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

The overall goal of this procedure is to present an optimized experimental approach to isolate embryonic tissues from the quail and chicken embryos. With this technique, we obtain pure embryonic tissues that can be used in gene expression studies and functional assays. Tissues from quail and chicken embryos are isolated by microsurgery and subject to in vitro enzymatic digestion while preserving its biological properties.

After isolation, the three-dimensional preserved tissues can be used for high resolution topographical gene expression patterns analysis. This approach is particularly useful for detailing in situ gene expression in embryonic territories otherwise inaccessible by conventional methods. In addition, transcriptome analysis approaches can also be applied in isolated tissues without requiring genetic markers, while providing a tissue-specific high throughput omic analysis.

Alternatively, the isolated tissues from both species can be associated in an in vitro organotypic assay for 48 hours. This approach allows to study tissues interactions, since quail and chicken cells can be discriminated by distinct nuclear features and molecular markers. The capacity of the culture tissues to form organs can be further tested by in ovo assay.

This method is therefore a useful tool for studying complex tissue interactions in developmental processes with highly dynamic spacial modifications and can also help to reveal the potential of specific embryonic territories to the formation of organs. As an example, herein is described the formation of quail-chicken chimeric thymus from isolated embryonic tissues. First, the endoderm of the third and fourth pharyngeal pouches, the prospective thymic rudiment is isolated from the pharyngeal region of quail embryos with three days of development.

Then, this tissue is associated with a mesenchyme capable of supporting its development, the somatopleura mesoderm. Finally, the heterospecific association of tissues is cultured in vitro and in ovo to form a chimeric thymus. Perform all egg manipulation procedures in sterile conditions using a horizontal laminar flow hood and sterilized instruments and materials.

Fertilized quail eggs were incubated with the air chamber facing up. To begin, remove the quail eggs from the incubator. Prepare a large borosilicate glass bowl filled with cold PBS.

With curved scissors, tap and cut a circular hole in the shell of a quail egg with three days of incubation. Make the hole in the opposite side of the egg blunt, and transfer the yolk to the bowl with cold PBS. Remove the embryo from the yolk by cutting the vitelline membrane externally to extraembryonic vessels.

With the help of thin forceps, transfer the embryo to a small bowl filled with cold PBS, then move the embryo to a Petri dish with black base with a skimmer. Remove the pharyngeal region containing the third and fourth pharyngeal pouches as described in previous Jove SPA ligation. Then, transfer the third and fourth pharyngeal arch region to a glass that is filled with cold pancreatin and incubate for one hour, on ice, for enzymatic digestion.

Note that the incubation period of enzymatic digestion depends on the stage of development. Place the glass dish under the stereo microscope and isolate the endoderm from the third and fourth pharyngeal arch region, using two micro-scalpel in a holder. First place the pharyngeal region with the dorsal side up, showing internally the endoderm and the ectoderm in the external surface.

Observe the ectoderm, endoderm, mesenchyme, heart tube, and ventral portion of the neural tube. Remove the neural tube and the mesoderm attached to the dorsal surface of the pharyngeal endoderm. Observe the endoderm of the pharynx, the fourth and third pharyngeal pouches, and the heart tube.

With the dorsal side up, carefully detach and remove the mesenchyme between the pharyngeal arches and expose the pharyngeal pouches. Perform this procedure in the other side of the pharyngeal region. Remove the mesenchyme attached to the most posterior part of the endoderm and observe the fourth pharyngeal pouch.

With posterior side up, observe both left and right fourth pharyngeal pouches. Continue by removing the mesenchyme attached to the most posterior part of the endoderm and to the second pouch. Remove the heart tube and the mesenchyme surrounding the interior pouches.

With the ventral side up, observe the pharynx endoderm, and the right side of the second pharyngeal arch. At this stage, the endoderm of the thyroid rudiment should be visible. Detach the ectoderm of the second and third pharyngeal arches and carefully remove the mesenchyme of the arches.

Observe the removal of the mesenchyme of the second pharyngeal arch on the right side. Note the location of the fourth and third pouches and thyroid rudiment. Remove the remains to mesenchymal cells attached to the pharyngeal pouches.

Observe the third and fourth pouches of the right side and the fourth pouch from the left side. Repeat the mesenchyme detachment of the left side. Observe now the third pouch of the left side.

Observe mesenchyme detachment of the second arch on the left side. Note that all surfaces and solutions should be kept cold during this procedure. Change to a new cold pancreatin solution and dish in case of taking a long time to dissect the tissues.

