We would like to thank Paul Poirier, the Media Producer, at Mount Saint Vincent University for his work in filming and editing the video portion of this manuscript. We acknowledge the Natural Science and Engineering Research Council of Canada for funding.

Note: All supplies are listed in Table 1.
1. Storing the Chicken Embryos
<ol>
	<li>Incubate chicken eggs of the strain Gallus gallus horizontally at 37oC with approximately 40% humidity and turn eggs once or twice daily. Turning eggs is important to prevent the embryo from adhering to the eggshell.</li>
	<li>Do not to turn the egg in the 24 hr&#160;prior to setting up the culture as otherwise the embryo will be located ventral to the yolk mass and will be damaged on opening the egg in step 3. In addition, keep the eggs at 4oC degrees for no more than one week before incubation to &#8220;halt&#8221; development but this is not ideal.</li>
</ol>
2. Staging Chicken Embryos
<ol>
	<li>Stage the chicken embryos using the Hamburger and Hamilton12 staging table. The ideal stage for setting up the ex-ovo culturing is HH stage 19-20 (around 3- days of incubation) because this is shortly after the head turns at 53 hpf. 
	Note: HH stage 19-20 is characterized by the following morphological traits: somites are extended into the majority of the tail, however the very end of the tail remains unsegmented, the tail bud is curled, the allantois is small and has limited vasculature, the leg-buds are larger than the wing-buds and the eyes are unpigmented or have a grayish hue.</li>
</ol>
3. Removing the Embryo from the Shell
<ol>
	<li>Before removing the embryo from the shell, spray the shell with 70% ethanol and allow it to dry. Do not turn the egg rapidly as this will damage the embryo.</li>
	<li>Being careful not to alter the orientation of the egg, carefully crack the egg on the lower side (i.e., the side that was ventral during incubation) and release the embryo into a sterile weigh boat (88 x 88 x 23 mm). Wipe down weigh boats with 70% ethanol. Inspect the embryo to ensure that the yolk sack is not damaged. If the yolk has broken, discard the embryo, as it will not survive.</li>
	<li>Observe the embryo to ensure that it is viable; the heart is beating, vasculature appears normal, and no obvious abnormalities are present. Ensure that the edges of the weigh boat are dry.</li>
	<li>Using a pipette, drop 40 &#956;l of penicillin/streptomycin (5,000 units penicillin, 5 mg streptomycin per ml) on top of the albumin to help prevent infection.</li>
</ol>
4. Preparing the Humidity Chamber
<ol>
	<li>Place a small stack of kim-wipes and/or an absorbent pad made of cotton in the bottom of a sterile plastic container (12 x 12 x 6 cm) wiped with 70% ethanol.</li>
	<li>Add sterile, distilled water to moisten the kim-wipes and/or cotton (approximately 150 ml of water).</li>
</ol>
5. Assembling the Ex Ovo Culture
<ol>
	<li>Place the weigh boat containing the embryo on top of the moist padding. Then, place half of a square sterile petri-dish (9.5 x 9.5 cm) on top of the weigh boat to form a loose lid.</li>
	<li>Cover the plastic container with its lid. Press down two corners of the lid, ensuring a partial seal that still allows for good airflow inside the chamber.</li>
	<li>Carefully place the setup into the 37oC incubator until desired stage.</li>
	<li>Place containers of water in the incubator to help control the humidity, if a controlled humidity incubator is unavailable. Sterilize all water in the humidity chambers and in the incubator and replenish as needed during the incubation period. 40% humidity is 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>

The authors have no competing financial interests in regards to the information presented in this manuscript.

