipiente de plástico con su tapa. Presione las dos esquinas de la tapa, asegurando un sello parcial que aún permite una buena circulación de aire dentro de la cámara. </li><li> Coloque cuidadosamente la configuración en la incubadora a 37 o hasta la etapa deseada. </li><li> Colocar los recipientes de agua en la incubadora para ayudar a controlar la humedad, si un incubador de humedad controlada no está disponible. Esterilizar todo el agua en las cámaras de humedad y en la incubadora y reponer cuando sea necesario durante el período de incubación. 40% de humedad es ideal. </li></ol>

<ol>
	<li>Auerbach, R., Kubai, L., Knighton, D., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+procedure+for+the+long-term+cultivation+of+chicken+embryos.">A simple procedure for the long-term cultivation of chicken embryos.</a> Dev. Biol. 41, 391-394 (1974).</li><li>Gennaro, L. D., Packard, D. S., Stach, R. W., Wagner, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+differentiation+of+chicken+embryos+in+simplified+shell-less+cultures+under+ordinary+conditions+of+incubation.">Growth and differentiation of chicken embryos in simplified shell-less cultures under ordinary conditions of incubation.</a> Growth. 44, 343-354 (1980).</li><li>Tufan, C. A., Akdogan, I., Adiguzel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shell-less+culture+of+the+chick+embryo+as+a+model+system+in+the+study+of+developmental+neurobiology.">Shell-less culture of the chick embryo as a model system in the study of developmental neurobiology.</a> Neuroanat. 3, 8-11 (2004).</li><li>Duench, K., Franz-Odendaal, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMP+and+Hedgehog+signaling+during+the+development+of+scleral+ossicles.">BMP and Hedgehog signaling during the development of scleral ossicles.</a> Dev. Biol. 365 (1), 251-258 (2012).</li><li>Datar, S., Bhonde, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shell-less+Chick+Embryo+Culture+as+an+Alternative+in+vitro+Model+to+Investigate+Glucose-Induced+Malformation+in+Mammalian+Embryos.">Shell-less Chick Embryo Culture as an Alternative in vitro Model to Investigate Glucose-Induced Malformation in Mammalian Embryos.</a> Rev Diabet Stud. 2 (4), 221-227 (2005).</li><li>Fisher, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chick+embryos+in+shell-less+culture.+Tested+studies+for+laboratory+teaching.">Chick embryos in shell-less culture. Tested studies for laboratory teaching.</a> , (1983).</li><li>Dunn, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technique+for+shell-less+culture+of+the+72-hour+avian+embryo).">Technique for shell-less culture of the 72-hour avian embryo).</a> Poultry Science. 53, 409-412 (1974).</li><li>Yalcin, H., Shekhar, A., Rane, A. A., Butcher, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ex-ovo+chicken+embryo+culture+system+suitable+for+imaging+and+microsurgery+applications.">An ex-ovo chicken embryo culture system suitable for imaging and microsurgery applications.</a> J. Vis. Exp. (44), (2010).</li><li>Silver, P. H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Special+problems+of+experimenting+in+ovo+on+the+early+chick+embryo,+and+a+solution.">Special problems of experimenting in ovo on the early chick embryo, and a solution.</a> J Embryol Exp Morph. 8 (4), 369-375 (1960).</li><li>Spurlin, J., Lwigale, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+technique+to+increase+accessibility+to+late-stage+chick+embryos+for+in+ovo+manipulations.">A technique to increase accessibility to late-stage chick embryos for in ovo manipulations.</a> Dev. Dyn. 242 (2), 148-154 (2012).</li><li>Dorrell, M. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+ovo+model+for+directly+visualizing+chicken+embryo+development.">Ex ovo model for directly visualizing chicken embryo development.</a> American Biology Teacher. 74 (9), (2012).</li><li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.">A series of normal stages in the development of the chick embryo.</a> J. Morph. 88, 49-92 (1951).</li><li>Drossopoulou, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+for+anteroposterior+patterning+of+the+vertebrate+limb+based+on+sequential+long-and+short-range+Shh+signaling+and+Bmp+signaling.">A model for anteroposterior patterning of the vertebrate limb based on sequential long-and short-range Shh signaling and Bmp signaling.</a> Development. , 127-1337 (2000).</li><li>Sys, G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+in+ovo+CAM-assay+as+a+xenograft+model+for+sarcoma.">The in ovo CAM-assay as a xenograft model for sarcoma.</a> J. Vis. Exp. (77), e50522 (2013).</li><li>Franz-Odendaal, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+understanding+the+development+of+scleral+ossicles+in+chicken,+Gallus+gallus.">Towards understanding the development of scleral ossicles in chicken, Gallus gallus.</a> Dev. Dyn. 237, 3240-3251 (2008).</li></ol>

