The authors are grateful to Ant&#243;nio Cidad&#227;o, Isabel Alcobia, and Leonor Parreira for the critical reading of the manuscript, to Padma Akkapeddi for video narration, and to Vitor Proa from the Histology Service of the Instituto de Histologia e Biologia do Desenvolvimento, Faculdade de Medicina de Lisboa, Universidade de Lisboa, for technical support. We are particularly indebted to Paulo Caeiro and Hugo Silva from the Unidade de audiovisuais (Audiovisual Unit), Faculdade de Medicina de Lisboa, Universidade de Lisboa for their outstanding commitment to the production of this video. We acknowledge Leica Microsystems for kindly providing a stereoscope equipped with video system and to Interaves - Sociedade Agro-Pecu&#225;ria, S.A for contributing with quail fertilized eggs. This work was supported by Faculdade de Medicina de Lisboa, Universidade de Lisboa (FMUL).

All these experiments follow the animal care and ethical guidelines of the Centro Acad&#233;mico de Medicina de Lisboa.
1. Incubation of Fertilized Quail and Chicken Eggs
<ol>
	<li>Incubate Japanese quail (Coturnix coturnix japonica) and chicken (Gallus gallus) fertilized eggs for 3 and 8 days, respectively.
	<ol>
		<li>Place the eggs with the air chamber (egg blunt end) facing up in a humidified incubator at 38 &#176;C.</li>
		<li>Use around 20 quail eggs and 40 chicken eggs to perform this experiment. 
		NOTE: These numbers should be doubled when establishing this procedure for the first time.</li>
	</ol>
	</li>
</ol>
2. Isolation of Quail Embryonic Region Containing the Presumptive Territory of Thymic and Parathyroid Rudiments
Note: Perform egg manipulation procedures in sterile conditions using a horizontal laminar flow hood and sterilized instruments and materials.
<ol>
	<li>Prepare a large borosilicate glass bowl about 3/4 filled with cold phosphate-buffered saline (PBS) solution.</li>
	<li>Open a quail egg after 3 days of incubation by tapping the shell and cutting a circular opening on the opposite side of its blunt end with curved scissors. Carefully remove pieces of shell and transfer the embryo to the glass bowl filled with cold PBS.</li>
	<li>Hold the quail (q) embryo at E3 (qE3) (the quail stage corresponding to the HH-stage 21 of the chicken) with the help of thin forceps. Make a cut into the vitelline membrane enveloping the yolk using curved scissors. Continue to cut around and externally to the circumference of extra-embryonic vessels.</li>
	<li>Transfer the embryo to a small bowl about 3/4 filled with cold PBS with the help of thin forceps. Thoroughly wash the embryo from the remaining attached yolk.</li>
	<li>Use a skimmer to transfer the embryo to a 100 mm glass Petri dish with black base (see Table of Materials) containing 10 mL of cold PBS.</li>
	<li>Place the Petri dish under a stereomicroscope. 
	Note: From this point forward, perform the microsurgery procedures under a stereomicroscope for progressive magnification. As an illumination source, it is advised to use LED lights incorporated in the stereomicroscope or in the optic fibers, considering the limited heat load.</li>
	<li>Hold the embryo to the bottom of the plate with thin insect pins. Place four pins forming a square shape in the extra-embryonic region.</li>
	<li>Remove the extra-embryonic membranes of the cephalic region with the help of thin forceps and place a fifth pin there. 
	Note: If the embryo is correctly positioned, then the otic vesicle, the heart tube, and the 1st, 2nd, and 3rd pharyngeal arches (PAs) should be visible.</li>
	<li>Dissect the embryonic region of interest, i.e., the 3rd and 4th pharyngeal arch region (3/4PAR), using Wecker eye scissors.
	<ol>
		<li>Start cutting longitudinally and parallel to the embryo axis, between the somite/neural tube area and the PAs.</li>