Ex ovo culturing and windowing both have advantages and challenges. Here we compare the advantages and challenges of the Styrofoam cup ex ovo method and the windowing method to our optimized ex ovo method shown here. Our method enables manipulation and easy observation of the chick embryo at late stages of development and our refinements to the traditional ex ovo method1, 2, 3 make it additionally very easy to use in undergraduate teaching laboratory classes.
Although many researchers prefer the windowing method to study chicken development because it is simple to perform and has excellent survivability to late stages of development (HH 41-42), it has limitations. The largest of which is the limited ability to view the entire embryo after HH stage 30. After stage HH 30, the window in the eggshell needs to be very large in order to view the growing, moving embryo. This large window is difficult to seal completely (leading to problems with sterility and survivability), and obtaining good lighting inside the egg can be challenging. In addition, advanced stage embryos are large and difficult to access (or manipulate) through the window.
The ex ovo method described here provides full uninhibited access to the embryo. Once the weigh boat is placed in the humidity chamber, the entire set-up is very stable. The entire humidity chamber can be placed under a dissecting microscope and high magnification can be used to observe the entire embryo. In comparison, the Styrofoam cup method has a humidity chamber that is tall (cup height) and this makes it very difficult to place under the microscope objectives. The cup design is also far less stable than the flat weigh boat and these cups can easily be knocked over. Our improvements to the ex ovo method make this method straightforward to use in a classroom or lab setting and enables the student to have full access to embryos from stage HH 19 through to HH 40; stages when major organogenesis is occurring. The biggest disadvantage of our ex ovo method is sterility. In order to prevent infection, our method includes the addition of antibiotics. Another dose of antibiotic can be given to the embryo at later stages. The antibiotics administered during culture set-up also does not appear to affect development of the embryo. Sterilizing containers with 70% ethanol prior to use can dramatically increase survivability.
Overall, the ex ovo method is ideal for viewing and manipulating embryos without limitations on access or stage of development. Previous research has shown that culturing the embryos ex ovo does not negatively affect patterning4,15. For example, we do not see any changes in the patterning or induction of the skeleton despite the absence of the eggshell. Our improved method solves multiple challenges that exist with the current traditional ex ovo method1,2, 3.

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.

This ex ovo method allows for the observation of embryos from early stages of development (HH 19/20) to late stages of development (HH 40-41) (Figure 1A and 1B). Setting up the culture at HH 19-20 increases survivability of the embryos in the culture. Prior to the head turning (before 53 hpf) survivability is very low in culture and after stage 21, the embryo tends to stick more to the shell on removal so fewer intact embryos are obtained. In general, survivability of the embryo in ex ovo culture is high up to HH 35-36 (90-100%), but it does drop in the more advanced stages of development (about 40% survive to HH 40-41). Additionally, although it has been shown that development appears normal at these late stages and patterning of the skeleton is unaffected 4,15, we have observed that the limbs are bent at these late stages suggesting that the degree or timing of ossification may be affected. This is not surprising considering the need for calcium from the shell for ossification. A detailed comparison of the development of the embryo compared to controls is underway and is beyond the scope of this methods paper.
Obtaining access to the embryo throughout development is important for two main reasons. First, researchers are able to view development on a daily basis without having to unseal and reseal a window. For example, individuals are able to observe limb and eye development at multiple stages (Figure 1C and 1D). Secondly, ex ovo culturing enables greater (unrestricted) access to the embryo, thereby enabling easier manipulations or surgeries.
Embryonic manipulations most often, need to be performed using a stereomicroscope. Using the ex ovo culturing method described here as opposed to the Styrofoam/plastic wrap method6,7,8 the embryo set-up is easily placed under the microscope, is less deep and will not tip over. This enables the researcher to position the humidity chamber precisely as needed and to rotate it to optimize access to the embryo, since there are no barriers (egg shell) obscuring the embryo and since the chamber is very stable. Overall, the support the embryo receives from the weigh boat allows gentle pressure to be applied to the embryo during manipulation without the set-up toppling over.
Several developmental biology methods can be conducted on the embryos in this culture system. These include detailed observations of organ (e.g., eye, brain etc) or tissue development (e.g., blood vessel growth) or application of teratogenic agents. Manipulations include grafting of tissues onto the chorioallantoic membranes to study the effect of combining different tissue layers or studying tumor growth and metastasis14. Another popular manipulation performed during development is bead or barrier implantation. Bead implantation allows a researcher to soak an inhibitor or factor onto the bead and then implant the bead locally to inhibit or induce genes locally in the area of interest. This is often done using affigel or heparin beads (Figure 2A). For example, bone morphogenetic proteins (BMPs) can be locally inhibited during the induction of neural crest derived bone4, 15, or in the developing limb bud13. After a bead is implanted, the embryo can be returned to the incubator and it will continue to develop. This allows researchers to view the downstream effects of the inhibition of a particular gene (in this case BMP), such as the inhibition of scleral ossicle formation in the sclerotic ring (Figure 2B and 2C)15. Similarly, barriers can easily be inserted between tissue layers in order to study tissue-to-tissue signaling and the microinjection of dyes can also easily be conducted.
<img alt="Figure 1" src="/files/ftp_upload/52129/52129fig1.jpg" /> 
Figure 1.&#160;Viewing the embryo ex ovo. (A) HH stage 20 chick embryo after removal from the shell, arrow points to the embryo with vascularized embryonic membranes. (B) A HH stage 40 embryo in an open humidity chamber. (C) HH stage 35 embryo showing early limb buds (arrow). (D) Stage HH 41 embryo showing later stage development of structures such as the eye (arrow). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52129/52129fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52129/52129fig2.jpg" /> 
Figure 2.&#160;Example of Noggin soaked bead Implantation Experiment4. (A) Embryo HH stage 35 exposed through a small hole torn in the overlying membranes in order to gain access to the underlying eye for bead implantation. High magnification of the affigel bead adjacent to a conjunctival papilla is shown in inset. Scale bar in C is 75 &#956;m. (B) Alkaline phosphatase stained right eye after Noggin bead implantation (arrow) showing gap in the ring of ossicles, HH 38 (C) AP stained left eye showing no disruption to the ring of ossicles (control), HH 38. Details of this experiment are provided in Duench and Franz-Odendaal4. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52129/52129fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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).