Los autores no tienen intereses financieros en competencia en lo que respecta a la información presentada en este manuscrito.

Ex ovo cultivo y de ventanas ambos tienen ventajas y desafíos. Aquí se comparan las ventajas y desafíos de la taza de espuma de poliestireno ex ovo método y el método de ventanas a nuestro ex optimizado ovo método que se muestra aquí. Nuestro método permite la manipulación y la fácil observación del embrión de pollo en las etapas finales de desarrollo y nuestros refinamientos al ex ovo tradicional método 1, 2, 3 lo hacen, además, muy fácil de utiliza...

Ex ovo culturing has played an important role in the study of development of the chicken1, 2. This culturing method has been used to study neurological diseases, limb development, craniofacial development, and as a model to investigate malformations associated with diabetes 3, 4, 5.
There are many variations to the ex ovo technique. The most common approach is to use a Styrofoam cup6,7,8 or a glass bowl5. In these methods, the cup or bowl is lined with plastic wrap to cradle the embryo, a lid is placed on the cup, and the embryo is then placed in an incubator with appropriate humidity6. This set up however, can be technically challenging. The first challenge is the plastic wrap that is used to cradle the embryo. This wrap is difficult to work with and often does not adhere to the cup very well. To solve this problem, an elastic band is placed around the cup to hold the wrap in place. Despite this, the wrap can still slip, which is fatal to the embryo. The plastic wrap has the potential to tear or get punctured by forceps or needles that may be used during embryo manipulations and observations. Finally, this set-up is not very stable and students can easily knock the cups over. The height of the cups also makes it very difficult to place the embryo under a stereomicroscope, which has a limited objective to stage height. These challenges make it difficult for undergraduate students to work with live chick embryos in teaching labs, such as advanced developmental biology courses. 
The above challenges in the ex ovo method has meant that researchers turn to the windowing method 9,10 to view embryonic chick development. In this technique, a hole or &#8220;window&#8221; is made in the eggshell overlying the embryo. The hole can be re-sealed with tape or wax9 to allow for further embryonic development. Although the windowing method has some advantages, such as the ability to view embryonic development and easy maintenance, this method also has several limitations. The first is that the window needs to be fairly large in order to view the entire embryo (especially at late stages). Secondly, large windows are difficult to seal; an improper seal will lead to sterility and survivability problems. Using molten wax as a sealant adds another inconvenient and messy step to the protocol. Therefore, although the windowing method may be ideal for chick embryos at young stages (HH 11 &#8211; HH 27), viewing the entire embryo at late stages is not easily accomplished. 
Here, we describe an improved and simple ex ovo culturing technique11 that avoids the need for high tech equipment, is easy to handle under a stereomicroscope, gives the embryo enough support to perform microscopic manipulations, and enables researchers to view the growth of the embryo in its entirety well into the later stages of development (up to HH 40-41). With these advances in the ex ovo technique, individuals gain access to a more complete understanding of embryonic development. For instance, growth into later stages allows individuals to observe developmental processes that do not occur until this time point, such as ossification, feather development, and advanced limb and eye development. The entire embryo and extraembryonic membranes and vasculature are clearly visible. More advanced research can also be performed, such as, embryonic manipulations (i.e., implanting beadssoaked in inhibitors or inserting barriers between tissue layers), and researchers are then able to observe the effect of the manipulations in later stage embryos.

