Name: an ex vivo culture system to study thyroid development
Uploaded: 2014-06-06T13:45:00
Description: Watch this Scientific Journal Video about An Ex vivo Culture System to Study Thyroid Development at JoVE.com

The work was supported by grants from the Universit&#233; catholique de Louvain (Action de recherch&#233; concert&#233;es) and the Fund for Scientific Medical Research (F.R.S.-FNRS, Belgium). A.-S.D. is a doctoral fellow from T&#233;l&#233;vie, A.-C.H. held a fellowship from the Fonds pour la formation &#224; la Recherche dans l'Industrie et dans l'Agriculture (Belgium), M.V. is supported by the de Duve Institute and C.E.P. is a senior research associate of the F.R.S.-FNRS (Belgium).

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.
<ol>
	<li>Coat plastic culture chambers with fibronectin one day prior to isolation of t...

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Biol. 381 (1), 227-240 (2013).</li><li>Toda, S., Koike, N., Sugihara, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+integration+of+thyrocytes+and+thyroid+folliculogenesis:+a+perspective+for+thyroid+tissue+regeneration+and+engineering.">Cellular integration of thyrocytes and thyroid folliculogenesis: a perspective for thyroid tissue regeneration and engineering.</a> Endocr. J. 48, 407-425 (2001).</li><li>Toda, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+models+for+studying+thyroid+biology+and+disorders.">Culture models for studying thyroid biology and disorders.</a> ISRN Endocrinol. , (2011).</li><li>Eggo, M. C., Quiney, V. 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This paper describes a method for dissecting and culturing thyroid explants (e12.5)&#160;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 ...

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.
During embryogenesis, thyrocytes progenitor originates as a thickening (the midline anlage) of the ventral wall of the foregut endoderm at embryonic day (e) 8.5 in the mouse embryo, while C-cells progenitors originate at e11.5 as droplet-shaped protrusions (the ultimobranchial bodies) of the fourth pharyngeal pouches2-6. The midline bud then detaches from the endoderm, expands bilaterally to fuse at e13.5 with the ultimobranchial bodies on each side of the trachea. Finally, thyrocytes organize into follicles and C-cells differentiate.
Current knowledge on thyroid formation mainly comes from histological analysis of fixed tissues, but the morphogenetic events involved in thyroid formation are highly dynamic and involve communications and interactions between different cell types and with the extracellular matrix. Recent work showed that thyrocyte progenitors produce high levels of VEGF to recruit endothelial cells to the developing thyroid, and, in turn, recruited endothelial cells promote follicle formation and C-cells differentiation7.
Most ex vivo studies on the thyroid have been performed on isolated adult thyroid-derived cells, grown either on 2D tissue culture plastic dishes or in 3D matrices. Using these types of cultures, differentiated follicular cells either remain polarized and organized as follicles or reacquire a 3D organization8-10. However, these pure epithelial cells, explanted from their physiological environment, and cultured in 2D, ignore interactions with extracellular matrix, cytokines, growth factors and with other cell types such as the endothelial or nerves cells that they normally encounter in vivo. A very nice study recently described a differentiation protocol of ES cells into thyroid follicles using a final culture step in 3D matrigel11. However, these 3D cultures lack contact with other cell types.
Based on previous expertise on pancreas and salivary glands organ culture12-14, a method for dissecting mouse embryonic thyroid anlagen and culturing the explants on semiporous filters or on microscopy plastic slides was developed.
When working with e12.5 embryos, the dual origin of the thyroid anlagen (the midline anlage and the two lateral ultimobranchial bodies) imposed the microdissection of a large fragment of tissue. This contained the trachea, but not the esophagus, and extended from the pharyngeal arch arteries up to the arytenoid swelling. When cultured on filters, the midline anlage extends laterally on each side of the trachea, where they fuse with the ultimobranchial bodies to form the two thyroid lobes, still connected by a narrow isthmus.
In culture, epithelial cells proliferate, organize into follicles and differentiate into thyrocytes and C-cells, depending on their origin. Endothelial cells contained in the microdissected tissue also proliferate and invade the thyroid lobes to finally associate closely with the epithelial follicular structure, independently of blood flow or circulating factors. As development of the explants faithfully recapitulates in vivo development, this culture system is optimal to study morphogenetic and differentiation events occurring during thyroid development.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos, and the culture system is amenable to loss- and gain-of-function experiments. Finally, time-lapse imaging of fluorescently labeled thyroid explants on microscopy plastic dishes could be exploited to better investigate the kinetics and continuity of morphogenetic movements that occur in vivo. Time-lapse imaging has already been used to study branching morphogenesis of the pancreas15,16 or the ureteric bud17.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">&#181;-slide 8 well, ibiTreat, tissue culture treated, sterile</td>
			<td style="width:85px;">proxylab</td>
			<td style="width:119px;">80826</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagen Type I, rat tail</td>
			<td style="width:85px;">Millipore</td>
			<td>08-115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fibronectin from human plasma</td>
			<td style="width:85px;">Invitrogen</td>
			<td># 33010-018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">M199</td>
			<td style="width:85px;">Invitrogen</td>
			<td>31150-022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">HBSS</td>
			<td style="width:85px;">Invitrogen</td>
			<td>14025-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Tungsten wire</td>
			<td style="width:85px;">Goodfellow</td>
			<td style="width:119px;">LS237450</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">culture plate insert (12 mm diameter)</td>
			<td style="width:85px;">Millipore</td>
			<td>PICM01250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">GlycoBlue</td>
			<td style="width:85px;">Invitrogen</td>
			<td style="width:119px;">AM9516</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">E-cadherin</td>
			<td style="width:85px;">BD Biosciences</td>
			<td style="width:119px;">610182</td>
			<td>1/1000</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Ezrin</td>
			<td style="width:85px;">Thermo Scientific</td>
			<td style="width:119px;">MS-661-P1</td>
			<td>1/400</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">PECAM</td>
			<td style="width:85px;">BD Biosciences</td>
			<td style="width:119px;">550274</td>
			<td>1/100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Hoechst</td>
			<td style="width:85px;">Sigma</td>
			<td style="width:119px;">B2261</td>
			<td></td>
		</tr>
	</tbody>
</table>

an ex vivo culture system to study thyroid development

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 &#8220;angio-follicular&#8221; 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.

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...

Watch this Scientific Journal Video about An Ex vivo Culture System to Study Thyroid Development at JoVE.com

An Ex vivo Culture System to Study Thyroid Development

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.

An Ex vivo Culture System to Study Thyroid Development

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 ...

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