This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Calì, G. Articles by Nitsch, L. Search for Related Content PubMed PubMed Citation Articles by Calì, G. Articles by Nitsch, L. Pubmed/NCBI databases Gene GEO Profiles HomoloGene UniGene Substance via MeSH

Conditional Inactivation of the E-Cadherin Gene in Thyroid Follicular Cells Affects Gland Development but Does Not Impair Junction Formation

Istituto di Endocrinologia e Oncologia Sperimentale-Consiglio Nazionale delle Ricerche (G.C., M.Z.), 80131 Napoli, Italy; MicroSCoBiO Research Center and IFOM Center of Cell Oncology and Ultrastructure, Department of Experimental Medicine, University of Genova (P.R., C.T.), 16145 Genova, Italy; Dipartimento di Biologia e Patologia Cellulare e Molecolare, University Federico II (B.D., D.T., M.D.F., L.N.), 80131 Napoli, Italy; BIOGEM: Biotechnology and Molecular Genetics in Southern Italy (A.A., E.A., R.D.L.), 83031 Ariano Irpino, Avellino, Italy; Department of Molecular Biology of the Cell I, Deutsches Krebsforschungszentrum (T.W., G.S.), D-69120 Heidelberg, Germany; Max-Planck Institute (O.B., R.K.), 80804 Freiburg, Germany; and Stazione Zoologica A. Dohrn (D.S.), 80121 Napoli, Italy

Address all correspondence and requests for reprints to: Professor Lucio Nitsch, Dipartimento di Biologia e Patologia Cellulare e Molecolare, University Federico II, Via S. Pansini 5, 80131 Napoli, Italy. E-mail: nitsch{at}unina.it.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
We have conditionally inactivated the E-cadherin gene in the thyroid follicular cells of mouse embryo to unravel its role in thyroid development. We used the Cre-loxP system in which the Cre-recombinase was expressed under the control of the tissue-specific thyroglobulin promoter that becomes active at embryonic d 15. At postnatal d 7, thyroid follicle lumens in the knockout mice were about 30% smaller with respect to control mice and had an irregular shape. E-cadherin was almost completely absent in thyrocytes, ß-catenin was significantly reduced, whereas no change in -catenin was detected. -Catenin was also reduced on the cell plasma membrane. Despite the dramatic loss of E-cadherin and ß-catenin, cell-cell junctions were not affected, the distribution of tight junction proteins was unaltered, and no increase of thyroglobulin circulating in the blood was observed. In addition, we found that other members of the cadherin family, the R-cadherin and the Ksp-cadherin, were expressed in thyrocytes and that their membrane distribution was not altered in the E-cadherin conditional knockout mouse. Our results indicate that E-cadherin has a role in the development of the thyroid gland and in the expression of ß-catenin, but it is not essential for the maintenance of follicular cell adhesion.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
CADHERINS ARE A large superfamily of adhesion molecules that are thought to play a central role in epithelial cell adhesion, a highly regulated process that is relevant to tissue organization and patterning (1, 2, 3). Classical type I cadherins are transmembrane proteins with a large extracellular domain that mediates interactions among neighboring cells, whereas the intracellular domain is responsible for the interaction with the actin cytoskeleton (4), which occurs through a number of cytoplasmic components among which catenins play a pivotal role (1, 2, 5, 6). E-cadherin, a major representative of the type I cadherins, mediates adhesion in epithelial cells and participates in the formation of adherens junctions. E-cadherin has been also implicated in the formation of other epithelial junctions, like the tight junctions (7). In vitro studies have demonstrated that E-cadherin is involved in the control of cell shape and migration and that it plays a major role in the establishment and maintenance of epithelial cell polarity (8). Loss of E-cadherin activity is relevant to tumor progression and is considered a hallmark of malignancy (9). The function of E-cadherin is essential to embryo development. Disruption of E-cadherin gene in murine embryonic stem cells leads to disruption of cell aggregation and is lethal in early embryogenesis (10). By the use of the Cre-loxP system (11),it has been possible to analyze the functional consequences of the lack of E-cadherin in specific epithelial cell types. In mouse mammary gland, the lack of E-cadherin causes increased premature cell death and alteration of the terminal differentiation program, thus preventing the mice from nurse-feeding their litters (12). In the epidermis it has been shown that overall tissue integrity is maintained and desmosomal structures preserved, but the renewal of hair follicles is impaired (13). In a similar model, it has also been found that a progressive hyperplasia develops with age (14). A more recent report indicates that lack of E-cadherin during development alters the tight junctions and impairs the function of the epidermal barrier (15).

