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 fi....

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 ....

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