		<li>Remove the ventrally positioned heart tube by cutting it. Keeping the scissors in the same position, rotate the Petri dish to reposition the embryo according to the direction of the cut.</li>
		<li>Cut between the 2nd and 3rd PAs and below the 4th PA.</li>
		<li>Detach the remaining membranes from the 3/4PAR with the help of thin forceps.</li>
	</ol>
	</li>
	<li>Aspirate the isolated tissues (the 3/4PAR) and transfer them to a glass dish 3/4 filled with cold PBS using a 2 mL sterile plastic pipette. 
	Note: Hereafter, tissues can be grown in vitro up to 48 h or be immediately grafted onto the CAM of a chicken embryo at E8.</li>
	<li>Keep the glass dish containing the isolated 3/4PAR on ice during the preparation of the in vitro assay.</li>
</ol>
3. In Vitro Organotypic Assay: Culture of the Embryonic Region Containing the Presumptive Territory of Thymic and Parathyroid Rudiments
<ol>
	<li>Prepare the culture medium with RPMI-1640 Medium supplemented with 10% FBS and 1% penicillin/streptomycin. 
	NOTE: Soluble pharmacological reagents can be added to the medium (for example, LY-411.575 (Ly) and di-benzazepine or cyclopamine and vismodegib, to inhibit Notch and Hedgehog (Hh) signaling pathways, respectively. For this assay, use 50-200 nM of Ly or 5-15 &#181;M of Di-benzazepine to inhibit Notch signaling in the 3/4PAR. Use 20 &#181;M of cyclopamine or 10 &#181;M of vismodegib to inhibit Hh signaling in the 3/4PAR5.</li>
	<li>Prepare the in vitro culture of the 3/4PP explant in a 6-well plate.
	<ol>
		<li>Fill one well from the 6-well plate with 5 mL of culture medium. Place a 24 mm Polycarbonate Membrane Insert (see Table of Materials) on the well with the help of thin forceps.</li>
		<li>Under the stereomicroscope, transfer the 3/4PAR explant from the glass dish to the membrane surface by gently sliding with the help of a transplantation spoon (or spatula) and thin forceps. Place the explants with the ventral side up and the dorsal side in contact with the membrane. Add up to seven explants per membrane insert. Proceed to step 3.4.</li>
	</ol>
	</li>
	<li>Alternatively, culture explants in floating membrane filters.
	<ol>
		<li>Prepare a 35 mm Petri dish with 5 mL of culture medium. With the help of thin forceps, float a membrane filter (see Table of Materials) and keep a dry surface in contact with air.</li>
		<li>Under the stereomicroscope, transfer the 3/4PAR explant from the glass dish to the membrane filter by gentle sliding with the help of a transplantation spoon (or spatula) and thin forceps. Place the explants with the ventral side up and the dorsal side in contact with the membrane. Add up to 8 explants per membrane filter.</li>
	</ol>
	</li>
	<li>Carefully place the explants prepared in steps 3.2 and 3.3 in a humidified incubator at 37 &#176;C with 5% CO2.</li>
	<li>After a 48 h incubation period, remove the 6-well plate and the 35 mm Petri dish from the incubator.
	<ol>
		<li>Collect the cultured explants from the membrane insert of the 6-well plate.
		<ol>
			<li>Add PBS at room temperature (RT) to the membrane insert.</li>
			<li>Detach the explants from the membrane by vigorous flushing using a 2 mL sterile plastic pipette.</li>
			<li>With the help of the spatula and thin forceps, transfer the cultured explants to a glass dish 3/4 filled with PBS at RT.</li>
		</ol>
		</li>
		<li>Similarly, collect the cultured explants from the floating membrane filter in the 35 mm Petri dish.
		<ol>
			<li>Transfer the membrane filter with thin forceps to a new 35 mm Petri dish filled with PBS at RT.</li>
			<li>Detach the explants from the membrane filter by vigorous pipetting using a 2 mL sterile plastic pipette.</li>
			<li>With the help of thin forceps, discharge the explant-free membrane filter after confirming that no explants remained attached to it.</li>
			<li>With a spatula and thin forceps, transfer the explants to a glass dish filled with PBS at RT.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Transfer the cultured explants with a spatula to 1 mL of a reagent for total RNA isolation and use for gene-expression studies. 
	CAUTION: Exposure to this reagent (see Table of Materials) can be a serious health hazard. Wear appropriate protective eyewear, clothing, and gloves. Follow the handling instructions and read the safety data sheets provided by the manufacturer.</li>
	<li>Alternatively, graft the cultured tissues onto CAM of chicken embryos at E8. Follow to step 4.</li>
</ol>
4. Preparation of the CAM
<ol>
	<li>Remove the chicken eggs with 8 days of embryonic development from the incubator. 
	NOTE: Eggs were incubated with air chamber facing upwards at 38 &#176;C in a humidified incubator.</li>
	<li>Cover the blunt end of the egg with clear plastic tape to prevent pieces of the shell from falling into the air chamber. Tap the shell and cut a circular opening in the egg with curved scissors. The air chamber should be visible.</li>
	<li>Remove with caution the white membrane of the air chamber with thin forceps. CAM is then visible and accessible for ectopic tissue transplantation. 
	Note: Do not use PBS to hydrate the CAM, before or after transplantation, since PBS promotes sliding and misplacement of the explants. If the membrane dries out, discard the egg.</li>
</ol>
5. Grafting of Cultured Explants onto the CAM
<ol>
	<li>Create small vascular lesions/wounds in the smaller vessels of the CAM with a microscalpel in a holder. 
	Note: Use the tip of a Pasteur pipette to remove blood by capillarity in the case of excess bleeding.</li>
	<li>Use a spatula and thin forceps to transfer the cultured explant to the wounded area of the CAM.</li>
	<li>Cut a piece of a filter paper slightly larger than the explant and place it on the top of the explant. 
	Note: The filter paper helps tracking the explant location after its development in the CAM. Also, it allows daily drug delivery to the explant during in ovo development, if necessary (described in step 5.6).</li>
	<li>Seal the egg window with clear plastic tape and identify it using a charcoal pencil. 
	Note: The plastic tape protects the embryo from dehydration during the incubation period.</li>
	<li>Incubate the manipulated egg for 10 days in a humidified incubator at 38 &#176;C. Follow to step 6.</li>
	<li>Optional Step: Daily drug administration during incubation period.
	<ol>
		<li>To access the filter, partially lift the plastic tape. Add 100 &#181;L of drug solution, drop by drop, on top of the paper. Re-seal the window and place the egg back in the humidified incubator at 38 &#176;C. 
		Note: For this assay, the dose of 20 &#181;M of cyclopamine will inhibit Hh signaling during in ovo development5.</li>
	</ol>
	</li>
</ol>
6. Ectopic Organ Formation in the CAM After 10 Days of In Ovo Development
<ol>
	<li>After 10 days of incubation, remove the egg from the incubator and carefully withdraw the plastic tape.</li>
	<li>Cut the CAM around the filter region using curved scissors and transfer the CAM-derived explant with filter paper to a small glass bowl about 3/4 filled with cold PBS.</li>
	<li>With the help of thin forceps transfer the CAM-derived explant to a 100 mm Petri dish with black base containing 10 mL of PBS. Gently remove the filter paper and the excess of membrane using Wecker eye scissors and thin forceps.</li>
	<li>Transfer the CAM-explant to fixative solution (3.7% PFA in PBS) with a skimmer. Euthanize the chicken embryos without removing them from the egg by making a precise cut in the neck region of the embryo with the help of large scissors.</li>
	<li>Assess the organ formation in paraffin sections of the CAM-derived explants by conventional histology and immunohistochemistry.</li>
</ol>

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The authors have nothing to disclose.

A crucial aspect for the success of this method is the quality of both the chicken and quail eggs. Considering the long incubation periods, particularly during the in ovo assay, a good quality of chicken eggs improves viability rates (up to 90%) by the end of the procedure. To achieve this, test eggs from different suppliers. Incubate unmanipulated eggs for long periods (up to 16-17 days) and check their development. To be considered a good quality batch, more than 80% of the embryos should present normal develo...

Avian embryos have been widely used in seminal developmental biology studies. The main advantages of the avian model include the possibility to open the egg, the relatively easy access to the embryo, and the ability to perform micromanipulation. Some examples comprise the classic quail-chicken chimera system for studying cell fate1, application of specific growth factors to the embryo2, and the growth of ectopic cellular structures in the CAM1,3,4.
To get new insights into distinct stages of organ formation, we have recently developed a method which combines grafting techniques with in vitro manipulation of embryonic tissues5. The two-step approach enables the discrimination and exploration of both early- and late-stages of organogenesis, which are often limited due to highly dynamic and complex tissue interactions2. Moreover, the lack of suitable tissue-specific markers frequently limits the use of genetically modified animal models6. This novel method of the two-step approach largely overcomes such limitations.
To study early-stages of organ formation, in the first step, the quail embryonic territory comprising the prospective organ rudiment is isolated and grown in an in vitro organotypic system for 48 h. During this period, pharmacological modulation of specific signaling pathways can be performed by adding drugs to the culture medium5,7. Additionally, cultured tissues can be collected at any stage of in vitro growth and probed for gene-expression (using methods such as qPCR, microarrays, etc.).
In the second step, 48 h-cultured tissues are then grafted onto the CAM of a chicken (c) embryo at embryonic day (E) 8 (cE8) (Hamburger and Hamilton (HH)-stages 33-35)8. The CAM behaves as a vascular supplier of nutrients and allows gas exchanges1,3,4 to grafted tissues enabling its development in ovo for longer periods of time. This experimental step is especially well suited to study late-stages of organogenesis, as fully formed organs can be obtained after 10 days of in ovo development5,9,10,11. Morphological analysis is easily performed by conventional histology to confirm proper organ formation and donor origin of cells can be identified by immunohistochemistry using species-specific antibodies (i.e., MAb Quail PeriNuclear (QCPN)). During the CAM incubation period, grafts can also be grown in the presence of pharmacological agents and collected at any stage of development to evaluate the progression of organogenesis.
The two-step approach, described here in depth, has already been employed in Figueiredo et al.5 to explore the avian parathyroid/thymus common primordium development. Accordingly, the inherent particularities of the embryonic territories and stages of development involved in the organogenesis of the thymus and parathyroid glands will be presented below.
The thymus and parathyroid glands epithelia, though functionally distinct, derive from the endoderm of the pharyngeal pouches (PP)12. In avian, the epithelia of these organs originate from the third and fourth PP endoderm (3/4PP)12, while in mammals the thymic epithelium derives from the 3PP and the epithelium of parathyroid glands derives from the 3PP and 3/4PP in mouse and human, respectively13,14.
One of the earliest stages in the formation of these organs is the emergence of discrete thymus and parathyroid domains in the common primordium. In chicken, these domains can be identified by in situ hybridization, with specific molecular markers, at E4.515. As development proceeds, these organ rudiments individualize and separate from the pharynx, while a thin mesenchymal capsule, formed by neural crest-derived cells, surrounds them (at E5; HH-stage 27). Later on, the thymic epithelium is colonized by hematopoietic progenitor cells (at E6.5; HH-stage 30)12.
As in classical quail-chicken studies1,12, the two-step approach is particularly useful to study the formation of hematopoietic/lymphoid organs, namely the thymus5. As the quail explant, with the organ rudiment, is grafted in the chicken embryo prior to hematopoietic progenitor cell colonization, a chimeric thymus is formed with chicken blood-borne progenitor cells infiltrating a quail thymic epithelial counterpart. This method is, therefore, a useful tool to explore the contribution of hematopoietic cells in the development of the avian hemato/lymphoid system.

<table><tbody><tr><td>Chicken fertilized eggs (&lt;em&gt;Gallus gallus&lt;/em&gt;)</td><td>Pintobar, Portugal</td><td></td><td>Poultry farm&amp;nbsp;</td></tr><tr><td>Quail fertilized eggs (&lt;em&gt;Coturnix coturnix&lt;/em&gt;)</td><td>Interaves, Portugal</td><td></td><td>Bird farm&amp;nbsp;</td></tr><tr><td>15 mL PP centrifuge tubes</td><td>Corning</td><td>430052</td><td></td></tr><tr><td>50 mL PP centrifuge tubes</td><td>Corning</td><td>430290</td><td></td></tr><tr><td>60 x 20 mm pyrex dishes</td><td>Duran group</td><td>21 755 41</td><td></td></tr><tr><td>100 x 20 mm pyrex dishes</td><td>Duran group</td><td>21 755 48</td><td></td></tr><tr><td>Polycarbonate Membrane Insert&amp;nbsp;</td><td>Corning</td><td>3412</td><td>24 mm transwell with 0.4 mm Pore Polycarbonate Membrane Insert&amp;nbsp;</td></tr><tr><td>Membrane filter</td><td>Millipore</td><td>DTTP01300</td><td>0.6 mm Isopore membrane filter</td></tr><tr><td>6-well culture plates</td><td>Nunc, Thermo Fisher Scientific</td><td>140675</td><td></td></tr><tr><td>Petri dish, 35 x 10 mm</td><td>Sigma-Aldrich</td><td>P5112&amp;nbsp;</td><td></td></tr><tr><td>Pyrex bowls</td><td></td><td></td><td>from supermarket&amp;nbsp;</td></tr><tr><td>Transfer pipettes</td><td>Samco Scientific, Thermo Fisher Scientific</td><td>2041S</td><td>2 mL plastic pipet</td></tr><tr><td>Glass pasteur pipette</td><td>normax</td><td>5426015</td><td></td></tr><tr><td>Whatman&amp;nbsp;qualitative filter paper</td><td>Sigma-Aldrich</td><td>WHA1001090</td><td>Filter paper</td></tr><tr><td>Clear plastic tape</td><td></td><td></td><td>from supermarket&amp;nbsp;</td></tr><tr><td>Cytokeratin (pan; acidic and basic, type I and II cytokeratins), clone Lu-5</td><td>BMA Biomedicals</td><td>T-1302</td><td></td></tr><tr><td>Cyclopamine hydrate</td><td>Sigma-Aldrich</td><td>C4116</td><td>Pharmacological inhibitor of Hh signalling</td></tr><tr><td>Fetal Bovine Serum</td><td>Invitrogen, Thermo Fisher Scientific</td><td></td><td>Standart FBS</td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Invitrogen, Thermo Fisher Scientific</td><td>15140-122</td><td></td></tr><tr><td>Phosphate-Buffered Saline (PBS)</td><td>GIBCO, Thermo Fisher Scientific</td><td>10010023</td><td></td></tr><tr><td>QCPN antibody</td><td>Developmental Studies Hybridoma Bank</td><td>QCPN</td><td></td></tr><tr><td>RPMI 1640 Medium, GlutaMAX Supplement&amp;nbsp;</td><td>GIBCO, Thermo Fisher Scientific</td><td>61870010</td><td></td></tr><tr><td>Bluesil RTV141A/B Silicone Elastomer 1.1Kg Kit</td><td>ELKEM/Silmid</td><td>RH141001KG</td><td>To prepare the back base for petri dish</td></tr><tr><td>Stemolecule LY411575</td><td>Stemgent</td><td>04-0054</td><td>Pharmacological inhibitor of Notch signalling</td></tr><tr><td>TRIzol Reagent</td><td>Invitrogen, Thermo Fisher Scientific</td><td>15596026</td><td>Reagent for total RNA isolation</td></tr><tr><td>Dumont #5 Forceps</td><td>Fine Science Tools</td><td>11251-30&amp;nbsp;</td><td>Thin forceps</td></tr><tr><td>Extra fine Bonn scissors, curved</td><td>Fine Science Tools</td><td>14085-08</td><td>Curved scissors</td></tr><tr><td>Insect pins&amp;nbsp;</td><td>Fine Science Tools</td><td>26001-30</td><td></td></tr><tr><td>Micro spatula&amp;nbsp;</td><td>Fine Science Tools</td><td>10087-12</td><td>Transplantation spoon</td></tr><tr><td>Minutien Pins</td><td>Fine Science Tools</td><td>26002-20</td><td>Microscalpel</td></tr><tr><td>Moria Nickel Plated Pin Holder</td><td>Fine Science Tools</td><td>26016-12</td><td>Holder</td></tr><tr><td>Moria Perforated Spoon</td><td>Fine Science Tools</td><td>10370-17</td><td>Skimmer</td></tr><tr><td>Wecker Eye Scissor</td><td>Fine Science Tools</td><td>15010-11</td><td></td></tr><tr><td>Camera</td><td>Leica Microsystems&amp;nbsp;</td><td>MC170 HD</td><td></td></tr><tr><td>Stereoscope</td><td>Leica Microsystems&amp;nbsp;</td><td>Leica M80</td><td></td></tr><tr><td>Microscope</td><td>Leica Microsystems&amp;nbsp;</td><td>DM2500</td><td></td></tr></tbody></table>

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.