The thyroid gland consists of individual structural and functional units, the thyroid follicles made of an epithelial cell monolayer that surrounds a colloid-filled lumen. The presence of junctional complexes, like apical tight junctions and adherens junctions, is thought to play an essential role in maintaining the integrity of the follicle structure and in preventing leakage of colloid from the lumen. Moreover, the polarized organization of the thyroid follicular cells is essential for the proper function of the gland. Several studies led to the proposal that E-cadherin is the principal molecule involved in cell-cell adhesion and cell polarization in the thyroid gland (16, 17). Moreover, this molecule appears to play an important role in human thyroid malignancies in which gene expression and posttranscriptional control of E-cadherin is frequently impaired (18, 19).

In this report we used the Cre-loxP system to evaluate the functional role of E-cadherin protein in the thyroid gland, in vivo. E-cadherin inactivation in the thyrocytes was achieved by the use of transgenic mice in which Cre-recombinase expression was under the control of the thyroglobulin (Tg) promoter that becomes active at about embryonic day (E) 15. We show that in these mice, at postnatal d 7, follicle lumens have a reduced size and acquire an unusual shape. Most notably, the disappearance of E-cadherin induces a significant loss of membrane catenins. However, no structural or functional alteration in cell-cell junctions is observed. Finally, we show that the thyroid gland expresses also the R-cadherin and the Ksp-cadherin, which might be responsible for the adhesive strength of E-cadherin-lacking thyrocytes.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Generation of Tg^Cre transgenic mice
Tg^Cre transgenic mice were generated as follows: a P1-derived artificial chromosome (PAC) harboring 100 kb from the mouse Tg gene was isolated from the RPCI21 mouse genomic PAC library using a mouse Tg cDNA probe. The coding sequence of the improved Cre (iCre) recombinase (20), followed by the bovine GH polyadenylation signal and an ampicillin resistance cassette (bla) flanked by frt sites (21) was amplified twice using nested PCR (Expand HighFidelitykit, conditions according to manufacturer’s instructions; Roche Diagnostics, Mannheim, Germany). Primer oligonucleotides were used that introduced sequences from the proximal promoter and first intron of the Tg gene to the very 5' and 3' ends of the PCR fragment. By homologous recombination in Escherichia coli, the PCR fragment was introduced into the Tg PAC in such a way that the ATG of the iCre matched the Tg ATG. The size of the resulting PAC and the proper integration of the Cre were analyzed by pulsed-field gel electrophoresis, Southern blot, and PAC sequencing (data not shown). The ampicillin cassette was removed by transiently expressing the Flpe recombinase in E. coli as described (21, 22). By microinjecting the 100-kb PAC insert harboring the transgenic construct into the male pronucleus of fertilized FVB/N oocytes, several transgenic lines were established carrying the TgiCre PAC in their genome, as verified by Southern blot. When Tg^Cre transgenic mice were crossed with the floxed lacZ⁺ reporter ROSA 26 line (23), only the thyroid was positive and, within the thyroid, only thyrocytes, and almost all of them were ß-gal positive (data not shown). All Cre-recombinase-positive mice were in apparent good health and fertile, although by 3–4 wk of age, they presented significant disorganization of the thyroid tissue and eventually became hypothyroid due to the toxicity of the Cre-recombinase expression (Calì, G., E. Amendola, R. Di Lauro, and L. Nitsch, in preparation). All subsequent experiments were performed on 7-d-old animals before any thyroid alteration was manifested.

Generation of conditional E-cadherin-deficient mice
E-cadherin null allele mice E-cad^–/wt were previously generated and described (10). Mice carrying the floxed E-cadherin gene (E-cad^flox/flox) were previously generated and described (12). Tg^Cre mice were intercrossed with E-cad^–/wt mice and Tg^Cre; E-cad^–/wt mice were obtained. These mice were crossed with mice homozygous for a floxed E-cadherin allele to obtain the Tg^cre; E-cad^–/flox mice. There was no evidence of embryonic lethality and the E-cadherin knockout (KO) mice were born with the expected frequencies. Tg^cre; E-cad^–/flox mice (mutant, KO) were compared with Tg^cre; E-cad^+/flox mice (control). Analyses of conditional E-cadherin KO were performed on CD1 genetic background.

Genotyping
A piece from the tail (both in the case of adult mice and embryos) was incubated overnight at 60 C with lysis buffer (50 mM Tris HCl, 100 mM EDTA, 100 mM NaCl, 1% sodium dodecyl sulfate, 0.5 mg/ml proteinase K), and genomic DNA was extracted by adding 0.3 vol 6 M NaCl and precipitated with isopropanol. Selected regions of the Cre-recombinase gene were amplified and analyzed by PCR. The primers used for the amplification, Cre forward (5'-CTGCCAGGGACATGGCCAGG-3') and Cre reverse (5'-GCACAGTCGAGGCTGATCAGC-3'), generate a product of 310 bp. E-cadherin mutants were genotyped as described (24).