<table border="0" cellpadding="0" cellspacing="0" width="658">
	<tbody>
		<tr height="78">
			<td height="78" style="height:79px;width:177px;">Penicillin/Streptomycin</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:152px;">P4458</td>
			<td style="width:192px;">Make small aliquots to avoid freeze/thaw events</td>
		</tr>
		<tr height="22">
			<td height="61" rowspan="3" style="height:61px;width:177px;">Square Petri Dish</td>
			<td rowspan="3" style="width:136px;">N/A</td>
			<td rowspan="3" style="width:152px;">N/A</td>
			<td rowspan="3" style="width:192px;">9.5 cm x 9.5 cm</td>
		</tr>
		<tr height="19">
		</tr>
		<tr height="20">
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:177px;">Weigh Boat</td>
			<td style="width:136px;">Fischer Scientific</td>
			<td align="right" style="width:152px;">8732113</td>
			<td style="width:192px;">88 x 88 x 23 mm</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:177px;">Ziplock container</td>
			<td style="width:136px;">Ziplock</td>
			<td style="width:152px;">N/A</td>
			<td style="width:192px;">12 cm x 12 cm x 6 cm</td>
		</tr>
	</tbody>
</table>

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.

Este ex ovo método permite la observación de los embriones desde las primeras etapas de desarrollo (HH 19/20) a las etapas finales de desarrollo (HH 40-41) (Figura 1A y 1B). La creación de la cultura en HH 19-20 aumenta la supervivencia de los embriones en la cultura. Antes de la cabeza de giro (antes de 53 hpf) de supervivencia es muy baja en cultivo y después de la etapa 21, el embrión tiende a pegarse más a la cáscara en la eliminación de por lo menos se obtienen emb...

Watch this Scientific Journal Video about Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development at JoVE.com

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).

Optimizado<em&gt; Ex-ovo</em&gt; El cultivo de embriones del polluelo a etapas avanzadas de desarrollo

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

optimized-ex-ovo-culturing-chick-embryos-to-advanced-stages

Research

JoVE Journal

Developmental Biology

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

13.5K vistas.  Mount Saint Vincent University. 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). 

Video: Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

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

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

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

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

Transferencia génica eficaz en polluelo retinas para estudios de cultivo celular primario: Un<em&gt; Ex-ovo</em&gt; Enfoque electroporación

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

Investigación

Biología del Desarrollo

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice &#8212; A High-throughput Screen in ES Cells

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely limits its potential. We describe here the use of mouse embryonic stem (ES) cells as surrogate cells to screen for a phenotype of interest and subsequently introduce these cells into a host embryo to develop into a living mouse carrying the phenotype. This method provides (1) a cost effective, high-throughput platform for genetic screen in mammalian cells; (2) a rapid way to identify the mutated genes and verify causality; and (3) a short-cut to develop mouse mutants directly from these selected ES cells for whole animal studies. We demonstrated the use of paraquat (PQ) to select resistant mutants and identify mutations that confer oxidative stress resistance. Other stressors or cytotoxic compounds may also be used to screen for resistant mutants to uncover novel genetic determinants of a variety of cellular stress resistance.

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice &amp;#8212; A High-throughput Screen in ES Cells

Stress resistance is one of the hallmarks for longevity and is known to be genetically governed. Here, we developed an unbiased high-throughput method to screen for mutations that confer stress resistance in ES cells with which to develop mouse models for longevity studies.

Enfoque genético adelante para descubrir Estrés Los genes de resistencia en ratones - Una pantalla de alto rendimiento en células madre embrionarias

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely ...

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 ...

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.

Un filtro de papel sumergido Sandwich para el largo plazo<em&gt; Ex Ovo</em&gt; Time-lapse de pollo temprano embriones

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 ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and zebrafish embryos. Our system comprises a chamber featuring a simple setup that is nevertheless robust to induce and maintain a specific oxygen concentration and temperature in any experimental solution of choice. The presented system is very cost-effective but highly functional, it allows induction and sustainment of hypoxia for direct experiments in vivo and for various time periods up to 48 h.
To monitor and study the effects of hypoxia, we have employed two methods - measurement of levels of hypoxia-inducible factor 1alpha (HIF-1&#945;) in whole embryos or specific tissues and determination of retinal stem cell proliferation by 5-ethynyl-2&#8242;-deoxyuridine (EdU) incorporation into the DNA. HIF-1&#945; levels can serve as a general hypoxia marker in the whole embryo or tissue of choice, here embryonic retina. EdU incorporation into the proliferating cells of embryonic retina is a specific output of hypoxia induction. Thus, we have shown that hypoxic embryonic retinal progenitors decrease proliferation within 1 h of incubation under 5% oxygen of both frog and zebrafish embryos.
Once mastered, our setup can be employed for use with small aquatic model organisms, for direct in vivo experiments, any given time period and under normal, hypoxic or hyperoxic oxygen concentration or under any other given gas mixture.