The above described protocol details a method that allows the investigation of both early- and late-stages of organogenesis, often limited by complex cellular and molecular interactions.
This method was previously employed in Figueiredo et al.5 to unravel the role of Notch and Hh signaling in the avian parathyroid/thymus common primordium development.
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Watch this Scientific Journal Video about Two-step Approach to Explore Early- and Late-stages of Organ Formation in the Avian Model: The Thymus and Parathyroid Glands Organogenesis Paradigm at JoVE.co

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

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 ...

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Research

JoVE Journal

Developmental Biology

7.4K Views.  Universidade de Lisboa. Dit artikel bevat een bijgewerkte aanpak van de klassieke kwartel-kip chimera systeem te bestuderen van de vorming van het orgel, door het combineren van nieuwe in vitro en in ovo experimentele procedures.

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

3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse this scaffold in a highly coordinated temporal and spatial order while sequentially passing obligatory check points, i.e., T cell lineage commitment, followed by T cell receptor repertoire generation and selection prior to their export into the periphery. The two major resident cell types forming this scaffold are cortical (cTECs) and medullary thymic epithelial cells (mTECs). A key feature of mTECs is the so-called promiscuous expression of numerous tissue-restricted antigens. These tissue-restricted antigens are presented to immature thymocytes directly or indirectly by mTECs or thymic dendritic cells, respectively resulting in self-tolerance.
Suitable in vitro models emulating the developmental pathways and functions of cTECs and mTECs are currently lacking. This lack of adequate experimental models has for instance hampered the analysis of promiscuous gene expression, which is still poorly understood at the cellular and molecular level. We adapted a 3D organotypic co-culture model to culture ex vivo isolated mTECs. This model was originally devised to cultivate keratinocytes in such a way as to generate a skin equivalent in vitro. The 3D model preserved key functional features of mTEC biology: (i) proliferation and terminal differentiation of CD80lo, Aire-negative into CD80hi, Aire-positive mTECs, (ii) responsiveness to RANKL, and (iii) sustained expression of FoxN1, Aire and tissue-restricted genes in CD80hi mTECs.

Studying medullary thymic epithelial cells in vitro has been largely unsuccessful, as current 2D culture systems do not mimic the in vivo scenario. The 3D culture system described herein - a modified skin organotypic culture model - has proven superior in recapitulating mTEC proliferation, differentiation and maintenance of promiscuous gene expression.

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse ...

Quantitative Analysis of Cancer Metastasis using an Avian Embryo Model

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex process that can involve many tissue types, span variable time periods, and often occur deep within organs, making it difficult to investigate and quantify. In addition, the efficacy of the metastatic process is influenced by multiple steps in the metastatic cascade making it difficult to evaluate the contribution of a single aspect of tumor cell behavior. As a consequence, metastasis assays are frequently performed in experimental animals to provide a necessarily realistic context in which to study metastasis. Unfortunately, these models are further complicated by their complex physiology. The chick embryo is a unique in vivo model that overcomes many limitations to studying metastasis, due to the accessibility of the chorioallantoic membrane (CAM), a well-vascularized extra-embryonic tissue located underneath the eggshell that is receptive to the xenografting of tumor cells (figure 1). Moreover, since the chick embryo is naturally immunodeficient, the CAM readily supports the engraftment of both normal and tumor tissues. Most importantly, the avian CAM successfully supports most cancer cell characteristics including growth, invasion, angiogenesis, and remodeling of the microenvironment. This makes the model exceptionally useful for the investigation of the pathways that lead to cancer metastasis and to predict the response of metastatic cancer to new potential therapeutics. The detection of disseminated cells by species-specific Alu PCR makes it possible to quantitatively assess metastasis in organs that are colonized by as few as 25 cells. Using the Human Epidermoid Carcinoma cell line (HEp3) we use this model to analyze spontaneous metastasis of cancer cells to distant organs, including the chick liver and lung. Furthermore, using the Alu-PCR protocol we demonstrate the sensitivity and reproducibility of the assay as a tool to analyze and quantitate intravasation, arrest, extravasation, and colonization as individual elements of metastasis.

Using quantitative PCR, we demonstrate how the well-established chick CAM model can be used to quantitatively analyze the metastasis of human tumor cells to distant organs.

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex ...