Mice handling, histology, and image analysis
Mice used in this study were housed and maintained in pathogen-free conditions. The day at which the vaginal plug was detected was designed as E0.5. Adult thyroid glands were obtained from 7-d-old mice. Mice were killed by CO₂ asphyxia. All animal experiments were performed according to approved institutional protocols.

Tissues were fixed in 4% paraformaldehyde in PBS overnight at 4 C, dehydrated with a graded alcohol series, embedded in paraffin, and cut in sections of 4 µm thicknesses that were stained with hematoxylin and eosin.

Morphometric analyses were performed on light microscopy images that had been captured by a digital camera (Olympus 4.5, Tokyo, Japan) and then analyzed on a Macintosh computer using the public domain NIH Image program (developed at the U.S. National Institutes of Health; available at http://rsb.info.nih.gov/nih-image/). Three nonadjacent sections for each thyroid (five E-cad^flox/flox, five Tg^cre; E-cad^+/flox, 15 Tg^cre; E-cad^–/flox) were analyzed. Result significance was calculated using Student’s t test with significance set at P < 0.05.

Hormone measurements
Blood samples were collected in microtubes without anticoagulant. After clot formation the samples were centrifuged and the serum was aspirated and stored at –20 C until assayed. TSH levels were determined on serum samples by a specific rat TSH RIA kit (GE Healthcare, Little Chalfont, Buckinghamshire, UK). Total T₄ and total T₃ were measured with Immulite analyzer using a commercial kit, as recommended by the manufacturer (Diagnostics Products Corp., Los Angeles, CA). Thyroglobulin serum levels were measured both with Immulite analyzer (Diagnostics Products) and ELECSYS analyzer (Roche Diagnostics) using commercial kits as recommended by the manufacturer.

Antibodies
The following antibodies were used: rabbit antiserum anti-Cre recombinase (1:500; BAbCo, Berkeley, CA); mouse monoclonal antibodies anti-E-cadherin [1:100 immunofluorescence (IF); 1:10,000 Western blot (WB), anti--catenin (1:100 IF; 1:10,000 WB), anti-ß-catenin (1:100 IF; 1:3,000 WB), and anti--catenin (1:100 IF; 1:10,000 WB) (BD Transduction Laboratories, Franklin Lakes, NJ); mouse monoclonal antibody anti--tubulin (1:5000) were from Sigma (St. Louis, MO). Rabbit polyclonal antibody anticlaudin-1 (1:100), antizonula occludens (ZO)-1 (1:100), mouse monoclonal antibody antioccludin (1:100), and anti-Ksp-cadherin (1:100) were from Zymed Lab, Inc. (San Francisco, CA). Rabbit polyclonal antithyroid transcription factor (TTF)-1/Nkx2.1 and antipaired box transcription factor (Pax)8 antibodies (1:100 IF; 1:5000 WB) were provided by RDL, and rabbit polyclonal anti-Tg (1:10,000) and mouse monoclonal anti-Tg antibodies (1:100) were from NeoMarkers (Fremont, CA). The rat monoclonal antibody MRCD5 anti-R-cadherin, developed by Takeichi and Matsunami (1), was obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the National Institute of Child Health and Human Development and maintained by the University of Iowa, Department of Biological Sciences (Iowa City, IA). Alexa Fluor 488 or 546 goat antirabbit or antimouse were from Molecular Probes (Leiden, The Netherlands).

Western blot analysis
Thyroids from 7-d-old mice were weighed and frozen in liquid nitrogen. Blood was collected and serum was separated from the clot. To detect serum Tg, 7 µl of serum were boiled for 5 min in sample buffer in the presence of protease inhibitors and separated on 6% SDS-PAGE. To detect E-cadherin and catenins, thyroids were homogenized in JS buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 150 mM MgCl2, EGTA 5 mM, glycerol 1%, Triton X-100 1%], and shakered for 20 min with iron ball on Retsch Mixer Mill MM300 (Retsch, Haan, Germany), and 30 µl of total lysate were boiled for 5 min in Laemmli sample buffer and analyzed on 7.5% SDS-PAGE.

Sodium dodecyl sulfate-polyacrylamide gels were then blotted either onto Immobilon-P (Millipore, Bedford, MA) or nitrocellulose transfer membrane (Schleicher and Schuell GmbH, Dassel, Germany) using a Bio-Rad apparatus (Bio-Rad Laboratories, Hercules, CA). The filters were washed extensively with TTBS [150 mM NaCl, 20 mM Tris-HCl (pH 7.5), 0.1% Tween 20] and Tris-buffered saline (TBS) [150 mM NaCl, 20 mM Tris-HCl (pH 7.5)], blocked at room temperature for 1 h with 5% nonfat dry milk in TBS or with 5% BSA in TTBS. After washing twice with TTBS, filters were incubated for 1 h at room temperature with the primary antibody. The filters were washed extensively with TTBS and incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibody (Amersham, Little Chalfont, Buckinghamshire, UK) diluted 1:30,000 in TTBS. The filters were then washed six times with TTBS and once with TBS and developed using a Super Signal West Femto detection method (Pierce, Rockford, IL).

