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

Summary

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.

Abstract

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.

Introduction

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 CAM^1,3,4.

To get new insights into distinct stages of organ for....

Protocol

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

1. Incubation of Fertilized Quail and Chicken Eggs

1. Incubate Japanese quail (Coturnix coturnix japonica) and chicken (Gallus gallus) fertilized eggs for 3 and 8 days, respectively.
   1. Place the eggs with the air chamber (egg blunt end) facing up in a humidified incubator at 38 °C.
   2. 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.
<....

Results

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.

Discussion

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.......

Materials

List of materials used in this article

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
Polycarbonate Membrane Insert	Corning	3412	24 mm transwell with 0.4 mm Pore Polycarbonate Membrane Insert
Membrane filter	Millipore	DTTP01300	0.6 mm Isopore membrane filter
6-well culture plates	Nunc, Thermo Fisher Scientific	140675
Petri dish, 35 x 10 mm	Sigma-Aldrich	P5112
Pyrex bowls			from supermarket
Transfer pipettes	Samco Scientific, Thermo Fisher Scientific	2041S	2 mL plastic pipet
Glass pasteur pipette	normax	5426015
Whatman qualitative filter paper	Sigma-Aldrich	WHA1001090	Filter paper
Clear plastic tape			from supermarket
Cytokeratin (pan; acidic and basic, type I and II cytokeratins), clone Lu-5	BMA Biomedicals	T-1302
Cyclopamine hydrate	Sigma-Aldrich	C4116	Pharmacological inhibitor of Hh signalling
Fetal Bovine Serum	Invitrogen, Thermo Fisher Scientific		Standart FBS
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
Stemolecule LY411575	Stemgent	04-0054	Pharmacological inhibitor of Notch signalling
TRIzol Reagent	Invitrogen, Thermo Fisher Scientific	15596026	Reagent for total RNA isolation
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
Micro spatula	Fine Science Tools	10087-12	Transplantation spoon
Minutien Pins	Fine Science Tools	26002-20	Microscalpel
Moria Nickel Plated Pin Holder	Fine Science Tools	26016-12	Holder
Moria Perforated Spoon	Fine Science Tools	10370-17	Skimmer
Wecker Eye Scissor	Fine Science Tools	15010-11
Camera	Leica Microsystems	MC170 HD
Stereoscope	Leica Microsystems	Leica M80
Microscope	Leica Microsystems	DM2500