Medicine

Preparation of 2-dGuo-Treated Thymus Organ Cultures

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play a key role in the development of a functionally competent T-cell pool. However, the complexity of T-cell development in the thymus in vivo can limit analysis of individual cellular components and particular stages of development. In vitro culture systems provide a readily accessible means to study multiple complex cellular processes. Thymus organ culture systems represent a widely used approach to study intrathymic development of T-cells under defined conditions in vitro. Here we describe a system in which mouse embryonic thymus lobes can be depleted of endogenous haemopoeitic elements by prior organ culture in 2-deoxyguanosine, a compound that is selectively toxic to haemopoeitic cells. As well as providing a readily accessible source of thymic stromal cells to investigate the role of thymic microenvironments in the development and selection of T-cells, this technique also underpins further experimental approaches that include the reconstitution of alymphoid thymus lobes in vitro with defined haemopoietic elements, the transplantation of alymphoid thymuses into recipient mice, and the formation of reaggregate thymus organ cultures. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).

This video demonstrates the dissection and removal of the fetal thymus as well the preparation of ex vivo cultures of 2-dGuo-treated thymus.

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play ...

Biology

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 ...

Creating Avian Forebrain Chimeras to Assess Facial Development

The avian embryo has been used as a model system for more than a century and has led to fundamental understanding of vertebrate development. One of the strengths of this model system is that the effect of, and interaction among, tissues can be directly assessed in chimeric embryos. We have previously shown that signals from the forebrain contribute to facial morphogenesis by regulating the shape of the expression domain of Sonic hedgehog (SHH) in the Frontonasal Ectodermal Zone (FEZ). In this article, the method of generating the forebrain chimeras and provide illustrations of the outcomes of these experiments is described.

This article describes a tissue transplantation technique that was designed to test the signaling and patterning properties of basal forebrain&#160;during craniofacial development.

The avian embryo has been used as a model system for more than a century and has led to fundamental understanding of vertebrate development. One of the ...

A Comprehensive Review of Avian Embryonic Skin Explant Culture Protocols

Using Avian Skin Explants to Study Tissue Patterning and Organogenesis

The developing avian skin during embryogenesis is a unique model that can provide valuable insights into tissue patterning. Here three variations on skin explant cultures to examine different aspects of skin development are described. First, ex vivo organ cultures and manipulations offer researchers opportunities to observe and study the development of feather buds directly. Skin explant culture can grow for 7 days enabling direct analysis of cellular behavior and 4D imaging at intervals during this growth period. This also allows for physical and molecular manipulations of culture conditions to visualize tissue response. For example, growth factor-coated beads can be applied locally to induce changes in feather patterning in a limited area. Alternatively, viral transduction can be delivered globally in the culture media to up or downregulate gene expression. Second, the skin recombination protocol allows researchers to investigate tissue interactions between the epidermis and mesenchyme that are derived from different skin regions, different life stages, or different species. This affords an opportunity to test the time window in which the epithelium is competent to respond to signals and its ability to form different skin appendages in response to signals from different mesenchymal sources. Third, skin reconstitution using dissociated dermal cells overlaid with intact epithelium resets skin development and enables the study of the initial processes of periodic patterning. This approach also enhances our ability to manipulate gene expression among the dissociated cells before creating the reconstituted skin explant. This paper provides the three culture protocols and exemplary experiments to demonstrate their utility.

Author Spotlight: Insight into the Current Experimental Avian Skin Explant Methodologies

Here we describe protocols for three types of avian embryonic skin explant cultures that can be used to examine tissue interactions, 4D imaging timelapse movie (3D plus time), global or local perturbation of molecular function, and systems biology characterization.

The developing avian skin during embryogenesis is a unique model that can provide valuable insights into tissue patterning. Here three variations on skin ...

