This work was supported by National Natural Science Foundation of China (Grant No. 91643203, 91543208, 81502835).

All procedures described were approved by the Institutional Animal Care and Use Committee (IACUC) of Qingdao University. In our lab, the eggs were incubated in two incubators. Eggs were held upright in the incubator and randomly placed on the shelves. The incubation conditions for the eggs were as follows: incubation temperature started at 37.9 &#176;C, and gradually decreased to 37.1 &#176;C as incubation proceeded; the humidity started at 50% and gradually increased to 70%.
1. Exposure methods
NOTE: Exposure of environmental contaminants to chicken embryos may be achieved in several ways. In this section, three routinely used methods are described in detail.
<ol>
	<li>Air cell injection (Figure 1) 
	NOTE: This is the classical exposure method to chicken embryos4,5,6, suitable for a wide range of materials, and may be performed at a very wide time window, from the beginning of development (embryonic day zero, ED0) all the way to the day prior to hatch (ED20). Sunflower oil is used as the vehicle. Previous studies have shown that no significant changes in mortality, hatchability, or body weight have been observed between untreated embryos and embryos injected with sunflower oil7.
	<ol>
		<li>Prepare the following necessary reagents/tools: 75% ethanol, tissue paper, metal probe (can substitute with any sharp metal needle/stick/awl), melted paraffin, brush, povidone iodide solution, pipette, pipette tips, candling lamp, dosing mixture. Prepare the dosing mixture with sunflower oil (recommended)4. To use other diluents, perform vehicle control (versus untreated embryos).</li>
		<li>Clean the surface of the eggs with povidone iodide solution (commercially available povidone iodide solution 1:5 diluted with deionized water), and dip-dry the egg shell with tissue paper without scrubbing. Scrubbing will break the protective layer coating the outside of the shell.</li>
		<li>Candle the eggs in a dark room and mark the air cell with pencil. Exclude eggs with cracks on the shell. Exclude eggs with air cells on the side instead of blunt tip, as those are highly unlikely to hatch normally.</li>
		<li>Sanitize the air cell area with 75% ethanol, and then drill a small hole at the center of air cell area with the metal probe. Do not stick the probe deep into the air cell or the inner membrane may be damaged, instead, only break the shell with the tip of the probe. If the hole is not large enough to fit in a fine pipette tip, break the shell again at the vicinity of existing hole, until the hole is large enough to allow insertion of 10 &#181;L pipette tip.</li>
		<li>Vortex the dosing mixture vigorously, and immediately draw solution to pipette tip. The recommended injection volume is 1 &#181;L per 10 gram of egg (e.g., 5 &#181;L injection volume for a 50-gram egg) as larger injection volumes may create hypoxic or anoxic conditions for the developing embryo. Calculate the concentration of the dosing solution for desired mg/egg kg dose.</li>
		<li>Insert the pipette tip into the hole, with the tip touching the inner membrane. Slowly eject the dosing mixture, hold for at least ten seconds (allow the viscous oil to be fully dispensed), and then remove the tip.</li>
		<li>Seal the hole with a brush and a drop of melted paraffin. Be careful not to drip melted paraffin onto the inner membrane.</li>
		<li>Once sealed, place the eggs in the incubator until they reach the desired embryonic stage. On already developing embryos, perform the whole process as soon as possible to prevent potential embryo loss due to low environmental temperatures.</li>
	</ol>
	</li>
	<li>Microinjection (Figure 2) 
	NOTE: This is a more direct exposure method, resulting in definitive exposure to the substance-of-interest, and especially suitable for compounds with a short duration of action (e.g., lentivirus), since the classical air cell injection requires time for the compounds to penetrate the inner membrane. This method may also be tried if satisfying results could not be achieved by air cell injection. This method is best suitable for early embryos (up to ED2), but also can be performed on older embryos (with higher risk of embryo loss).
	<ol>
		<li>Prepare the following necessary reagents/tools: 75% ethanol, povidone iodide solution, micro-injector (5 &#181;L), metal probe (can substitute with any sharp metal needle/stick/awl should work), fine forceps, tape. Prepare the dosing mixture with sterile saline, which also serves as an injection control without affecting hatchability significantly. Ensure the sterility of the saline, as a contaminated injection will dramatically increase mortality.</li>
		<li>Clean the eggs as described in 1.1.2.</li>
		<li>Candle the eggs as described in 1.1.3.</li>