For reprobing, the nitrocellulose filters were rehydrated and stripped for 20 min at 37 C in Restore Western blotting stripping buffer (Pierce) and washed extensively with TTBS.

Immunohistochemistry
Animals were killed by cervical dislocation. Staged mouse embryos were obtained by dissection of pregnant females. Embryos were fixed overnight at 4 C in 4% paraformaldehyde in PBS (pH 7.2), dehydrated through ethanol series, cleared in xylene, and embedded in paraffin, and 7-µm sections were cut.

For immunohistochemical analyses, serial sections from dissected Cre embryos and wild-type embryos were dewaxed by standard techniques. Heating was performed to retrieve the antigen sites. To quench endogenous peroxidases, sections were treated with 1.5% hydrogen peroxide in methanol at room temperature. Sections were incubated for 1 h at room temperature with blocking buffer (3% BSA, 5% goat serum, 20 mM MgCl₂, 0.3% Tween 20 in PBS) and then with primary antibodies overnight at 4 C. Staining procedures and chromogenic reactions were carried out according to the Vectastain ABC kit protocol (Vector Laboratories, Burlingame, CA).

Immunofluorescence and confocal microscopy
Tissue sections were deparaffinized and hydrated through xylenes and graded alcohol series followed by antigen retrieval in sodium citrate buffer [0.01 M (pH 6.0)]. Sections were microwaved for 15 min, washed in PBS and PBS containing 0.2% Triton X-100 for 5 min, and incubated for 1 h with blocking buffer. Tissue sections were then incubated overnight at 4 C with primary antibody diluted in blocking buffer, washed in PBS containing 0.2% Triton X-100 for 5 min and PBS, incubated with the secondary antibody for 1 h at room temperature, washed in PBS containing 0.2% Triton X-100 for 5 min and PBS, and finally mounted in PBS/glycerol (1:1).

Immunofluorescence analysis was performed at a confocal laser scanner microscope (LSM 510; Zeiss, Göttingen, Germany). The lambda of the argon ion laser was set at 488 nm, and that of the HeNe laser was set at 543 nm. Fluorescence emission was revealed by BP 505–530 band pass filter for Alexa Fluor 488 and by BP 560–615 band pass filter for Alexa Fluor 546. Double-staining immunofluorescence images were acquired simultaneously in the green and red channels at a resolution of 1024 x 1024 pixels.

Electron microscopy
Mouse thyroid tissues were processed for electron microscopy as described (25). Briefly samples were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3), postfixed in 1% osmium tetroxide in 0.1 M cacodylate buffer (pH 7.3), and dehydrated in ascending concentration of ethanol, followed by infiltration in propylene oxide and propylene oxide/EPON 1:1. Samples were then embedded in Polybed 812 (Polysciences, Inc., Warrington, PA). Ultrathin sections were obtained with an Ultracut E (Reichert-Jung, Heidelberg, Germany), stained with uranyl acetate and lead citrate, and examined with a CM 10 (Philips, Best, The Netherlands) or FEI Tecnai 12G2 (FEI, Eindhoven, The Netherlands) electron microscopes.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Lack of expression of the E-cadherin gene in the thyroid gland of conditional KO mice
We preliminarily analyzed by immunohistochemistry the Tg^cre mouse embryo to determine in which tissues the Cre-recombinase was expressed (Fig. 1). By using an anti-Cre-recombinase-specific antiserum (Fig. 1, A and B) and an anti-Tg monoclonal antibody (Fig. 1, C and D) on serial sections of paraffin-embedded E14.5 Tg^cre mice, we assessed that the Cre-recombinase was exclusively detectable in the thyroid gland. No peroxidase staining was seen in the thyroids of control mice stained by the anti-Cre-recombinase antiserum (Fig. 1, E and F).

View larger version (113K): [in this window] [in a new window]   FIG. 1. Cre-recombinase is expressed only in the thyroid gland of Tgcre mouse embryos. Serial sections of paraffin-embedded E14.5 Tgcre transgenic mice were stained by immunoperoxidase with rabbit anti-Cre recombinase antiserum (A and B) or mouse monoclonal anti-Tg antibodies (C and D). In either case, peroxidase staining is detected only on the thyroid gland (arrows). Paraffin sections of E14.5 control embryos were also stained by immunoperoxidase with rabbit anti-Cre recombinase antiserum (E and F). Peroxidase staining is detected on neither the thyroid gland (arrow) or any other tissue. Bars, 1 mm (A, C, and E); 100 µm (B, D, and F).

Cre-recombinase and E-cadherin protein expression and distribution were detected by double immunofluorescence on paraffin sections of the thyroid gland of control Tg^cre; E-cad^+/flox mice at E18 (data not shown) and at postnatal d 7 (Fig. 2A). The Cre-recombinase was expressed all over the thyroid gland and was not expressed in the adjacent tissues (Fig. 2B). In addition, the Cre-recombinase was clearly localized in the nuclei of thyrocytes (Fig. 2C) that were identified on the basis of their positivity to antibody staining of the thyroid-specific transcription factors TTF-1/Nkx2.1 and Pax8 (data not shown). Cre-recombinase staining was overall rather intense, although some variability was observed in different areas of the sections. E-cadherin was also expressed all over the thyroid gland but not in nearby nonepithelial tissues (Fig. 2D). Staining was limited to the plasma membrane of thyrocytes at sites of cell-cell contacts. The expression of Cre-recombinase, therefore, was confined to thyroid follicular cells, and it did not affect the expression of E-cadherin in control Tg^cre; E-cad^+/flox mice.

View larger version (71K): [in this window] [in a new window]   FIG. 2. Conditional KO mice do not express E-cadherin in the thyroid gland. A, Hematoxylin and eosin staining of a control (Tgcre; E-cad+/flox) mouse at postnatal d 7. B, immunofluorescence staining of an adjacent section of the thyroid gland with the anti-Cre-recombinase antibody. The expression of Cre-recombinase is confined to the thyroid gland; neighboring tissues are Cre-recombinase negative. C and D, Double-immunofluorescence staining of control thyroid gland with anti-Cre-recombinase polyclonal antibodies (C) and anti-E-cadherin monoclonal antibodies (D). Cre-recombinase expression is all over the gland and is confined to the nuclei of thyrocytes, whereas E-cadherin expression is localized at the cell surface. E and F, Double-immunofluorescence staining of the thyroid gland of conditional KO mice at postnatal d 7 with anti-Cre-recombinase (E) and anti-E-cadherin antibodies (F). Cre-recombinase is present virtually in all nuclei (E); expression of E-cadherin is completely abolished in the thyroid (F) but not in the parathyroid (pT) in which Cre-recombinase is not expressed (compare E and F). T, Thyroid; Tr, trachea. Bars, 100 µm (A and B); 50 µm (C and D); 25 µm (E and F).

These data indicate that the expression of the Cre-recombinase in the thyrocytes of the mutant mice causes the ablation of the E-cadherin gene and the lack of the corresponding protein.

Morphological defects in the thyroid of KO mice
Thyroids from control and KO mice were examined after hematoxylin and eosin staining of sections of paraffin embedded glands. Both control (Fig. 3A) and KO (Fig. 3B) mice at postnatal d 7 were characterized by differentiated thyroids displaying follicles of different size. However, the follicle lumens of the KO-mouse thyroid appeared to be of smaller size and of irregular shape, compared with controls. Morphometric measurements confirmed that the surface area of lumens of KO mouse thyroids was about 33% smaller with respect to controls (Table 1). We could not evidence, however, a significant difference in thyroid weight between control and KO mice (Table 1). Moreover, whereas follicle lumen profiles in control-mouse sections were roughly circular, in KO mice they had a rather irregular shape.

View larger version (58K): [in this window] [in a new window]   FIG. 3. Lumens of conditional KO mice are smaller and have an irregular shape. Thyroid sections from control (A) and conditional KO mice (B) were stained with hematoxylin and eosin at postnatal d 7. No gross alteration in the overall organization of the gland was observed. However, lumens of the KO mouse thyroid (B) have a smaller size and an irregular shape (arrowheads) if compared with the lumen of controls (A, arrows). Bar, 30 µm (A and B).

View larger version (77K): [in this window] [in a new window]   FIG. 4. Thyroid ultrastructure in control and conditional KO mice. Electron microscopy images of thyroid sections were obtained from Tgcre; E-cad+/flox control mice (A) and Tgcre; E-cad–/flox conditional KO (B) mice at postnatal d 7. A, Follicle architecture in control mice resembles that of nontransgenic mice. Tight junctions (TJ) and microvilli (M) are present at the luminal side of polarized thyrocytes. Inset, Plasma membranes of adjacent cells are in close contact (white arrowhead), and numerous junctional complexes (black arrow) are present. B, In conditional KO mice, thyroid lumens are smaller and frequently have an irregular, nonround shape. Tight junctions (TJ) and microvilli (M) are clearly detected at the apical cell border. Inset, Membranes of neighboring cells closely adhere (white arrowhead) and many junctions (black arrow) are present. Bars, 5 µm (A and B); 0.5 µm (insets).

View larger version (49K): [in this window] [in a new window]   FIG. 5. Distribution of catenins in the thyroid gland from control and KO mice. Thyroid sections of control mice (A, C, and E) and KO mice (B, D, and F) at postnatal d 7 were stained by immunofluorescence with monoclonal antibodies anti-- (A and B), ß- (C and D), and - (E and F) catenin. In KO mice the staining intensity of -catenin (B) on the plasma membrane is reduced with respect to controls (A). The staining for ß-catenin (C and D) is dramatically reduced on thyrocytes plasma membrane, is not detected in the cell cytoplasm or in the nucleus, but can still be observed on blood vessels (D, white arrowhead). The expression of -catenin is comparable between control and KO mice (E and F). L, Follicle lumen. Bars, 10 µm (A–F).

View larger version (69K): [in this window] [in a new window]   FIG. 6. Expression of E-cadherin and catenins in the thyroid gland from control and KO mice. Thyroid cell lysates from controls and from KO mice at postnatal d 7 were separated on 7.5% SDS-PAGE, and the following proteins were detected by Western blot analysis: E-cadherin (E-cad), -catenin (-cat), ß-catenin (ß-cat), -catenin (-cat), and -tubulin.

Tight junction proteins are present at the apical border of KO mouse thyrocytes
Morphological data indicated that follicle lumens of KO mice had smaller size and more irregular shape with respect to control mice (Fig. 3 and Table 1). A simple explanation for this result is that follicle lumens are somehow leaky and that loss of colloid content might occur due to an alteration of the tight junctions of thyrocytes. It has been, in fact, shown that inactivation of E-cadherin can lead to a perturbation of the organization of the tight junctions in epithelial cells in culture (8). Moreover, E-cadherin is specifically required for tight junctions formation in mouse epidermal cells (15). However, electron microscopy results indicated the integrity of the tight junctions at the apical border of follicular cells (Fig. 4). Nevertheless, we investigated by immunofluorescence and confocal microscopy the expression and localization on thyroid sections of some of the main protein constituents of tight junctions, i.e. claudin-1, ZO-1, and occludin. In both control and KO mice, all three antibodies equally stained the apical cell domain of thyrocytes at sites of cell-cell contacts (Fig. 7). Furthermore, we investigated the potential leakiness of the tight junctions by determining the level of Tg present in the blood by both chemiluminescent immunoassay and Western blot. Whereas by the former method, we could not detect any Tg in the serum (Table 2), the Western blot analysis indicated that the amount of Tg in a blood sample was not different in the KO mice with respect to controls (Fig. 8).

View larger version (35K): [in this window] [in a new window]   FIG. 7. Tight junction proteins are expressed in the thyroid follicles of KO mice. Thyroid sections of control (A, C, and E) and KO (B, D, and F) mice at postnatal d 7 were stained by immunofluorescence with anti-ZO-1 (A and B), anticlaudin-1 (C and D), and antioccludin (E and F) antibodies. Alexa Fluor 488 secondary antibodies (A–D) or Alexa Fluor 543 (E and F) antibodies were used. The three tight junction proteins are all expressed, and their staining is localized at the apical side of thyrocytes (white arrowheads). L, Follicle lumen. Bar, 5 µm (A–F).

View larger version (51K): [in this window] [in a new window]   FIG. 8. Detection of Tg in the blood of control and KO mice. Serum from controls and KO mice at postnatal d 7 was run on an 6% SDS-PAGE, and Tg was detected by Western blot analysis. Small amounts of Tg are present in the serum of control mice, as expected. Nonincrease of Tg in the serum of KO mice is observed.

Additional cadherins are present in the thyroid gland
Our data indicated that the ablation of E-cadherin in thyroid follicular cells did not disrupt cell-cell adhesion, and junctions between neighboring cells persisted. We therefore investigated whether other cadherins, which could substitute for the lack of E-cadherin, were expressed on the lateral plasma membrane of the thyrocytes of KO mice.