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

13.5K Views.  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.

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

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.

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.

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).

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.

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.

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 ...

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells (HSPCs) in Developing Zebrafish Embryos

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell lineages that comprise mature blood. Isolation and identification of these HSPCs is difficult because they are defined ex post facto; they can only be defined after their differentiation into specific cell lineages. Over the past few decades, the zebrafish (Danio rerio) has become a model organism to study hematopoiesis. Zebrafish embryos develop ex utero, and by 48 h post-fertilization (hpf) have generated definitive HSPCs. Assays to assess HSPC differentiation and proliferation capabilities have been developed, utilizing transplantation and subsequent reconstitution of the hematopoietic system in addition to visualizing specialized transgenic lines with confocal microscopy. However, these assays are cost prohibitive, technically difficult, and time consuming for many laboratories. Development of an in vitro model to assess HSPCs would be cost effective, quicker, and present fewer difficulties compared to previously described methods, allowing laboratories to quickly assess mutagenesis and drug screens that affect HSPC biology. This novel in vitro assay to assess HSPCs is performed by plating dissociated whole zebrafish embryos and adding exogenous factors that promote only HSPC differentiation and proliferation. Embryos are dissociated into single cells and plated with HSPC-supportive colony stimulating factors that cause them to generate colony forming units (CFUs) that arise from a single progenitor cell. These assays should allow more careful examination of the molecular pathways responsible for HSPC proliferation, differentiation, and regulation, which will allow researchers to understand the underpinnings of vertebrate hematopoiesis and its dysregulation during disease.

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos

Here, we present a simple method to quantitate hematopoietic stem and progenitor cells (HSPCs) in embryonic zebrafish. HSPCs from dissociated zebrafish are plated in methylcellulose with supportive factors, differentiating into mature blood. This allows the detection of blood defects and allows drug screening to be easily conducted.

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell ...

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.

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

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...

A Simple and Effective Transplantation Device for Zebrafish Embryos

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex developmental processes. Zebrafish embryos are ideally suited for these manipulations since they are easily accessible, relatively large in size, and transparent. However, previously developed devices for cell removal and transplantation are cumbersome to use or expensive to purchase. In contrast, the transplantation device presented here is economical, easy to assemble, and simple to use. In this protocol, we first introduce the handling of the transplantation device as well as its assembly from commercially and widely available parts. We then present three applications for its use: generation of ectopic clones to study signal dispersal from localized sources, extirpation of cells to produce size-reduced embryos, and germline transplantation to generate maternal-zygotic mutants. Finally, we show that the tool can also be used for embryological manipulations in other species such as the Japanese rice fish medaka.

Embryological manipulations such as extirpation and transplantation of cells are important tools to study early development. This protocol describes a simple and effective transplantation device to perform these manipulations in zebrafish embryos.

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex ...