Funding for the development of current protocol is particularly supported by &#8220;Aim for the Top University Plan&#8221; of the National Taiwan University, Taiwan (grant ID-104R350144), as well as the Ministry of Science and Technology of Taiwan (grant ID: MOST 104-2313-B-002-039-MY3). We especially thank the Center for Biotechnology of National Taiwan University for providing an animal room and laboratory space for the current study.

NOTE: This procedure is a modification of a chicken model culture protocol developed by Bauer et al. 2013 and Nakazawa et al., 2011. 2,9
1. Prepare Healthy Embryonic Day 5 Embryos from Japanese Quail
<ol>
	<li>Place 1 male and 3 female sexually mature Japanese quail together in the same cage. Supply feed and water ad libitum. Adjust the light in the animal room to 14 hr of light and 10 hr of darkness.</li>
	<li>Collect fertilized eggs in plastic bags every day in the afternoon and keep them in a 16 &#176;C refrigerator to retard the growth rate of embryos.</li>
	<li>After collecting a sufficient number of eggs within a two-week period, incubate the fertilized eggs in a 37 &#176;C incubator with good ventilation for five days.</li>
</ol>
2. Prepare Basal Medium and Wash Solutions
<ol>
	<li>Use DMEM/ F-12 medium (adjusted to pH 7.2) as the growth medium, and supplement it with 10% new born calf serum (NBCS) and 1% Pen-Strep Ampho. Solution (PSA; Penicillin, 105 units/L; Streptomycin, 100 mg/L; Amphotericin B, 0.25 mg/L).</li>
	<li>Prepare phosphate buffered saline (PBS) solution at a 10x concentration for storage; use 1x PBS solution (containing NaCl, 137.93 mM; KCl, 2.667 mM; KH2PO4, 1.471 mM; Na2HPO4-7H2O, 8.06 mM; adjust to pH 7.2) to wash and moisten the yolk sac membrane while separating the endoderm from egg yolk and albumin.</li>
	<li>Use DMEM medium (adjusted to pH 7.2) to re-suspend endoderm pellets.</li>
</ol>
3. Collection of Primary EECs from Japanese Quail YSMs
<ol>
	<li>Incubate the collected eggs for 5 days (step 1.3). Examine the eggs by an egg candler to confirm normal embryonic development. 
	NOTE: On embryonic day 5, the boundary of YSM mesoderm is very clear and the 3 embryonic layers are less tightly-linked together, so it is easy to obtain endoderm. Only use the eggs showing normal capillary circulation development for YSM collections.</li>
	<li>Clean the eggshell carefully using fresh water and 75% ethanol. Open the eggshell from the air cell position by a pair of scissors, gently peel out and collect the whole YSM by forceps and place in a 10 cm culture dish filled with 37&#176;C 1x PBS.</li>
	<li>Using a pair of scissors remove the embryo, yolk and egg-white, and gently wash the YSM with 1x PBS three times and place YSM in PBS solution in a 10 cm culture dish.</li>
	<li>Use forceps and a pair of scissors to remove the ectoderm from YSM. Use 2 forceps, one to firmly hold the endodermal cell layer, and the other to hold the capillary mesoderm.</li>
	<li>Under a dissecting microscope, pull apart and separate the endoderm from the edge of the mesoderm in the direction towards the embryo. Collect the light-yellow endoderm in 50 ml centrifuge tubes using a dropper and place in 15 ml of growth medium in a 37 &#176;C water bath until all tissue collections are completed.</li>
</ol>
4. Digestion of the Endoderm Slices by Collagenase Digestion
<ol>
	<li>Freshly dissolve 6.5 units of type IV collagenase in 10 ml of DMEM.</li>
	<li>Centrifuge the 50 ml tubes (3.4) at 130 x g for 3 min at RT. Aspirate the medium and place all the endoderm in a 6 cm culture dish using a pipette. Add 1 ml collagenase solution to this culture dish.</li>
	<li>Slice the endoderm collected from 2 quail embryos with a pair of curved scissors until the size of each slice is approximately 2 to 3 mm. Use a dropper, collect all the slices into a 50 ml centrifuge tube along with 9 ml of fresh collagenase solution.</li>
	<li>Digest the tissue with collagenase for 30 min in a 37 &#176;C shaking water bath at 175 rpm. This partial digestion with collagenase is used to improve the explant cell proliferation.</li>
	<li>Centrifuge at 130 x g for 3 min at RT and gently remove the supernatant fraction using a pipette. Wash the pellet by suspension in 20 ml DMEM and remove the supernatant fraction after 130 x g centrifugation for 3 min.</li>
	<li>Re-suspend the pellet with 12 ml of growth medium (DMEM/F12 with 10% NBCS and 1% PSA) and seed gently and equally into a 24-well plate (each well with 500 &#181;l cell solution).</li>
	<li>Incubate tissue explants at 37 &#176;C in 5% CO2 in air for 2 days to allow cells to proliferate out of the tissues.</li>
	<li>Incubate cells for another 2 days and characterize for cell viability by a cell viability assay protocol.15 
	NOTE: Living cells maintain a reducing environment within the cytosol. Resazurin, the active ingredient, is a non-toxic, cell permeable compound that is blue in color and virtually non-fluorescent. When added to cell cultures, resazurin is reduced to resorufin in the cytosol by mitochondrial enzymes. Resorufin is red in color and highly fluorescent. Viable cells continuously convert resazurin to resorufin, increasing the overall fluorescence and color of the culture medium surrounding cells.</li>
	<li>Perform endoderm epithelial cell total RNA isolation, reverse transcription and quantitative real-time PCR of marker gene mRNA expression as described. 16,17
	<ol>
		<li>Use reverse transcriptase PCR to detect SOAT1 mRNA (sense: 5&#39;-GAAGGGGCCTATCTGGAACG-3&#39;; antisense: 5&#39;-ATCTGCACGTGACATGACCA-3&#39;; PCR product = 168 bp) as a specific marker for the EEC cells. Use &#946;-Actin mRNA (sense: 5&#39;- TGGTGAAGCTGTAGCCTCTC-3&#39;; antisense: 5&#39;- GTGATGGACTCTGGTGATGG-3&#39;; PCR product = 151 bp) as a housekeeping gene and internal control.</li>
		<li>Use gel electrophoresis to detect the product size of RT-qPCR. Prepare 1% agarose gel, and mix 10 &#956;l qPCR product with 2 &#956;l loading dye. Load 10 &#956;l mixture into each well in the 1% agarose gel, and run the gel by 100V for 30 min.</li>
	</ol>
	</li>
</ol>

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There is no competing financial interests or other conflicts of interest. We did not get any funding from any private company.

Because the previous culture system has only limited success, a better culture system is needed. The Japanese quail YSM endoderm requires treatment with proteolytic enzymes such as collagenase to loosen the cell-cell junctions to achieve better growth performance in the ex-vivo explants. Our data show that cell numbers from partial digestion treatment were greater than from the undigested tissue culture after seeding for 2 days (Figure 1). Therefore, the partial proteolytic digestion is a critical step to improve cell yield from the YSM.
The critical modification in the current culture protocol is the partial proteolytic digestion. This treatment greatly enhanced the cell yield and the successful rate of obtaining live EECs. The digestion time is very critical because over-digestion will separate cells from the tissue and cells will not attach to the plate, whereas under-digestion resulted in less cell yield from the tissue explants. We found that 30 to 40 min digestion using the conditions described above is optimal.
The current protocol requires high technical training to obtain EECs. The time point of collecting EECs is restricted, for example, we only collect EECs at embryonic day 5. The reason is that as avian embryos grow, the connections between layers in the yolk sac membrane are more complex and it decreases the successful rate of collecting EECs. The protocol can be used to obtain EECs of early stage embryos but not late stage embryos. It limits the possibility of direct research on the late stage EECs.
The expression of SOAT1 and lipoprotein lipase, and lipid accumulation are essential characteristics of the YSM 4,5,11, especially the EECs. In the current study, SOAT1, LPL, and perilipin 2 (PLN2) mRNA were expressed highly in the EECs, suggesting the culture system can generate cells with the correct physiological characteristics. The current technique produced a large quantity of primary EECs. When we used the previous described procedure9, the success rate of EEC culture was approximately 60% in cell culture plates of 24-well, whereas the current protocol generated EECs at nearly 100% success rate.
Yolk sac membrane is the main tissue to absorb proteins, lipids, and cholesterol from yolk, thus functioning to transfer nutrients from yolk to embryos during development. The main enzyme for cholesterol ester formation is SOAT1.11 Because SOAT1 increases dramatically in the late stages of avian embryonic development when massive cholesterol esterification occurs4, we used SOAT1 mRNA expression as the marker gene for evaluating the characteristics of cultured EECs. We demonstrated that this primary EEC culture system produced expected physiological characteristics. Therefore, the EEC culture system can be a valuable tool to study embryonic lipid transportation and to clarify the role of genes involved in mediating nutrient utilization in YSM during avian embryonic development.

The major nutritional resource of the avian embryo is yolk, composed of 33% lipids, 17% protein, and 1% ash.1 During embryonic development, the yolk sac membrane (YSM) grows from within the embryonic abdominal cavity and gradually covers the yolk surface. Beginning at embryonic day 2, gene expression associated with lipid metabolism and angiogenesis is gradually increased in YSM, and the YSM slowly develops villus-like projections.8,9 These projections increase absorption of yolk nutrients to support embryonic development. The YSM is an extraembryonic tissue that contains three germ layers, endoderm, mesoderm and ectoderm.14 The yolk sac ectoderm faces the albumen and links with the vitelline membrane to gently cover the yolk sac. The endodermal epithelial cells are faced directly toward the egg yolk and serve as nutrient utilization portals.6 As the YSM expands, endodermal epithelial cell (EECs) can be divided by shape and functionality into two groups, area vitelline and area vasculosa.7
Area vitelline is composed of endodermal cells and is distant from the embryo; area vasculosa is composed of mesodermal cells and covers differentiated EECs with blood vessels and connective tissues. By embryonic day 5, the yolk is totally covered by ectoderm and endoderm of YSM and the vascular area has grown rapidly. The YSM absorbs, recomposes and releases lipids (as yolk-derived very low density lipoprotein) and proteins into the embryonic circulatory system.9, 2 Therefore, we established a primary Japanese quail embryonic endodermal epithelial cell culture system, to study the mechanisms of lipid utilization in YSM during avian embryonic development.
Lipids such as triacylglycerol, lecithin, phospholipid and cholesterol ester (CE) are the primary energy sources for avian embryos. At the early stages of development, yolk lipids are composed of only 1.3% CE and it rises to 10-15% at mid-term of avian embryonic development 3, 11. Cholesterol ester is synthesized from cholesterol by sterol O-acyltransferase 1 (SOAT1) in avian embryo YSM.4
The storage form of cholesterol is CE, CE is carried in lipoproteins, and lipoproteins are transported by circulation to tissues.13 A week before hatch there is rapid growth of avian embryos. Approximately 68% of the remaining lipid contents in yolk are absorbed during this stage.10 The mechanism by which yolk lipids are utilized can be clarified by an EEC research model. A chicken EEC culture protocol was established to achieve this research goal.2, 9 However, due to the low success rate of tissue explants, an improved EEC cell culture procedure is needed to study the function of certain enzymes and proteins in mediating nutrient utilization by avian embryos during development.

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagle Medium</td><td>Gibco by Life technologies</td><td>12800-017 10 X 1 L</td><td>For wash the EECs pellets</td></tr><tr><td>D-MEM/F-12</td><td>Gibco by Life technologies</td><td>12400-024 10 X 1 L</td><td>As the basal medium in culturing EECs</td></tr><tr><td>NBCS</td><td>Gibco by Life technologies</td><td>16010-159</td><td>As the supplyment serum in culturing EECs</td></tr><tr><td>Pen-Strep Ampho. Solution</td><td>BI (Biological Industries)</td><td>03-033-1B 100 ml</td><td>For attenuating the possible infection&amp;nbsp;</td></tr><tr><td>Collagenase Type IV</td><td>Gibco by Life technologies</td><td>17104-019 1 g</td><td>&amp;nbsp;Collagenase is a protease with specificity for the bond between a neutral amino acid (X) and glycine in the sequence Pro-XGly-Pro. As the protease for dissociation of cells from primary tissue.</td></tr><tr><td>24-well plate</td><td>FALCON&lt;sup&gt;&amp;reg;&lt;/sup&gt;</td><td>REF-353047</td><td>For EECs to attach and extension</td></tr><tr><td>50 ml PP centrifuge tubes</td><td>Corning&lt;sup&gt;&amp;reg;&lt;/sup&gt; CentriStar&lt;sup&gt;TM&lt;/sup&gt;</td><td>430829</td><td>For transportion of membranes and enzyme digestion</td></tr><tr><td>50 ml Conical bottomed Tube with Cap</td><td>PRO TECH</td><td>CT-50-PL-TW</td><td>For transportion of membranes and enzyme digestion</td></tr><tr><td>Reciprocal shaking bath</td><td>DEAGLE</td><td>SB302</td><td>For better enzymatic digestion on membranes</td></tr></tbody></table>

primary endodermal epithelial cell culture from the yolk sac membrane of japanese quail embryos

We established an endodermal epithelial cell culture model (EEC) for studying the function of certain enzymes and proteins in mediating nutrient utilization by avian embryos during development. Fertilized Japanese quail eggs were incubated at 37 &#176;C for 5 days and then yolk sac membranes (YSM) were collected to establish the EEC culture system. We isolated the embryonic endoderm layer from YSM, and sliced the membrane into 2 - 3 mm pieces and partially digested with collagenase before seeding in 24-well culture plates. The EECs proliferate out of the tissue and are ready for cell culture studies. We found that the EECs had typical characteristics of YSM in vivo, for example, accumulation of lipid droplets, expression of sterol O-acyltransferase and lipoprotein lipase. The partial digestion treatment significantly increased the successful rate of EEC culture. Utilizing the EECs, we demonstrated that the expression of SOAT1 was regulated by the cAMP dependent protein kinase A related pathway. This primary Japanese quail EEC culture system is a useful tool to study embryonic lipid transportation and to clarify the role of genes involved in mediating nutrient utilization in YSM during avian embryonic development.

In order to achieve the goal of establishing a consistent and useful cell model, we need to extend and stabilize the proliferation rate and performance of avian EECs. We compared direct incubation of the endoderm with no enzyme digestion with endoderm partially digested with proteolytic enzymes, such as collagenase or collagenase plus 0.6 U of dispase. Dispase is an amino-endopeptidase that hydrolyzes the N-terminal peptide bonds of non-polar amino acid residues. Whereas protease digestion treatments increased cell proliferation compared with no digestion (Figure 1), cell growth was not markedly different between the two enzyme treatments after five days incubation (Figure 2). However, the collagenase treatment resulted in a better growth curve and showed that EECs proliferated steadily for 6-9 days (Figure 2). Thus, partial collagenase digestion improved growth of EECs from the ex-vivo explants and is recommended to obtain sufficient and functional EEC.
After 4 days incubation, EECs had an adipocyte-like appearance detected after formalin fixation and Oil-Red O staining (Figure 3), suggesting correct physiological characteristics of the cultured cells.
We hypothesized that cyclic adenosine monophosphate (cAMP) is a transcription regulatory agent, the cAMP phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX; to decrease degradation of cAMP) and the adenyl cyclase activator, forskolin (to increase synthesis of cAMP) both stimulated SOAT1 mRNA expression after 24 hr treatment (Figure 4). These treatments also increased the expression of SREBP2, a cholesterol synthesis regulatory transcription factor, and forskolin enhanced the expression of perilipin-2 (PLIN2), the surface protein marker of lipid droplets (Figure 5). The EEC mRNA profiles for lipogenic genes quantified by real-time PCR are similar to adipocytes (Figure 5).
<img alt="Figure 1" src="/files/ftp_upload/53624/53624fig1.jpg" /> 
Figure 1. Effect of Partial Collagenase Digestion on Endodermal Epithelium Cells. Tissue explants were incubated for 2 days to allow cells to proliferate out of the tissue. We observed and calculated the numbers of the successfully grown EECs. Numbers were calculated for cells with no digestion and cells partially digested with collagenase; both were grown in 24-well plates. The Y-axis is well number of successfully proliferated EECs in cell culture plate of 24-well. Data were presented as Mean &#177; SEM. Analysis were determined by paired t test. P&#60; 0.0001. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53624/53624fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53624/53624fig2.jpg" /> 
Figure 2. The Cell Growth Performance of EECs After Partial Enzyme Digestion. The proliferation rate was detected by cell viability assay. The assay contained resazurin, a blue compound that has weak fluorescence and resazurin is reduced to resorufin upon entering cells. Viable cells continuously convert resazurin to resorufin, increasing the overall fluorescence and color of the media surrounding cells. Viable cells were detected by absorbance at 570 nm with normalization at 600 nm. Data were presented as Mean &#177; SEM. Analysis were determined by two-way ANOVA with Bonferroni post-test (collagenase group n=10; collagenase + dispase group n=11). * P &#60; 0.05, ** P&#60;0.01, *** P&#60;0.001. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53624/53624fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53624/53624fig3.jpg" /> 
Figure 3. The Morphology of Cultured EECs. After 4 days incubation, EECs had an adipocyte-like appearance detected after formalin fixation and Oil-Red O staining. From left to right: EEC&#39;s in bright field with 100X magnification, in bright field with 200X magnification and in 200X magnification after staining with Oil-red O and hematoxylin. The dark spot on the left of the far left figure is the partially digested endoderm (membrane). Scale bar represents 200 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53624/53624fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53624/53624fig4.jpg" /> 
Figure 4. The SOAT1 Transcription of EECs Treated with cAMP Activators. We treated EECs with increasing concentrations of IBMX or forskolin at day 5 for 24 hr. We extracted mRNAs from treated cells and converted the RNA into cDNA by reverse transcription PCR. We analyzed transcription levels by real-time quantitative PCR. &#946;-actin was used as the housekeeping gene. Data were presented as Mean &#177; SEM. Analyses were by one-way ANOVA with Tukey&#39;s post-test (n = 6) (*P&#8804; 0.05, ** P&#8804; 0.01). IBMX is a competitive nonselective phosphodiesterase inhibitor which raises intracellular cAMP, activates PKA, inhibits TNF-alpha and leukotriene synthesis, reduces inflammation and innate immunity and is a nonselective adenosine receptor antagonist. Forskolin is a ubiquitous activator of eukaryotic adenylyl cyclase, commonly used to raise levels of cAMP. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53624/53624fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/53624/53624fig5.jpg" /> 
Figure 5. The mRNA Expressions of EEC&#39;s Grown for Five Days, Then Treated with cAMP Activators for 24 hr. The cDNA&#39;s in Figure 4 were used to analyze gene expression levels by real-time quantitative PCR. &#946;-actin was used as the housekeeping gene and served as the internal control. Data were presented as Mean &#177; SEM. Analyses were by one-way ANOVA with Tukey&#39;s post-test (n&#8804; 6) to compare treatments to the control. SREBP2, sterol regulatory element-binding protein 2; LPL, lipoprotein lipase; PLIN2, perilipin-2. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53624/53624fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos at JoVE.com

Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos

To study the mechanism of lipid utilization in yolk sac membranes during the late stages of avian embryonic development, we established a primary Japanese quail embryonic endodermal epithelial cell culture system.

We established an endodermal epithelial cell culture model (EEC) for studying the function of certain enzymes and proteins in mediating nutrient ...

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Nanyang Technological University

Research

JoVE Journal

Developmental Biology

8.8K Views.  National Taiwan University. To study the mechanism of lipid utilization in yolk sac membranes during the late stages of avian embryonic development, we established a primary Japanese quail embryonic endodermal epithelial cell culture system.

Video: Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos

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 ...

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Biology

Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. Chimeric avian embryos, in which quail donor tissue is transplanted into a chick embryo in ovo, combine the power of indelible genetic labeling of cell populations with the ease of manipulation presented by the avian embryo.
Quail-chick chimeras are a classical tool for tracing migratory neural crest cells (NCCs)1-3. NCCs are a transient migratory population of cells in the embryo, which originate in the dorsal region of the developing neural tube4. They undergo an epithelial to mesenchymal transition and subsequently migrate to other regions of the embryo, where they differentiate into various cell types including cartilage5-13, melanocytes11,14-20, neurons and glia21-32. NCCs are multipotent, and their ultimate fate is influenced by 1) the region of the neural tube in which they originate along the rostro-caudal axis of the embryo11,33-37, 2) signals from neighboring cells as they migrate38-44, and 3) the microenvironment of their ultimate destination within the embryo45,46. Tracing these cells from their point of origin at the neural tube, to their final position and fate within the embryo, provides important insight into the developmental processes that regulate patterning and organogenesis.
Transplantation of complementary regions of donor neural tube (homotopic grafting) or different regions of donor neural tube (heterotopic grafting) can reveal differences in pre-specification of NCCs along the rostro-caudal axis2,47. This technique can be further adapted to transplant a unilateral compartment of the neural tube, such that one side is derived from donor tissue, and the contralateral side remains unperturbed in the host embryo, yielding an internal control within the same sample2,47. It can also be adapted for transplantation of brain segments in later embryos, after HH10, when the anterior neural tube has closed47.
Here we report techniques for generating quail-chick chimeras via neural tube transplantation, which allow for tracing of migratory NCCs derived from a discrete segment of the neural tube. Species-specific labeling of the donor-derived cells with the quail-specific QCPN antibody48-56 allows the researcher to distinguish donor and host cells at the experimental end point. This technique is straightforward, inexpensive, and has many applications, including fate-mapping, cell lineage tracing, and identifying pre-patterning events along the rostro-caudal axis45. Because of the ease of access to the avian embryo, the quail-chick graft technique may be combined with other manipulations, including but not limited to lens ablation40, injection of inhibitory molecules57,58, or genetic manipulation via electroporation of expression plasmids59-61, to identify the response of particular migratory streams of NCCs to perturbations in the embryo's developmental program. Furthermore, this grafting technique may also be used to generate other interspecific chimeric embryos such as quail-duck chimeras to study NCC contribution to craniofacial morphogenesis, or mouse-chick chimeras to combine the power of mouse genetics with the ease of manipulation of the avian embryo.62

An approach for analyzing migration and eventual fate of avian neural crest cells in quail-chick chimeric embryos is described. This method is a simple and straightforward technique for tracing neural crest cells during migration and differentiation that are otherwise difficult to distinguish within an unmanipulated chick embryo.

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. ...

Neuroscience

Two-step Approach to Explore Early- and Late-stages of Organ Formation in the Avian Model: The Thymus and Parathyroid Glands Organogenesis Paradigm

The avian embryo, as an experimental model, has been of utmost importance for seminal discoveries in developmental biology. Among several approaches, the formation of quail-chicken chimeras and the use of the chorioallantoic membrane (CAM) to sustain the development of ectopic tissues date back to the last century. Nowadays, the combination of these classical techniques with recent in vitro methodologies offers novel prospects to further explore organ formation.
Here we describe a two-step approach to study early- and late-stages of organogenesis. Briefly, the embryonic region containing the presumptive territory of the organ is isolated from quail embryos and grown in vitro in an organotypic system (up to 48 h). Cultured tissues are subsequently grafted onto the CAM of a chicken embryo. After 10 days of in ovo development, fully formed organs are obtained from grafted tissues. This method also allows the modulation of signaling pathways by the regular administration of pharmacological agents and tissue genetic manipulation throughout in vitro and in ovo developmental steps. Additionally, developing tissues can be collected at any time-window to analyze their gene-expression profile (using quantitative PCR (qPCR), microarrays, etc.) and morphology (assessed with conventional histology and immunochemistry).
The described experimental procedure can be used as a tool to follow organ formation outside the avian embryo, from the early stages of organogenesis to fully formed and functional organs.

This article provides an updated approach to the classical quail-chicken chimera system to study organ formation, by combining novel in vitro and in ovo experimental procedures.

The avian embryo, as an experimental model, has been of utmost importance for seminal discoveries in developmental biology. Among several approaches, the ...

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 ...

Quail Chorioallantoic Membrane - A Tool for Photodynamic Diagnosis and Therapy

The chorioallantoic membrane (CAM) of an avian embryo is a thin, extraembryonic membrane that functions as a primary respiratory organ. Its properties make it an excellent in vivo experimental model to study angiogenesis, tumor growth, drug delivery systems, or photodynamic diagnosis (PDD) and photodynamic therapy (PDT). At the same time, this model addresses the requirement for the replacement of experimental animals with a suitable alternative. Ex ovo cultivated embryo allows easy substance application, access, monitoring, and documentation. The most frequently used is chick CAM; however, this article describes the advantages of the Japanese quail CAM as a low-cost and high-throughput model. Another advantage is the shorter embryonic development, which allows higher experimental turnover. The suitability of quail CAM for PDD and PDT of cancer and microbial infections is explored here. As an example, the use of the photosensitizer hypericin in combination with lipoproteins or nanoparticles as a delivery system is described. The damage score from images in white light and changes in fluorescence intensity of the CAM tissue under violet light (405 nm) was determined, together with analysis of histological sections. The quail CAM clearly showed the effect of PDT on the vasculature and tissue. Moreover, changes like capillary hemorrhage, thrombosis, lysis of small vessels, and bleeding of larger vessels could be observed. Japanese quail CAM is a promising in vivo model for photodynamic diagnosis and therapy research, with applications in studies of tumor angiogenesis, as well as antivascular and antimicrobial therapy.

