An Ex vivo Culture System to Study Thyroid Development

Summary

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.

Abstract

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 “angio-follicular” 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.

Introduction

The thyroid gland is a collection of independent epithelial spheres, called follicles, surrounded by a dense network of endothelial capillaries. This organization allows thyroid function: endothelial capillaries provide thyrocytes with iodine, required for T3 and T4 hormone synthesis, and distribute these latter to the whole body. Scattered in between the follicles and capillaries, C-cells produce the hypocalcemic hormone calcitonin¹. Although adult thyroid architecture and functions are well known, the cellular and molecular mechanisms involved in thyroid embryonic development (follicle formation and differentiation) are far from being understood.

Protocol

Mice were raised and treated according to the principles of laboratory animal care of the University Animal Welfare Committee. All procedures and protocols were approved by this Committee.

1. Coating of Microscopy Plastic Culture Chambers

NOTE: Perform all the following steps under sterile conditions in a laminar flow hood. The two types of coating are functionally equivalent.

1. Coat plastic culture chambers with fibronectin one day prior to isolation of thyroid tissues:
   1. Dilute sterile fibronectin to a 50 μg/ml final concentration in M199 medium supplemented with 100 U/ml penicillin, 10....

Results

Thyroid anlages (midline and UB, together with surrounding tissues), and thyroid lobes are dissected from mouse embryos at e12.5 and e13.5/e14.5, respectively (Figure 1). After one day in culture on filter, the midline anlage is visible (Figure 2A) as an elongated tissue spanning on top of the trachea. It progressively forms two lobes on each side of the trachea. If isolated thyroid lobes (e13.5 or e14.5) are cultured, they will expand, undergo morphogenesis and epithelial cells will org.......

Discussion

This paper describes a method for dissecting and culturing thyroid explants (e12.5) or lobes (e14.5) in order to study and better understand the complex events leading to thyroid formation. Due to the dual origin of the thyroid anlagen (the midline anlage and the two lateral ultimobranchial bodies), and their small size, a fragment of e12.5 tissue localized rostral to the pharyngeal arch arteries, and containing the trachea, but not the esophagus is microdissected. As illustrated, within a period of four days, this .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
µ-slide 8 well, ibiTreat, tissue culture treated, sterile	proxylab	80826
Collagen Type I, rat tail	Millipore	08-115
Fibronectin from human plasma	Invitrogen	# 33010-018
M199	Invitrogen	31150-022
HBSS	Invitrogen	14025-100
Tungsten wire	Goodfellow	LS237450
culture plate insert (12 mm diameter)	Millipore	PICM01250
GlycoBlue	Invitrogen	AM9516
E-cadherin	BD Biosciences	610182	1/1,000
Ezrin	Thermo Scientific	MS-661-P1	1/400
PECAM	BD Biosciences	550274	1/100
Hoechst	Sigma	B2261