We introduce a novel hypoxic chamber system for use with aquatic organisms such as frog and zebrafish embryos. Our system is simple, robust, cost-effective and allows the induction and sustainment of hypoxia in vivo and for up to 48 h. We present 2 reproducible methods to monitor the effectiveness of hypoxia.

Inducción de la hipoxia en los sapos vivos y los embriones de pez cebra

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in zebrafish during different developmental stages. To investigate the role of endoglin(eng) in vessel formation during the development of hereditary hemorrhagic telangiectasia&#160;(HHT), morpholino-mediated targeted knockdown of eng in zebrafish are used to study its temporal expression and associated functions. Here, whole-mount in situ RNA hybridization (WISH) is employed for the analysis of eng and its downstream genes in zebrafish embryos. Also, tube formation assays are performed in HHT patient-derived induced pluripotent stem cell-differentiated&#160;endothelial cells (iPSC-ECs; with eng mutations). A specific signal amplifying system using the whole amount In Situ Hybridization &#8211; WISH provides higher resolution and lower background results compared to traditional methods. To obtain a better signal, the post-fixation time is adjusted to 30 min after probe hybridization. Because fluorescence staining is not sensitive in zebrafish embryos, it is replaced with diaminobezidine (DAB) staining here. In this protocol, HHT&#160;patient-derived iPSC lines containing an eng mutation are differentiated into endothelial cells. After coating a plate with basement membrane matrix for 30 min at 37 &#176;C, iPSC-ECs are seeded as a monolayer into wells and kept at 37 &#176;C for 3 h. Then, the tube length and number of branches are calculated using microscopic images. Thus, with this improved WISH protocol, it is shown that reduced eng expression affects endothelial progenitor formation in zebrafish embryos. This is further confirmed by tube formation assays using iPSC-ECs derived from a patient with HHT. These assays confirm the role for eng in early vascular development.

Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

Hibridación en situ de montaje completo en embriones de pez cebra y ensayo de formación de tubos en iPSC-CE para estudiar el papel de la endoglina en el desarrollo vascular

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

Postimplantation mammalian embryo culture methods have been generally inefficient and limited to brief periods after dissection out of the uterus. Platforms have been recently developed for highly robust and prolonged ex utero culture of mouse embryos from egg-cylinder stages until advanced organogenesis. These platforms enable appropriate and faithful development of pregastrulating embryos (E5.5) until the hind limb formation stage (E11). Late gastrulating embryos (E7.5) are grown in rotating bottles in these settings, while extended culture from pregastrulation stages (E5.5 or E6.5) requires a combination of static and rotating bottle cultures. In addition, sensitive regulation of O2 and CO2 concentration, gas pressure, glucose levels, and the use of a specific ex utero culture medium are critical for proper embryo development. Here, a detailed step-by-step protocol for extended ex utero mouse embryo culture is provided. The ability to grow normal mouse embryos ex utero from gastrulation to organogenesis represents a valuable tool for characterizing the effect of different experimental perturbations during embryonic development.

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

An enhanced platform for whole-embryo culture allows continuous and robust ex utero development of postimplantation mouse embryos for up to six days, from pregastrulation stages until advanced organogenesis. In this protocol, we detail the standard procedure for successful embryo culture using static plates and rotating bottle systems.

Ex Utero Cultivo de embriones de ratón desde la pregastrulación hasta la organogénesis avanzada

Postimplantation mammalian embryo culture methods have been generally inefficient and limited to brief periods after dissection out of the uterus. ...

Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures

During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like the craniofacial skeleton and the peripheral nervous system. While it is critical to understand NCC migration in the context of a 3D embryo, isolating migratory cells in 2D culture facilitates visualization and functional characterization, complementing embryonic studies. The present protocol demonstrates a method for isolating chick cranial neural folds to generate primary NCC cultures. Migratory NCCs emerge from neural fold explants plated onto a fibronectin-coated substrate. This results in dispersed, adherent NCC populations that can be assessed by staining and quantitative morphological analyses. This simplified culture approach is highly adaptable and can be combined with other techniques. For example, NCC emigration and migratory behaviors can be evaluated by time-lapse imaging or functionally queried by including inhibitors or experimental manipulations of gene expression (e.g., DNA, morpholino, or CRISPR electroporation). Because of its versatility, this method provides a powerful system for investigating cranial NCC development.

This versatile protocol describes the isolation of premigratory neural crest cells (NCCs) through the excision of cranial neural folds from chick embryos. Upon plating and incubation, migratory NCCs emerge from neural fold explants, allowing for assessment of cell morphology and migration in a simplified 2D environment.

Preparación y análisis morfológico de cultivos de células de cresta neural craneal de pollo

During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like ...

Isolation of Preadipocytes from Broiler Chick Embryos

Primary preadipocytes are a valuable experimental system for understanding the molecular pathways that control adipocyte differentiation and metabolism. Chicken embryos provide the opportunity to isolate preadipocytes from the earliest stage of adipose development. This primary cell can be used to identify factors influencing preadipocyte proliferation and adipogenic differentiation, making them a valuable model for studies related to childhood obesity and control of excess fat deposition in poultry. The rapid growth of postnatal adipose tissue effectively wastes feed by allocating it away from muscle growth in broiler chickens. Therefore, methods to understand the earliest stages of adipose tissue development may provide clues to regulate this tendency and identify ways to limit adipose expansion early in life. The present study was designed to develop an efficient method for isolation, primary culture, and adipogenic differentiation of preadipocytes isolated from developing adipose tissue of commercial broiler (meat-type) chick embryos. The procedure has been optimized to yield cells with high viability (~98%) and increased capacity to differentiate into mature adipocytes. This simple method of embryonic preadipocyte isolation, culture, and differentiation supports functional analyses of fat growth and development in early life.

The present protocol describes a simple method for isolating preadipocytes from adipose tissue in broiler embryos. This method enables isolation with high yield, primary culture, and adipogenic differentiation of preadipocytes. Oil Red O staining and lipid/DNA stain measured the adipogenic ability of isolated cells induced with differentiation media.

Aislamiento de preadipocitos de embriones de pollos de engorde

Primary preadipocytes are a valuable experimental system for understanding the molecular pathways that control adipocyte differentiation and metabolism. ...

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as the hair cell. Basic research in the inner ear has led to a deep understanding of how the hair cell functions, and how dysregulation can lead to hearing loss and vertigo. For this research, the mouse has been the pre-eminent model system. However, mice, like all mammals, have lost the ability to replace hair cells. Thus, when trying to understand cellular therapies for restoring inner ear function, complementary studies in other vertebrate species could provide further insights. The auditory epithelium of birds, the basilar papilla (BP), is a sheet of epithelium composed of mechanosensory hair cells (HCs) intercalated by supporting cells (SCs). Although the anatomical architecture of the basilar papilla and the mammalian cochlea differ, the molecular mechanisms of inner ear development and hearing are similar. This makes the basilar papilla a useful system for not only comparative studies but also to understand regeneration. Here, we describe dissection and manipulation techniques for the chicken inner ear. The technique shows genetic and small molecule inhibition methods, which offer a potent tool for studying the molecular mechanisms of inner ear development. In this paper, we discuss in ovo electroporation techniques to genetically perturb the basilar papilla using CRIPSR-Cas9 deletions, followed by dissection of the basilar papilla. We also demonstrate the BP organ culture and optimal use of culture matrices, to observe the development of the epithelium and the hair cells.

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The chick is a cost-effective, accessible, and widely available model organism for a variety of studies. Here, a series of protocols is detailed to understand the molecular mechanisms underlying avian inner ear development and regeneration.

Métodos In Ovo y Ex Ovo para estudiar el desarrollo del oído interno aviar

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as ...