Finally, make a transversal cut between the second and third pharyngeal pouches to remove the second pouches and the thyroid rudiment. At this point, the isolated endoderm is composed of the third and fourth pharyngeal pouches and the posterior tip of the pharynx. Remove the remains detached mesenchyme with the help of two micro-scalpels.

Transfer the isolated endoderm to a glass dish filled with cold fetal bovine serum and keep it on ice during the preparation of the in vitro assay. Eggs were placed in horizontal position and the upper side marked for using a charcoal. To begin, remove the eggs from the incubator.

With a curved scissor, open a small hole in the shell, insert a needle, and aspirate two milliliters of albumin with a 10 milliliter syringe. Open a circular hole in the marked region of the shell and cut the vitelline membrane externally to the extra embryonic vessels while holding the embryo with thin forceps. Under a stereo microscope, place the embryo in a Petri dish with black base containing cold PBS.

Use four insect pins to hold the embryo to the bottom of the plate. Place the pins in the extraembryonic region forming a square shape. Perform two cuts between the somites 19 and 24 transversally to the embryo axis and crossing the embryo.

Release this section by cutting the marginal embryonic edges. Observe the lateral plate, neural tube, and somites. Aspirate and transfer the tissues to a glass dish filled with cold pancreatin.

Incubate for 30 minutes on ice for enzymatic digestion. Under the stereo microscope, isolate the mesoderm from the surrounding tissues using two micro-scalpels in holders. Observe the somatopleura mesoderm, the splanchnopleura, neural tube, and ectoderm.

First, remove the ectoderm at the surface. Observe the somatopleura mesoderm dissociated from the ectoderm. Then, carefully detach the ventrally located splanchnopleura from the somatopleura.

Observe the right lateral mesoderm of the somatopleura. Release it by cutting it in a parallel motion to the neural tube. Note that all surfaces and solutions should be kept cold during this procedure.

Change to a new cold pancreatin solution and dish in case of taking a long time to dissect the tissues. Observe the left lateral mesoderm of the somatopleura, somites, neural tube, and ectoderm. Repeat the mesoderm release of this side.

Make slow movements with the micro-scalpel. The exposed extracellular matrix proteins sit through tissues and instruments, preventing fluid movements. Carefully cut the somatopleura in a parallel motion to the neural tube.

Transfer the isolated mesoderm to a glass dish filled with cold fetal bovine serum and keep it on ice during the preparation of in vitro assay. Place a fine meshed metal grate in a Petri dish and fill with culture medium. Remove the excessive liquids to level the medium surface with the top of the grate.

With the help of thin forceps, dip a membrane filter into the culture medium and carefully place it on the top of the grate to have a surface in contact with her. Under the stereo microscope, transfer the isolated endoderm from the glass dish to the membrane filter by gentle sliding with the help of spatula and thin forceps. Repeat this procedure for the isolated mesoderm.

With the help of a micro-scalpel, mix the tissues to maximize it's association. Carefully place the associated tissues in a humidified incubator at 37 degrees with 5%CO2 for 48 hours. After the incubation period, graft the culture tissues onto the coriolan type membrane and allow it to develop in oval for further 10 days to complete organ formation as previously described.

Here is the reticular method that allows the isolation of avian embryonic tissues to be used in distinct experimental approaches. As an example, the expression of genes known to be involved in the formation of the pharyngeal pouches were also evaluated an isolated endoderm containing the second, third, and fourth pharyngeal pouches from chicken's embryos at embryonic day three and a half by whole mount in situ hybridization. The expression of Sonic Hedgehog was detected in the endoderm of the central pharynx and excluded from the pouches, while BMP7 was expressed in the endoderm of the second and third pharyngeal pouches and excluded from the central pharynx and fourth pharyngeal pouch.

Isolated tissues can also be vitro specifically associated in vitro for 48 hours, graft onto the coriolan type membrane, and allowed to develop for further 10 days. Explants can be analyzed by conventional histology and immunohistochemistry. Some representative results of the association of quail endoderm from the third and fourth pharyngeal pouches with chicken somatopleura mesoderm are the following-Grafts showed a fully formed chimeric thymus with quail derived thymic epithelial cells positive for QCPN and chicken lymphoid cells.

Additionally, cytokeratin positive thymic epithelium showed normal morphological features, displaying a typical reticular architecture. After watching this video, you should have a good understanding how to isolate quail and chicken embryonic tissues that can be used in gene expression studies and by forming chimeric organs can also help deciphering the complexity of organ genetics.