The chorioallantoic membrane (CAM) of the avian embryo is a very useful and applicable tool for various areas of research. A special ex ovo model of Japanese quail CAM is suitable for photodynamic treatment investigation.

The chorioallantoic membrane (CAM) of an avian embryo is a thin, extraembryonic membrane that functions as a primary respiratory organ. Its properties ...

Cancer Research

Isolation of Embryonic Tissues and Formation of Quail-Chicken Chimeric Organs Using The Thymus Example

The capacity to isolate embryonic tissues was an essential step for establishing the quail-chicken chimera system, which in turn has provided undisputed contributions to unveiling key processes in developmental biology.
Herein is described an optimized method to isolate embryonic tissues from quail and chickens by microsurgery and enzymatic digestion while preserving its biological properties. After isolation, tissues from both species are associated in an in vitro organotypic assay for 48 h. Quail and chicken tissues can be discriminated by distinct nuclear features and molecular markers allowing the study of the cellular cross-talk between heterospecific association of tissues. This approach is, therefore, a useful tool for studying complex tissue interactions in developmental processes with highly dynamic spatial modifications, such as those occurring during pharyngeal morphogenesis and the formation of the foregut endoderm-derived organs. This experimental approach was first developed to study the epithelial-mesenchymal interactions during early-stages of thymus formation. In this, the endoderm-derived prospective thymic rudiment and mesoderm-derived mesenchyme, were isolated from quail and chicken embryos, respectively.
The capacity of the associated tissues to generate organs can be further tested by grafting them onto the chorioallantoic membrane (CAM) of a chicken embryo. The CAM provides nutrients and allows gas exchanges to the explanted tissues. After 10 days of in ovo development, the chimeric organs can be analyzed in the harvested explants by conventional morphological methods. This procedure also allows studying tissue-specific contributions during organ formation, from its initial development (in vitro development) to the final stages of organogenesis (in ovo development).
Finally, the improved isolation method also provides three-dimensionally (3D) preserved embryonic tissues, that can also be used for high-resolution topographical analysis of tissue-specific gene-expression patterns.

This article provides a method to isolate pure embryonic tissues from quail and chicken embryos that can be combined to form ex vivo chimeric organs.

The capacity to isolate embryonic tissues was an essential step for establishing the quail-chicken chimera system, which in turn has provided undisputed ...

In Vitro Culture of Epithelial Cells from Different Anatomical Regions of the Human Amniotic Membrane

Several protocols have been reported in the literature for the isolation and culture of human amniotic epithelial cells (HAEC). However, these assume that the amniotic epithelium is a homogeneous layer. The human amnion can be divided into three anatomical regions: reflected, placental, and umbilical. Each region has different physiological roles, such as in pathological conditions. Here, we describe a protocol to dissect human amnion tissue in three sections and maintain it in vitro. In culture, cells derived from the reflected amnion displayed a cuboidal morphology, while cells from both placental and umbilical regions were squamous. Nonetheless, all the cells obtained have an epithelial phenotype, demonstrated by the immunodetection of E-cadherin. Thus, because the placental and reflected regions in situ differ in cellular components and molecular functions, it may be necessary for in vitro studies to consider these differences, because they could have physiological implications for the use of HAEC in biomedical research and the promising application of these cells in regenerative medicine.

This protocol describes the isolation of epithelial cells from different anatomical regions of the human amniotic membrane to determine their heterogeneity and functional properties for possible application in clinical and physiopathological models.

Several protocols have been reported in the literature for the isolation and culture of human amniotic epithelial cells (HAEC). However, these assume that ...

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

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.

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

Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos

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