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

Summary

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.

Abstract

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.

Introduction

In the early 1970s, an elegant quail-chicken chimera system was developed by Le Douarin, opening new avenues to understand the role of cell migration and cellular interactions during development^1,2. The model was devised on the premise that cell exchange between the two species would not significantly disturb embryogenesis, later confirmed when used to study numerous developmental processes, including the formation of the nervous and the hematopoietic systems¹. Taking the latter as an example, the cyclic waves of hematopoietic progenitors colonizing the thymic epithelial rudiment was first observed using the quail-chicken chimera system³. For that, the prospective territory of the thymus, the endoderm of the third and fourth pharyngeal pouches (3/4PP), was mechanically and enzymatically isolated from quail (q) embryos at 15 to 30-somite stage [embryonic day (E) 1.5- E2.5]. These stages correspond to chicken Hamburger and Hamilton⁴ (HH) - stages 12-17. The isolation procedures started with the use of trypsin to enzymatically dissociate the endoderm from the attached mesenchyme. The isolated endoderm was grafted into the somatopleura region of a host chicken (c) embryo at E3-E3.5 (HH-stages 20-21). This heterologous mesenchyme was considered "permissive" to thymic epithelium development contributing also to the organ formation³. Afterward, successive waves of chicken host blood-borne progenitor cells infiltrated the quail donor thymic epithelial counterpart contributing to thymus formation in the host embryo³.

More recently, a modified version of this approach was also proven to be important for studying epithelial-mesenchymal interactions during early-stages of thymus formation⁵. In this respect, the tissues involved in the formation of the ectopic thymus in chimeric embryos³ were isolated, both from donor and host embryos, and associated ex vivo. An improved protocol was used to isolate the quail 3/4PP endoderm (E2.5-E3) and the chicken somatopleura mesoderm (E2.5-E3). Briefly, embryonic tissues were isolated by microsurgery and subject to in vitro pancreatin digestion. Also, the conditions of enzymatic digestion, temperature and time of incubation were optimized according to tissue-type and developmental stage (Table 1).

Next, the isolated tissues were associated in an organotypic in vitro system for 48 h, as previously reported^5,6. The in vitro association of tissues mimics the local cellular interactions in the embryo, overcoming some restrictions of the in vivo manipulation. This system is particularly useful to study cellular interactions in complex morphogenic events, such as the development of the pharyngeal apparatus.

The contribution of each tissue in thymus histogenesis, as well as the ability of the heterospecific association to generate a thymus can be further explored using the CAM methodology, previously detailed^5,7,8. Succinctly, the cultured tissues were grafted onto the CAM of cE8 embryos and allowed to develop in ovo for 10 days. Then, thymus formation was evaluated by morphological analysis in the harvested explants. As in the classical quail-chicken studies³, the quail thymic epithelium was colonized by hematopoietic progenitor cells (HPCs) derived from the chicken embryo, which was later shown to contribute to organ development^9,10. The HPCs migrated from the embryo to the ectopic chimeric thymus through the highly vascularized CAM^5,7,8. Quail derived thymic epithelium can be identified by immunohistochemistry using species-specific antibodies (i.e., QCPN- MAb Quail PeriNuclear), overcoming the need for tissue-specific molecular markers.

This experimental method, as the two-step approach reported in previous publication⁸, allows the modulation of signalling pathways by regular administration of pharmacological agents during in vitro and in ovo development. Also, explants can be harvested at any time-point of the course of the experiment⁸.

Lastly, the isolation protocol here detailed allows the preservation of the natural properties and 3D-architecture of embryonic tissues, particularly useful for detailing in situ gene-expression patterns of embryonic territories otherwise inaccessible by conventional methods. In addition, transcriptome analysis approaches, including RNA-seq or microarrays, can also be applied in isolated tissues without requiring genetic markers while providing a tissue-specific high throughput "omics" analysis.

Protocol

All these experiments follow the animal care and ethical guidelines of the Centro Académico de Medicina de Lisboa.

1. Fertilized quail and chicken egg incubation

1. Place fertilized eggs of Japanese quail (Coturnix coturnix japonica) in a 38 °C humidified incubator for 3 days. Incubate the eggs (egg blunt end) facing up in the air chamber.
   NOTE: The humidified environment is achieved by placing a water container at the bottom of the incubator.
2. Incubate fertilized eggs of chicken (Gallus gallus) for 2.5 days in a 38 °C humidified incubator. Incubate the eggs in a horizontal position and mark the upper side using a piece of charcoal to identify the embryo location.
   NOTE: Start with 40 quail eggs and 60 chicken eggs when establishing this experiment.

2. Isolation of quail endoderm containing the prospective domain of the thymic rudiment

NOTE: Use a horizontal laminar flow hood and sterilized instruments and materials for egg manipulation procedures in sterile conditions.

1. Remove the embryonic region containing the presumptive territory of thymic rudiment, the pharyngeal arch region containing the 3^rd and 4^th arches (3/4PAR), as described^7,8.
   1. Fill a large borosilicate glass bowl (100 mm x 50 mm; 100 cm³) with 60 mL of cold phosphate-buffered saline solution (PBS).
   2. With the help of curved scissors, tap and cut a circular hole in the shell of a quail egg that has been incubated for 3 days. Make the hole on the opposite side of the egg blunt and transfer the yolk (with the embryo) to the bowl with cold PBS.
   3. Remove the embryo from the yolk by cutting the vitelline membrane externally to extra-embryonic vessels using curved scissors.
   4. With the help of thin forceps, transfer the embryo to a small bowl (60 mm x 30 mm; 15 cm³) filled with 10 mL of cold PBS.
   5. With a skimmer, move the embryo to a 100 mm Petri dish with a black base (see Table of Materials) containing 10 mL of cold PBS and place it under a stereomicroscope.
   6. Dissect the 3/4PAR, as previously described⁸.
   7. Aspirate the 3/4PAR and transfer to a glass dish three-quarters filled with cold PBS using a 2 mL sterile Pasteur pipette.
2. Isolate the endoderm containing the presumptive territory of thymic rudiment (the 3/4PP endoderm) by enzymatic digestion with pancreatin.
   1. With the help of spatula and thin forceps, transfer the 3/4PAR to a glass dish three-quarters filled with cold pancreatin (8 mg/mL; 1:3 dilution of 25 mg/mL with cold PBS).
   2. Incubate for 1 h on ice for enzymatic digestion.
      NOTE: The time of enzymatic digestion depends of the stage of development (Table 1).
   3. Place the glass dish under the stereomicroscope (40x-60x magnification) to isolate the endoderm from the 3/4PAR.
      NOTE: Keep all surfaces and solutions cold during this procedure. Change to a new cold pancreatin solution if taking a long time to dissect the tissues (>15 min). As an illumination source, use LED lights incorporated in the stereomicroscope or in the optic fibers, considering the limited heat load.
   4. To isolate the endoderm from the surrounding tissues, use two stainless steel microscalpels in pin holders.
      NOTE: Use microscalpels with a diameter between 0.1 mm and 0.2 mm and nickel pin holders with a jaw opening diameter of 0 mm to 1 mm.
      1. First remove the neural tube and mesoderm attached to the dorsal surface of the pharyngeal endoderm.
      2. With the dorsal side up, carefully detach and remove the mesenchyme between the pharyngeal arches and expose the pharyngeal pouches. Perform this procedure on both sides of the 3/4PAR.
      3. Remove the heart tube and the mesenchyme surrounding the anterior pouches.
      4. With the ventral side up, cut the ectoderm of the 2^nd and 3^rd pharyngeal arches and carefully remove the mesenchyme attached to the pouches. Repeat this procedure on the other side of the 3/4PAR. At this stage the thyroid rudiment should be visible.
      5. Remove any remaining mesenchymal cells attached to the pharyngeal endoderm with the two microscalpels.
      6. Make a transversal cut between the 2^nd and 3^rd PP, dissociating the pharyngeal endoderm containing the 3^rd and 4^th pouches from the anterior part of the endoderm having the thyroid rudiment and 2^nd pharyngeal pouch.
      7. With the help of spatula and thin forceps, transfer the isolated 3/4PP endoderm to a glass dish three-quarters filled with 100% cold fetal bovine serum (FBS).
3. Keep the glass dish with the isolated tissues on ice during the preparation of in vitro assay. Alternatively, the isolated tissues can be three-dimensionally preserved and in situ analyzed for gene-expression.

3. Isolation of chicken somatopleura mesoderm

NOTE: Perform egg manipulation procedures in sterile conditions using a horizontal laminar flow hood and sterilized instruments and materials.

1. Remove the embryonic territory containing the somatopleura mesoderm at the level of somites 19-24 (ss19-24).
   1. Remove the chicken egg from the incubator after 2.5 days of incubation.
   2. With curved scissors, open a small hole in the shell. Insert a needle and aspirate 2 mL of albumin with a 10 mL syringe to lower albumin volume inside the egg and prevent damage of the embryo (located below the marked region of the shell). Discard the aspirated albumin.
   3. Cut a circular hole (up to two-thirds of the top surface area) in the marked region of the shell using curved scissors.
   4. Cut the vitelline membrane externally to the extraembryonic vessels while holding the embryo with thin forceps.
   5. Under a stereomicroscope, place the embryo in a 100 mm Petri dish with a black base containing 10 mL of cold PBS.
      NOTE: Use a stereomicroscope from this point forward for progressive magnification of microsurgery procedures.
   6. Use four thin insect pins to hold the embryo to the bottom of the plate. Place the pins in the extraembryonic region forming a square shape.
   7. Perform two cuts between the somites 19 and 24 transversely to the embryo axis and crossing all embryo territory, using wecker eye scissors.
   8. Release the embryo section, ss19-24, by cutting marginal embryonic edges.
   9. Aspirate the ss19-24 tissues and transfer to a glass dish three-quarters filled with cold PBS using a 2 mL sterile Pasteur pipette.
2. Isolate the lateral mesoderm from somatopleura region (ss19-24) by enzymatic digestion with pancreatin (8 mg/mL; 1:3 dilution of 25 mg/mL with cold PBS).
   1. With the help of spatula and thin forceps, transfer the ss19-24 tissues to a glass dish three-quarters filled with cold pancreatin solution.
   2. Incubate for 30 min on ice for enzymatic digestion.
   3. Under the stereomicroscope, isolate the mesoderm from the surrounding tissues using two microscalpels in a holder.
      NOTE: Keep all surfaces and solutions cold during this procedure. Change to a new cold pancreatin solution if taking a long time to dissect the tissues (>10 min.). As an illumination source, use LED lights incorporated in the stereomicroscope or in the optic fibers, considering the limited heat load.
   4. During mesoderm isolation, first remove the ectoderm at the surface followed by careful detachment of the ventrally located splancnopleura tissues.
   5. Release the right lateral mesoderm of the somatopleura by cutting it in a parallel motion to the neural tube.
   6. Repeat the mesoderm separation of the left side of the embryo.
      NOTE: Make slow microscalpel movements during this procedure. The exposed extra-cellular matrix proteins stick to tissues and instruments preventing fluid movements.
   7. With the help of spatula and thin forceps transfer the isolated mesoderm to a glass dish three-quarters filled with cold FBS.
3. Keep the glass dish with the isolated tissues on ice during the preparation of in vitro assay.

4. In vitro organotypic assay: heterospecific association of quail 3/4PP endoderm and chicken somatopleura mesoderm

1. Prepare the culture medium with RPMI-1640 medium supplemented with 10% FBS and 1% Pen/Strep^3,5.
2. Place a metal grid in a 35 mm Petri dish with 5 mL of culture medium.
   NOTE: Remove the excess of liquid to level the medium surface with the top of the grid.
3. With the help of thin forceps, dip a membrane filter into the culture medium and then place it on the top of the grid to have one surface in contact with air.
   NOTE: One-quarter of the membrane area (with 13 mm diameter) is adequate for the tissue association.
4. Under the stereomicroscope, associate the isolated tissues on the top of the membrane filter. First transfer the 3/4PP endoderm (step 2) from the glass dish by gentle sliding with the help of a transplantation spoon (or spatula) and thin forceps. Repeat this procedure for the isolated mesoderm (step 3).
   NOTE: With the help of a microscalpel, mix the tissues to maximize its association.
5. Carefully place the associated tissues in a humidified incubator at 37 °C with 5% CO₂ for 48 h. Cultured tissues can be grafted onto the chorioallantoic membrane (CAM) .
   NOTE: Ectopic organ formation in the CAM was previously detailed⁸.

Results

The protocol details a method to isolate avian embryonic tissues to be used in several cellular and developmental biology technical approaches. This method was previously employed to study epithelial-mesenchymal interactions during early stages of thymus formation⁵. Herein, new results are shown in Figure 1 and Figure 2, using similar approaches.

Discussion

The embryonic tissue isolation procedure detailed here was improved from previous techniques to produce quail-chicken chimeric embryos in different biological contexts^3,5,6.

This approach is suitable to isolate pure embryonic tissues without requiring genetic manipulation or the use of tissue-specific markers, which are often undetermined, limiting the use of genetically modified animal models. It can...

Materials

Name	Company	Catalog Number	Comments
Chicken fertilized eggs (Gallus gallus)	Pintobar, Portugal		Poultry farm
Quail fertilized eggs (Coturnix coturnix)	Interaves, Portugal		Bird farm
15 mL PP centrifuge tubes	Corning	430052
50 mL PP centrifuge tubes	Corning	430290
60 x 20 mm pyrex dishes	Duran group	21 755 41
100 x 20 mm pyrex dishes	Duran group	21 755 48
Metal grid	Goodfellows		fine meshed  stainless steel grid
Membrane filter	Millipore	DTTP01300	0.6 mm Isopore membrane filter
Petri dish, 35 x 10 mm	Sigma-Aldrich	P5112
60 x 30 mm pyrex bowls (Small size)			from supermarket
100 x 50 mm pyrex bowls (Large size)			from supermarket
Transfer pipettes	Samco Scientific, Thermo Fisher Scientific	2041S	2 mL plastic pipet
Glass pasteur pipette	Normax	5426015
Clear plastic tape			from supermarket
Cytokeratin (pan; acidic and basic, type I and II cytokeratins), clone Lu-5	BMA Biomedicals	T-1302
Fetal Bovine Serum	Invitrogen, Thermo Fisher Scientific		Standart FBS
Pancreatin	Sigma-Aldrich	P-3292	Prepare a 25 mg/mL solution according to manufacturer's instructions; centrifuge and filter prior to aliquote and store at -20ºC. Aliquots can be kept frozen for several years.
Paraformaldehyde	Sigma-Aldrich	P6148
Penicillin-Streptomycin	Invitrogen, Thermo Fisher Scientific	15140-122
Phosphate-Buffered Saline (PBS)	GIBCO, Thermo Fisher Scientific	10010023
QCPN antibody	Developmental Studies Hybridoma Bank	QCPN
RPMI 1640 Medium, GlutaMAX Supplement	GIBCO, Thermo Fisher Scientific	61870010
Bluesil RTV141A/B Silicone Elastomer 1.1Kg Kit	ELKEM/Silmid	RH141001KG	To prepare the back base for petri dish
Dumont #5 Forceps	Fine Science Tools	11251-30	Thin forceps
Extra fine Bonn scissors, curved	Fine Science Tools	14085-08	Curved scissors
Insect pins	Fine Science Tools	26001-30	0.3 mm Stainless steel pin
Micro spatula	Fine Science Tools	10087-12	Transplantation spoon
Minutien Pins	Fine Science Tools	26002-20	0.2 mm Stainless steel microscalpel
Minutien Pins	Fine Science Tools	26002-10	0.1 mm Stainless steel microscalpel
Moria Nickel Plated Pin Holder	Fine Science Tools	26016-12	Nickel plated pin holder
Moria Perforated Spoon	Fine Science Tools	10370-17	Skimmer
Wecker Eye Scissor	Fine Science Tools	15010-11
Camera	Leica Microsystems	MC170 HD
Microscope	Leica Microsystems	DM2500
NanoZoomer S360 Digital slide scanner	Hamamatsu Photonics	C13220-01
Stereoscope	Leica Microsystems	Leica M80