The global gene expression profile in E18 thyroids dissected from C57/BL mouse embryos was determined by microarray analysis using the Affymetrix mouse expression set, MOE430 (data not shown). Several cadherins were expressed in the embryonic mouse thyroid gland. Other than E-cadherin, two of them were likely to be expressed by follicular epithelial cells, the Ksp-cadherin and the R-cadherin. Ksp-cadherin has been reported to be a tissue-specific cadherin that is coexpressed with E-cadherin in the kidney and developing genitourinary tract (27, 28). R-cadherin has been demonstrated to be expressed in the follicular cells of the mouse thyroid gland starting from E15 and in the adult (29). We tested whether Ksp-cadherin was indeed present in the thyroid follicles of control and KO mice, at postnatal d 7. Immunofluorescence staining and confocal microscopy analysis with a specific anti-Ksp-cadherin antibody gave a positive signal in the thyroid gland of control mice (Fig. 9A) and KO mice (Fig. 9B). The membrane distribution of Ksp-cadherin was superimposable to that of E-cadherin (Fig. 9C) at the basolateral plasma membrane. The specificity of the Ksp antibody was further demonstrated by the absence of E-cadherin staining in the KO mice (Fig. 9D). We finally examined whether the R-cadherin was equally expressed on the plasma membrane of KO and control mice. We found no difference in the level of fluorescence signal and the membrane distribution (Fig. 9, E and F). R-cadherin antibodies were not suitable to perform immunoprecipitation or Western blot analysis.

View larger version (56K): [in this window] [in a new window]   FIG. 9. Ksp-cadherin and R-cadherin are expressed in the thyrocytes of KO mice. Adjacent thyroid sections of 7-d-old control mice (A, C, and E) and KO mice (B, D, and F) were stained by immunofluorescence with anti-Ksp-cadherin antibodies (A and B), anti-E-cadherin antibodies (C and D), or anti-R-cadherin antibodies (E and F). In control mice all three antibodies stain the basolateral plasma membrane of thyroid follicular cells. In KO mice staining of the plasma membrane is detected with the anti-Ksp-cadherin and the anti-R-cadherin antibodies only. L, Follicle lumen. Bar, 10 µm (A–D); 10 µm (E and F).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We tested here the hypothesis that E-cadherin is the main cell adhesion molecule responsible for the formation of follicles and the organization of junctional complexes in the thyroid gland. We conditionally inactivated the E-cadherin gene in thyrocytes by the use of a Cre-loxP system in which the Cre-recombinase was put under the control of the thyroid-specific Tg promoter. The synthesis of Cre-recombinase around E15, and the consequent ablation of the E-cadherin gene, almost completely abolished the synthesis of the E-cadherin protein in thyroid follicular cells. At postnatal d 7, the follicular organization was maintained but significant changes in lumen size and lumen shape had occurred, which might be a consequence of the modification of the junctions between cells. E-cadherin is, in fact, considered to be the principal component of adhesion junctions of epithelial cells (30), and it has been reported to be responsible for the correct formation of the tight junctions. Desmosome formation has also been proposed to depend on E-cadherin expression (7). However, transmission electron microscopy and immunofluorescence localization of tight junction proteins did not reveal any major change in the junctional complexes among thyrocytes after E-cadherin gene ablation. The presence of tight junctions at the thyrocyte apical border is consistent with the finding that the amount of Tg in the circulating blood is not increased. This suggests that the reduction in lumen size is not due leakage of Tg through the tight junctions. It does not indicate, however, the absolute tightness of the tight junctions because a morphologically intact junction can still be permeable to small-molecular-weight proteins (31). Moreover, because a significant amount of circulating Tg can also derive from basolateral secretion by intact follicles, it cannot be ruled out that the Tg detected in the serum of KO mice might reflect, at the same time, a decreased basolateral secretion and a partial leakage through less tight junctions.

Integrity of the junctions is unlikely to be due to inefficient E-cadherin inactivation. By Western blot analysis, we determined that there is a dramatic reduction of E-cadherin. Moreover, by immunofluorescence, we could directly detect, at a single cell level, lack of expression of E-cadherin on the cell plasma membrane. Because one of the two E-cadherin alleles is a KO allele and the other is a floxed allele, the result of the Cre-recombinase activation could only lead, in each cell, to the disappearance of the protein. Indeed, we detected very few follicular cells in which E-cadherin was still expressed. Interestingly, those cells in which the gene had not been inactivated did not express TTF-1/Nkx2.1 and Pax8, two transcription factors that are responsible for the activity of the Tg promoter. It is conceivable that they might belong to a type of follicular cell that is different from the principal thyroid epithelial cell and that has been described several years ago (32).

Because E-cadherin was completely absent at cell-cell contact sites in KO mice, a reduction of ß-catenin in that same location was expected. We observed, by immunofluorescence, an almost complete loss of ß-catenin on the lateral plasma membrane of follicular cells. The reduction of ß-catenin was less dramatic when examined by Western blot analysis. An explanation for the apparent discrepancy is that cells other than thyrocytes, like vascular endothelial cells and mesenchymal cells, can synthesize ß-catenin. Moreover, small amounts of ß-catenin, which we cannot detect by immunofluorescence, might be located within the cytosol or in the nucleus, in which the protein translocates and might act as a transcription factor (33). It is likely that the ß-catenin that does not bind to the E-cadherin is largely degraded intracellularly by the ubiquitin-proteasome pathway (34). It has been proposed that ß-catenin associates to the cytosolic tail of E-cadherin, whereas it is still in transit through the rough endoplasmic reticulum, i.e. largely before it reaches the plasma membrane (35). Lack of association could perhaps lead to more rapid degradation.