		<li>Sanitize the air cell area with 75% ethanol, and then drill a small hole at the center of air cell area with the metal probe. Do not stick the probe deep into the air cell or the inner membrane may be damaged, instead, only break the shell with the tip of the probe. Then use the fine forceps to carefully enlarge the hole until the diameter is approximately 2 mm, allowing visual confirmation of the inner membrane.</li>
		<li>Load the solution into microinjector (maximum injection volume: 0.5 &#181;L/10 g egg. (e.g., 2.5 &#181;L for a 50 g egg) and carefully insert the needle through the hole into the inner membrane for approximately 2-3 mm. Gently dispense the solution and remove the needle. Keep the needle as perpendicular to the membrane as possible.</li>
		<li>Seal the hole with a small piece of tape. Completely cover the hole to prevent embryo dehydration and death during subsequent incubation. Nevertheless, avoid pieces of tape that are too large to prevent hypoxia.</li>
		<li>Once sealed, place the eggs in the incubator until they reach the desired embryonic stage. On already developing embryos, perform the whole process as soon as possible to prevent potential embryo loss due to low environmental temperature.</li>
	</ol>
	</li>
	<li>Air cell inhalation (Figure 3) 
	NOTE: This is a novel inhalation method taking advantage of the air cell, from which the late-stage chicken embryo will start to breathe air. It is suitable for gas or aerosol exposures and may achieve very early-in-life inhalation exposures, and fill the lungs with the target gas/aerosol when they open for the first time in life.
	<ol>
		<li>Prepare the following necessary reagents/tools: Sampling bag (PVF bag, for storage of gas/aerosol sample before exposure), catheter needle, syringe, metal probe (can substitute with any sharp metal needle/stick/awl should work), tape, fume hood.</li>
		<li>Clean the eggs as described in 1.1.2 and candle them as described in 1.1.3 (it is not necessary to mark air cell prior to incubation), and then incubate eggs without treatment until ED17.</li>
		<li>Candle the eggs at ED17 to mark the air cell area.</li>
		<li>At ED18, take an egg out from the incubator, sanitize the air cell area with 75% ethanol, and then carefully drill two small holes on two sides of the air cell. One is for injection of gas/aerosol, the other is for expelling of air. Carefully control the size of the holes so that the size of the injection hole is just enough for the catheter needle to be inserted, while the diameter of the expelling hole is a bit larger (approximately 1 mm).</li>
		<li>Gently inject 10 mL of target gas/aerosol from the injection hole with a syringe attached to the catheter needle. Inject air for an inhalation control group, which should have no significant differences to a negative control group8. Apply pressure against the catheter needle (with appropriate amount of pressure the elastic needle can be pushed against the shell) to minimize the leakage from injection hole. Seal both holes immediately with tape afterwards, and return the egg to incubator. 
		&#8203;NOTE: This procedure should be performed in a fume hood to prevent inhalation of gas/aerosol by the operator.</li>
		<li>Repeat the described procedure after one hour to further ensure the whole air cell is filled up with target gas/aerosol (optional).</li>
		<li>Repeat the described procedure at ED19 again (optional). Repeating the exposure helps to ensure consistent exposure until hatch. Record the hatch time for an approximate estimation of exposure duration.</li>
		<li>Once desired exposures have been performed and sealed, place the eggs in the incubator for hatching. Minimize the time the egg spends outside of the incubator to prevent death from low environmental temperature.</li>
	</ol>
	</li>
</ol>
2. Endpoint assessment methods
NOTE: Following exposure of contaminant-of-interest to the developing embryo, several toxicity parameters can be evaluated, including cardiotoxicity. In this section, two frequently used specific methods are described in detail.
<ol>
	<li>Electrocardiography (Figure 4) 
	NOTE: It is impossible to do non-invasive electrocardiography in hatchling chickens due to the presence of feathers. Thus, subcutaneous implantation of electrodes is essential, requiring anesthesia. The dose used in the lab is 33 mg/kg pentobarbital via intraperitoneal injection (some chickens may require up to 50% dose increase). This method will result in stable electrocardiography in over 90% of animals, allowing for analyzation of heart rate.
	<ol>
		<li>Prepare the following necessary reagents/tools: 1% (10 mg/mL) pentobarbital solution in saline, syringe, electrical balance, heater (if necessary), an electrocardiography instrument attached (e.g., BL-420E+) with two-channel metal needle electrodes.</li>
		<li>Weigh the chickens with a balance and calculate the necessary volume of pentobarbital solution and inject the chickens. For a chicken that weighs 30 g, 0.1 mL of pentobarbital solution is needed. Make sure that the injection is done on the lateral side of the abdomen, as the yolk is located at the middle and injection there may not be effective.</li>
		<li>Wait until the injected chickens are anesthetized (hold the chicken in hand, if the neck has no tension and the head can be swung freely, the anesthetization is sufficient). Place chickens on the operation table (heaters are necessary if room temperature is below 20 &#176;C).</li>
		<li>Insert two needle electrodes from two sides of the abdomen, subcutaneously. Make sure that the needles do not enter the abdominal cavity by lifting the skin a little bit and inserting needle from there. Once inserted, carefully push the needle forward until it reaches the side of chest cavity. Make sure that the needle does not go deep into the body or stick out from the skin.</li>
		<li>Make the measurements with the electrocardiography instrument. Other similar instruments capable of electrocardiography can be used.</li>
		<li>If the chickens are to be sacrificed, perform euthanasia after the electrocardiography since they are already under anesthesia. If chickens are to survive, place them back into their cages and warm until waking up. Returning them to the incubator is another option.</li>
	</ol>
	</li>
	<li>Histomorphometry (Figure 5) 
	NOTE: A specific method is developed to assess the right ventricular wall thickness in transverse sections of the heart. Morphological assessment of right ventricle dimension via echocardiography is not 100% accurate due to the irregular shape of right ventricle, and this method may serve as a good supplement in the morphological assessments for the right ventricle.
	<ol>
		<li>Prepare the following necessary reagents/tools: 4% phosphate buffered formaldehyde, sharp blade, phosphate buffered saline, paper towel, electrical balance, small scissors, general histological processing agents (graded ethanol, xylene, paraffin).</li>
		<li>Once animals are sacrificed, use water to wet the feathers. This is to minimize the potential contamination due to flying feathers while opening up the chest.</li>
		<li>Open the chest cavity carefully without damaging the heart. Use small scissors to cut the vasculature and gently remove the heart from the chest cavity. Leave a small piece (approximately 1-2 mm) of vasculature attached to the heart as this may be convenient for subsequent handling of the heart without damaging the heart.</li>
		<li>Once removed, rinse the heart in cold phosphate buffered saline to remove blood and relax the muscle. Then dip dry the heart on paper towel before weighing for accurate weight reading. Put the heart into 10x volume fixative (4% phosphate buffered formaldehyde) for 24 hours at room temperature. Fixed tissues may be subsequently processed to paraffin blocks, or be stored at 4 &#176;C for years (not recommended if immunohistochemistry is planned).</li>
		<li>Before embedding, cut the tissues at approximately 60% length of the heart counting from the apex (Figure 5A), for easier subsequent processing. A microtome blade is recommended for a quick and vertical clean cut. For chickens older than one day, make another cut at approximately 25-30% length from the apex for easier paraffin penetration and to allow the tissue to fit in tissue cassettes.</li>
		<li>Process the tissues with the following conditions (adjust as needed): 70% ethanol for 1 h, 80% ethanol for 1 h, 95% ethanol for 1 h x2, 100% ethanol for 30 min x2, xylene for 5 min x2, paraffin (melting point 52-54 &#176;C) for 1.5 h, paraffin (melting point 62-64 &#176;C) for 1 h, and then embed the tissues in a 3:1 mixture of paraffin (melting point 62-64 &#176;C) and paraffin (melting point 52-54 &#176;C).</li>
		<li>Section the tissue at 6 &#181;m thickness. Carefully maintain identical relative positions of the cross-sections by confirming the presence and size of an anatomical landmark (septomarginal trabecula) in the right ventricle. Confirm a landmark with moderate length on each section (Figure 5B, arrow).</li>
		<li>Make two electronic rulers with Logo programmer: Ruler 1 is a straight line with 7 radius measure lines attached to the middle point, with 22.5&#176; in between two adjacent measure lines. Ruler 2 is just two perpendicular lines in a T shape (Figure 5B).</li>
		<li>Measure with two software programs: Adobe Photoshop and ImageJ.
		<ol>
			<li>In Photoshop, resize ruler 1 (NO reshaping) to place the two ends of the ruler on the two ends of free right ventricular wall, so that the seven measure lines on ruler 1 will each meet the inner right ventricular wall. Then use ruler 2 to make perpendicular measurements from the inner to external ventricular wall (Figure 5B).</li>
			<li>Use ImageJ to make the seven measurements for each heart.</li>
		</ol>
		</li>
		<li>Depending on specific needs, analyze the seven measurements or average for one representative right ventricular wall thickness. Normalize to whole heart weight for specific ventricular wall thickness changes.</li>
	</ol>
	</li>
</ol>

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The authors declare no conflict of interest.

The chicken embryo has been a classical model in developmental studies for 200 years1. Our methods presented in this manuscript have been used in the assessment of several environmental contaminants, including perfluorooctanoic acid, particulate matter, and diesel exhaust with success5, 7,8,9,10,11<sup...

The chicken embryo is a classic developmental model, which has been used for over two hundred years1. The chicken embryo model has various advantages compared to traditional models. First of all, as early as over 70 years ago, the normal development of the chicken embryo had been illustrated very clearly in the Hamburger-Hamilton staging guide2, in which a total of 46 stages during chicken embryo development were defined with precise time and morphological characteristics, facilitating detections of abnormal development. Additionally, the chicken embryo model has other features such as being relatively low-cost and redundant in quantity, relatively accurate exposure-dose controls, an independent, closed system within the shell and easy manipulation of the developing embryo, all of which guarantees its potential to be used as a powerful toxicological assessment model. 
In cardiotoxicity, the chicken embryo features a four chambered heart, similar to mammalian hearts but with thicker walls, allowing easier morphological assessments. Additionally, the chicken embryo allows for developmental inhalation exposure, which is not possible in mammalian models: during the later stage of development, the chicken embryo will transition from internal respiration to external respiration (getting oxygen via the lung); the latter requires that the embryo penetrates the air cell membrane with the beak, and starts to breathe air3, making the air cell a mini-inhalation chamber. Utilizing this phenomenon, the toxicological effects of gas contaminants on the heart (and other organs) may be assessed without the need of dedicated inhalation chamber instruments. 
In this manuscript, several exposure/endpoint assessment methods are described, all of which serve to make the chicken embryo a powerful tool in the assessment of development cardiotoxicity following exposure to environmental contaminants.

<table><tbody><tr><td>4% phosphate buffered formaldehydefixative</td><td>Biosharp, Hefei, China</td><td>REF: BL539A</td><td></td></tr><tr><td>75% ethanol</td><td>Guoyao,Shanghai,China</td><td>CAS:64-17-5</td><td></td></tr><tr><td>Biosignaling monitor BL-420E+</td><td>Taimeng, Chengdu, China</td><td>BL-420E&lt;sup&gt;+&lt;/sup&gt;</td><td></td></tr><tr><td>Candling lamp</td><td>Zhenwei, Dezhou, China</td><td>WZ-001</td><td></td></tr><tr><td>Disposable syringe</td><td>Zhiyu, Jiangsu, China</td><td></td><td></td></tr><tr><td>Egg incubator</td><td>Keyu,Dezhou, China</td><td>KFX</td><td></td></tr><tr><td>Electrical balance</td><td>OHAUS, Shanghai, China</td><td>AR 224CN</td><td></td></tr><tr><td>Electro-thermal incubator</td><td>Shenxian, Shanghai, China</td><td>DHP-9022</td><td></td></tr><tr><td>Ethanol absolute</td><td>Guoyao,Shanghai,China</td><td>CAS:64-17-5</td><td></td></tr><tr><td>Fertile chicken egg</td><td>Jianuo, Jining, China</td><td></td><td></td></tr><tr><td>Hematoxylin and Eosin Staining Kit</td><td>Beyotime, Bejing, China</td><td>C0105</td><td></td></tr><tr><td>Histology paraffin</td><td>Aladdin, Shanghai, China</td><td>P100928-500g</td><td>Melt point 52~54&amp;deg;C</td></tr><tr><td>Histology paraffin</td><td>Aladdin, Shanghai, China</td><td>P100936-500g</td><td>Melt point 62~64&amp;deg;C</td></tr><tr><td>IV catheter</td><td>KDL, Zhejiang, China</td><td></td><td>The catheters have to be soft, plastic ones.</td></tr><tr><td>Lentivirus</td><td>Genechem, Shanghai, China</td><td></td><td>The lentivirus were individually designed/synthesized by Genechem.</td></tr><tr><td>Masson&#039;s trichrome staining kit</td><td>Solarbio, Beijing, China</td><td>G1340</td><td></td></tr><tr><td>Metal probe</td><td>Jinuotai, Beijing, China</td><td></td><td></td></tr><tr><td>Microinjector (5 uL)</td><td>Anting,Shanghai, China</td><td></td><td></td></tr><tr><td>Microscope</td><td>CAIKON, Shanghai, China</td><td>XSP-500</td><td></td></tr><tr><td>Microtome</td><td>Leica, Germany</td><td>HistoCore BIOCUT</td><td></td></tr><tr><td>Microtome blade</td><td>Leica,Germany</td><td>Leica 819</td><td></td></tr><tr><td>Pentobarbitual sodium</td><td>Yitai Technology Co. Ltd.,&amp;nbsp; Wuhan, China</td><td>CAS: 57-33-0</td><td></td></tr><tr><td>Pipetter(10ul)</td><td>Sartorius, Germany</td><td></td><td></td></tr><tr><td>Povidone iodide</td><td>Longyuquan, Taian, China</td><td></td><td></td></tr><tr><td>Scissor</td><td>Anqisheng,Suzhou, China</td><td></td><td></td></tr><tr><td>Sterile saline</td><td>Kelun,Chengdu, China</td><td></td><td></td></tr><tr><td>Sunflower oil</td><td>Mighty Jiage, Jiangsu, China</td><td></td><td>Any commerical sunflower oil for human consumption should work</td></tr><tr><td>Tape</td><td>M&amp;amp;G, Shanghai, China</td><td></td><td></td></tr><tr><td>Tedlar PVF Bag (5L)</td><td>Delin, Dalian, China</td><td></td><td></td></tr><tr><td>Vortex mixer</td><td>SCILOGEX, Rocky Hill, CT, US</td><td>MX-F</td><td></td></tr><tr><td>Xylene</td><td>Guoyao,Shanghai,China</td><td>CAS:1330-20-7</td><td></td></tr></tbody></table>

using chicken embryo as a powerful tool in assessment of developmental cardiotoxicities

Chicken embryos are a classical model in developmental studies. During the development of chicken embryos, the time window of heart development is well-defined, and it is relatively easy to achieve precise and timely exposure via multiple methods. Moreover, the process of heart development in chicken embryos is similar to mammals, also resulting in a four-chambered heart, making it a valuable alternative model in the assessment of developmental cardiotoxicities. In our lab, the chicken embryo model is routinely used in the assessment of developmental cardiotoxicities following exposure to various environmental pollutants, including per- and polyfluoroalkyl substances (PFAS), particulate matter (PMs), diesel exhaust (DE) and nano materials. The exposure time can be freely selected based on the need, from the beginning of development (embryonic day 0, ED0) all the way to the day prior to hatch. The major exposure methods include air-cell injection, direct microinjection, and air-cell inhalation (originally developed in our lab), and the currently available endpoints include cardiac function (electrocardiography), morphology (histological assessments) and molecular biological assessments (immunohistochemistry, qRT-PCR, western blotting, etc.). Of course, the chicken embryo model has its own limitations, such as limited availability of antibodies. Nevertheless, with more laboratories starting to utilize this model, it can be used to make significant contributions to the study of developmental cardiotoxicities.

Exposure results 
Air cell injection 
Air cell injection can effectively expose developing chicken embryos to various agents, which may be subsequently detected in the collected samples (serum, tissue, etc.) of embryos/hatchling chickens. Here is an example, in which perfluorooctanoic acid (PFOA) was air-cell injected, and serum PFOA concentrations were then determined with Ultra-performance liquid chromatography-mass spectrometry. The serum concentrations corresponded with the injected doses, indicating the ef...

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Using Chicken Embryo as a Powerful Tool in Assessment of Developmental Cardiotoxicities

Chicken embryos, as a classical developmental model, are used in our lab to assess developmental cardiotoxicities following exposure to various environmental contaminants. Exposure methods and morphological/functional assessment methods established are described in this manuscript.

Chicken embryos are a classical model in developmental studies. During the development of chicken embryos, the time window of heart development is ...

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Developmental Biology

4.2K Views.  Qingdao University. Chicken embryos, as a classical developmental model, are used in our lab to assess developmental cardiotoxicities following exposure to various environmental contaminants. Exposure methods and morphological/functional assessment methods established are described in this manuscript. 

Video: Using Chicken Embryo as a Powerful Tool in Assessment of Developmental Cardiotoxicities

Grafting of Beads into Developing Chicken Embryo Limbs to Identify Signal Transduction Pathways Affecting Gene Expression

Using chicken embryos it is possible to test directly the effects of either growth factors or specific inhibitors of signaling pathways on gene expression and activation of signal transduction pathways. This technique allows the delivery of signaling molecules at precisely defined developmental stages for specific times. After this embryos can be harvested and gene expression examined, for example by in situ hybridization, or activation of signal transduction pathways observed with immunostaining.
In this video heparin beads soaked in FGF18 or AG 1-X2 beads soaked in U0126, a MEK inhibitor, are grafted into the limb bud in ovo. This shows that FGF18 induces expression of MyoD and ERK phosphorylation and both endogenous and FGF18 induced MyoD expression is inhibited by U0126. Beads soaked in a retinoic acid antagonist can potentiate premature MyoD induction by FGF18.
This approach can be used with a wide range of different growth factors and inhibitors and is easily adapted to other tissues in the developing embryo.

By grafting beads soaked in growth factors or specific inhibitors of signaling pathways into developing embryos it is possible to directly test their effects in vivo. In this protocol beads are grafted into the limb bud to determine the effects of these molecules on gene expression and signal transduction.

Using chicken embryos it is possible to test directly the effects of either growth factors or specific inhibitors of signaling pathways on gene expression ...

In Ovo Electroporations of HH Stage 10 Chicken Embryos

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of molecular biological techniques, the chick has become a useful system in which to study gene regulation and function.  By electroporating DNA or RNA constructs into the developing chicken embryo, genes can be expressed or knocked down in order to analyze in vivo gene function.  Similarly, reporter constructs can be used for fate mapping or to examine putative gene regulatory elements.  Compared to similar experiments in mouse, chick electroporation has the advantages of being quick, easy and inexpensive.  This video demonstrates first how to make a window in the eggshell to manipulate the embryo.  Next, the embryo is visualized with a dilute solution of India ink injected below the embryo.  A glass needle and pipette are used to inject DNA and Fast Green dye into the developing neural tube, then platinum electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator.  Finally, the egg is sealed with tape and placed back into an incubator for further development.  Additionally, the video shows proper egg storage and handling and discusses possible causes of embryo loss following electroporation.

Chick in ovo electroporation is a technique which allows genetic manipulation of the avian embryo. Common applications of this technique include functional analysis of genes and putative enhancer elements. This video demonstrates neural tube electroporation in HH 10 chick embryos. Injection technique and proper egg handling are discussed.

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of ...

Biology

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 ...

Using the Chicken Chorioallantoic Membrane In Vivo Model to Study Gynecological and Urological Cancers

Mouse models are the benchmark tests for in vivo cancer studies. However, cost, time, and ethical considerations have led to calls for alternative in vivo cancer models. The chicken chorioallantoic membrane (CAM) model provides an inexpensive, rapid alternative that permits direct visualization of tumor development and is suitable for in vivo imaging. As such, we sought to develop an optimized protocol for engrafting gynecological and urological tumors into this model, which we present here. Approximately 7 days postfertilization, the air cell is moved to the vascularized side of the egg, where an opening is created in the shell. Tumors from murine and human cell lines and primary tissues can then be engrafted. These are typically seeded in a mixture of extracellular matrix and medium to avoid cellular dispersal and provide nutrient support until the cells recruit a vascular supply. Tumors may then grow for up to an additional 14 days prior to the eggs hatching. By implanting cells stably transduced with firefly luciferase, bioluminescence imaging can be used for the sensitive detection of tumor growth on the membrane and cancer cell spread throughout the embryo. This model can potentially be used to study tumorigenicity, invasion, metastasis, and therapeutic effectiveness. The chicken CAM model requires significantly less time and financial resources compared to traditional murine models. Because the eggs are immunocompromised and immune tolerant, tissues from any organism can potentially be implanted without costly transgenic animals (e.g., mice) required for implantation of human tissues. However, many of the advantages of this model could potentially also be limitations, including the short tumor generation time and immunocompromised/immune tolerant status. Additionally, although all tumor types presented here engraft in the chicken chorioallantoic membrane model, they do so with varying degrees of tumor growth.

We present the chicken chorioallantoic membrane model as an alternative, transplantable, in vivo model for the engraftment of gynecological and urological cancer cell lines and patient-derived tumors.

Mouse models are the benchmark tests for in vivo cancer studies. However, cost, time, and ethical considerations have led to calls for alternative in vivo ...

Cancer Research

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

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

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a general challenge in developmental biology research. In the embryonic heart, this can be particularly difficult as regional differences in the expression of transcriptional regulators, paracrine/juxtacrine signaling cues, and hemodynamic force are all known to influence cardiomyocyte maturation. A simplified method to alter a developing cardiomyocyte's molecular and biomechanical microenvironment would, therefore, serve as a powerful technique to examine how local conditions influence cell fate and function. To address this, we have optimized a method to physically transplant juvenile cardiomyocytes into ectopic locations in the heart or the surrounding embryonic tissue. This allows us to examine how microenvironmental conditions influence cardiomyocyte fate transitions at single cell resolution within the intact embryo. Here, we describe a protocol in which embryonic myocytes can be isolated from a variety of cardiac sub-domains, dissociated, fluorescently labeled, and microinjected into host embryos with high precision. Cells can then be directly analyzed in situ using a variety of imaging and histological techniques. This protocol is a powerful alternative to traditional grafting experiments that can be prohibitively difficult in a moving tissue such as the heart. The general outline of this method can also be adapted to a variety of donor tissues and host environments, and its ease of use, low cost, and speed make it a potentially useful application for a variety of developmental studies.

In this method, embryonic cardiac tissues are surgically microdissected, dissociated, fluorescently labeled, and implanted into host embryonic tissues. This provides a platform for studying the individual or tissue level developmental organization under ectopic hemodynamic conditions, and/or altered paracrine/juxtacrine environments.

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a ...

Xenotransplantation of Human Stem Cells into the Chicken Embryo

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the behavior of cells in a standardized in vivo environment. The main advantages of the chicken embryo include low cost, high accessibility, ease of surgical manipulation and lack of a fully developed immune system. Xenotransplantation into chicken embryos can provide valuable information about cell proliferation, differentiation and behavior, the responses of cells to signals in defined embryonic tissue niches, and tumorigenic potential. Transplanting cells into chicken embryos can also be a step towards transplantation experiments in other animal models. Recently the chicken embryo has been used to evaluate the neurogenic potential of human stem and progenitor cells following implantation into neural anlage1-6. In this video we document the entire procedure for transplanting human stem cells into the developing central nervous system of the chicken embryo. The procedure starts with incubation of fertilized eggs until embryos of the desired age have developed. The eggshell is then opened, and the embryo contrasted by injecting dye between the embryo and the yolk. Small lesions are made in the neural tube using microsurgery, creating a regenerative site for cell deposition that promotes subsequent integration into the host tissue. We demonstrate injections of human stem cells into such lesions made in the part of the neural tube that forms the hindbrain and the spinal cord, and into the lumen of the part of the neural tube that forms the brain. Systemic injections into extraembryonic veins and arteries are also demonstrated as an alternative way to deliver cells to vascularized tissues including the central nervous system. Finally we show how to remove the embryo from the egg after several days of further development and how to dissect the spinal cord free for subsequent physiological, histological or biochemical analyses.