Recreating the Thymic Microenvironment with iPSC-Derived Organoids

Formation of Human Thymus Organoids in Three-Dimensional Fibrin Hydrogels

Generation of a functional and self-tolerant T cell repertoire is a complex process dependent on the thymic microenvironment and, primarily, on the properties of its extracellular matrix (ECM). Thymic epithelial cells (TECs) are crucial in thymopoiesis, nurturing and selecting developing T cells by filtering self-reactive clones. TECs have been empirically demonstrated to be particularly sensitive to physical and chemical clues supplied by the ECM and classical monolayer cell culture leads to a quick loss of functionality until their death. Because of this delicate maintenance combined with relative rarity, and despite the high stakes in modeling thymus biology in vitro, models able to faithfully mimic the TEC niche at scale and over time are still lacking. Here, we describe the formation of a multicellular human thymic organoid model, in which the TEC compartment is derived from human induced pluripotent stem cells (iPSC) and reaggregated with primary early thymocyte progenitors in a three-dimensional (3D) fibrin-based hydrogel. This model answers current needs for a scalable culture system that reproduces the thymic microenvironment ex vivo and demonstrates functionality, i.e., the ability to produce T cells and to support thymus organoid growth over several weeks. Thus, we propose a practical in vitro model of thymus functionality through iPSC-derived organoids that would benefit research on TEC biology and T cell generation ex vivo.

Author Spotlight: Advancing Thymic Epithelial Cells and T-Cell Research with Human Thymic Organoids

Here, we describe a protocol for the formation of human iPSC-derived thymus organoids grown in 3D fibrin hydrogels aiming to support thymic epithelial cell (TEC) maturation and prolonged maintenance, as well as thymopoiesis in vitro.

Generation of a functional and self-tolerant T cell repertoire is a complex process dependent on the thymic microenvironment and, primarily, on the ...

Two Techniques to Create Hypoparathyroid Mice: Parathyroidectomy Using GFP Glands and Diphtheria-Toxin-Mediated Parathyroid Ablation

Hypoparathyroidism (HP) is a disorder characterized by low levels of PTH which lead to hypocalcemia, hyperphosphatemia, and low bone turnover. The most common cause of the disease is accidental removal of the parathyroid glands during thyroid surgery. Novel therapies for HP are needed, but testing them requires reliable animal models of acquired HP. 
Here, we demonstrate the generation of two mouse models of acquired HP. In the GFP-PTX model, mice with green fluorescent protein (GFP) expressed specifically in the parathyroids (PTHcre-mTmG) were created by crossing PTHcre+ mice with Rosa-mTmGfl/fl mice. Green fluorescing parathyroid glands are easily identified under a fluorescence dissecting microscope and parathyroidectomy is performed in less than 20 min. After fluorescence-guided surgery, mice are profoundly hypocalcemic. Contrary to the traditional thyro-parathyroidectomy, this precise surgical approach leaves thyroid glands and thyroid function intact. The second model, which does not require surgery, is based on a diphtheria-toxin approach. PTHcre-iDTR mice, which express the diphtheria toxin (DT) receptor specifically in the parathyroids, were generated by crossing the inducible DTR mouse with the PTHcre mouse. Parathyroid cells are thus rendered sensitive to diphtheria toxin (DT) and can be selectively destroyed by systemically injecting mice with DT. The resulting hypocalcemic phenotype is stable.

Mice with acquired hypoparathyroidism would be useful for studying novel drug therapies for hypoparathyroidism. Two procedures to create such mice are demonstrated. The GFP-PTX mouse is generated by surgical parathyroidectomy guided by green fluorescing parathyroid glands. A second, non-surgical approach is based on parathyroid-specific expression of the diphtheria toxin receptor.

Hypoparathyroidism (HP) is a disorder characterized by low levels of PTH which lead to hypocalcemia, hyperphosphatemia, and low bone turnover. The most ...

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the &#8220;angio-follicular&#8221; units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.
This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.
In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

Development of the Chick

The chicken embryo (Gallus gallus domesticus) provides an economical and accessible model for developmental biology research. Chicks develop rapidly and are amenable to genetic and physiological manipulations, allowing researchers to investigate developmental pathways down to the cell and molecular levels.
This video review of chick development begins by describing the process of egg fertilization and formation within the chicken reproductive tract. Next, the most commonly used chick staging nomenclature, the Hamburger Hamilton staging series, is introduced. Major events in chick development are then outlined, including the dramatic cellular movements known as gastrulation that form the three major cell layers: The ectoderm, mesoderm, and endoderm. Cells from these layers go on to generate all the tissues within the organism, as well as extraembryonic membranes, which are necessary for the transport of gases, nutrients, and wastes within the eggshell. To conclude the discussion, some exciting techniques will be presented as strategies for studying chick development in greater detail.

Development of the Chick: Egg Formation and Key Events in Development

The chicken embryo (Gallus gallus domesticus) provides an economical and accessible model for developmental biology research. Chicks develop rapidly and ...

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

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