In this paper we present a method for transplanting human stem cells into various regions of the central nervous system of the chicken embryo. This provides an in vivo model for assessing the proliferation and differentiation of various types of human stem cells in embryonic tissue environments.

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the ...

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

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

In Ovo Intravascular Injection in Chicken Embryos

As a classical model system of embryo biology, the chicken embryo has been used to investigate embryonic development and differentiation. Delivering exogenous materials into chicken embryos has a great advantage for studying gene function, transgenic breeding, and chimera preparation during embryonic development. Here we show the method of in ovo intravascular injection whereby exogenous materials such as plasmid vectors or modified primordial germ cells (PGCs) can be transferred into donor chicken embryos at early developmental stages. The results show that the intravascular injection through the dorsal aorta and head allows injected materials to diffuse into the whole embryo through the blood circulatory system. In the presented protocol, the efficacy of exogenous plasmid and lentiviral vector introduction, and the colonization of injected exogenous PGCs in the recipient gonad, were determined by observing fluorescence in the embryos. This article describes detailed procedures of this method, thereby providing an excellent approach to studying gene function, embryo and developmental biology, and gonad-chimeric chicken production. In conclusion, this article will allow researchers to perform in ovo intravascular injection of exogenous materials into chicken embryos with great success and reproducibility.

In Ovo Intravascular Injection in Chicken Embryos

The overall goal of this paper is to describe how to perform in ovo intracellular injection of exogenous materials into chicken embryos. This approach is very useful to study the developmental biology of chicken embryos.

As a classical model system of embryo biology, the chicken embryo has been used to investigate embryonic development and differentiation. Delivering ...

The Preparation of Chicken Ex Ovo Embryos and Chorioallantoic Membrane Vessels as In Vivo Model for Contrast-Enhanced Ultrasound Imaging and Microbubble-Mediated Drug Delivery Studies

The chicken embryo and the blood-vessel rich chorioallantoic membrane (CAM) is a valuable in vivo model to investigate biomedical processes, new ultrasound pulsing schemes, or novel transducers for contrast-enhanced ultrasound imaging and microbubble-mediated drug delivery. The reasons for this are the accessibility of the embryo and vessel network of the CAM as well as the low costs of the model. An important step to get access to the embryo and CAM vessels is to take the egg content out of the eggshell. In this protocol, three methods for taking the content out of the eggshell between day 5 and&#160;8 of incubation are described thus allowing the embryos to develop inside the eggshell up to these days. The described methods only require simple tools and equipment and yield a higher survival success rate of 90% for 5-day, 75% for 6-day, 50% for 7-day, and 60% for 8-day old incubated eggs in comparison to ex ovo cultured embryos (~50%). The protocol also describes how to inject cavitation nuclei, such as microbubbles, into the CAM vascular system, how to separate the membrane containing the embryo and CAM from the rest of the egg content for optically transparent studies, and how to use the chicken embryo and CAM in a variety of short-term ultrasound experiments. The in vivo&#160;chicken embryo and CAM model is extremely relevant to investigate novel imaging protocols, ultrasound contrast agents, and ultrasound pulsing schemes for contrast-enhanced ultrasound imaging, and to unravel the mechanisms of ultrasound-mediated drug delivery.

This protocol describes three methods on how to obtain and use 5 to 8-day old chicken embryos and their chorioallantoic membrane (CAM) as an in vivo&#160;model to study contrast-enhanced ultrasound imaging and microbubble-mediated drug delivery.

Using Chicken Embryo as a Powerful Tool in Assessment of Developmental Cardiotoxicities

Summary

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Grafting of Beads into Developing Chicken Embryo Limbs to Identify Signal Transduction Pathways Affecting Gene Expression

In Ovo Electroporations of HH Stage 10 Chicken Embryos

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

Using the Chicken Chorioallantoic Membrane In Vivo Model to Study Gynecological and Urological Cancers

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

Xenotransplantation of Human Stem Cells into the Chicken Embryo

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In Ovo Intravascular Injection in Chicken Embryos

The Preparation of Chicken Ex Ovo Embryos and Chorioallantoic Membrane Vessels as In Vivo Model for Contrast-Enhanced Ultrasound Imaging and Microbubble-Mediated Drug Delivery Studies