The lack of ß-catenin on the plasma membrane of thyrocytes does not impair follicle formation. This is in agreement with our preliminary observations that the conditional KO of ß-catenin in the thyroid gland does not affect follicle formation (Calì, G., E. Amendola, R. Di Lauro, and L. Nitsch, unpublished observations). Although ß-catenin was strongly reduced on the thyrocyte membrane of mutant mice, -catenin was instead unchanged. This might be due to the fact that it plays different roles within the cell, other than mediating the interaction between the type I cadherin cytosolic tail and the actin cytoskeleton. -Catenin is, in fact, a normal constituent of desmosomal complexes in which it binds the cytoplasmic domain of desmosomal cadherins (36). -Catenin expression, as detected by Western blot, is also unchanged, but its staining intensity on the plasma membrane is significantly decreased. This could indicate that some -catenin is displaced from the plasma membrane to different intracellular locations. It is known that -catenin also has binding sites for other molecules, among which is the ZO-1 protein (37).

The presence of - and -catenins on the plasma membrane of conditional KO mice led us to consider also the possibility that thyrocytes express other cadherins that might substitute, at least in part, for the lack of E-cadherin. Data from microarray analysis indicated that mouse thyroids express several cadherins, including the R-cadherin (a type I cadherin) and the Ksp-cadherin. This finding is in agreement with a report showing that R-cadherin has been detected in the mouse embryonic thyroid starting from E15 and that its distribution within the gland is superimposable to that of E-cadherin (29). It is puzzling, however, that in E-cadherin conditional KO mice, the R-cadherin could exert its role in the absence of ß-catenin. It can be hypothesized that very low levels of ß-catenin are sufficient to R-cadherin for its adhesive function and/or that R-cadherin is preferentially binding to -catenin. This would also explain why no appreciable reduction of -catenin on the plasma membrane could be seen.

Moreover, we demonstrate here, for the first time, that mouse thyrocytes express on their plasma membrane the Ksp-cadherin, which was known to be expressed by kidney and genitourinary tract cells only. Ksp-cadherin does not belong to the family of type I cadherins, lacks a ß-catenin binding site (27), and likely does not interact with -catenin. However, an adhesive role of Ksp-cadherin in thyrocytes is conceivable because it has been shown, in vitro, that it has the same quantitative adhesive effect (measured by the aggregation index) as E-cadherin (38).

Conditional inactivation of E-cadherin in epithelial tissues has been previously obtained in the mouse mammary gland (12) and the epidermis (13, 14, 15). In the mouse mammary gland, it has been shown that E-cadherin has an essential role in determining survival and differentiation of alveolar epithelial cells (12). The inactivation of E-cadherin in the epidermis after birth results in a mild phenotype, with reduced tightness among cells and progressive loss of skin hair follicles but with substantial maintenance of the overall tissue integrity (13). A more severe phenotype, with perinatal cell death and loss of functional water barrier, is observed when inactivation occurs during development (15). It is interesting to note that, similarly to what we describe here, lack of E-cadherin is followed by lack of ß-catenin and significant reduction of -catenin but no alteration in desmosome formation. However, different from what we find in the thyroid gland, the inactivation of E-cadherin in the epidermis causes altered targeting of essential components to tight junctions resulting in their malfunctioning and consequent increase in tissue permeability (15).

In conclusion, our data indicate that lack of E-cadherin in the thyroid gland in late embryogenesis causes a change in lumen size and shape, affects the level of expression of ß-catenin and the distribution of -catenin, but does not impair follicle formation. We suggest that other cadherins be important in follicular cell adhesion. E-cadherin might play a fundamental role in the initial junction formation that occurs at very early embryonic stage, whereas the Ksp-cadherin, the R-cadherin, and possibly some others might play a modulatory role at later stages of development (39). The overall picture that is emerging is that the maintenance of tissue integrity possibly results from the interplay of more than one type of cadherin molecule.

	Acknowledgments

 
This work is dedicated to the memory of Professor Antonio Calì. We thank Professor Guido Pettinato for helpful discussion and Dr. Ernesto Mezza, Armando Coppola, Ciro Sociale, and Pasquale Signoriello for technical assistance with this work.

	Footnotes

 
First Published Online March 8, 2007

Abbreviations: E, Embryonic day; iCre, improved Cre; IF, immunofluorescence; Ksp, kidney-specific cadherin; KO, knockout; PAC, P1-derived bacterial artificial chromosome; Pax, paired box transcription factor; Tg, thyroglobulin; TBS, Tris-buffered saline; TTBS, TBS and Tween 20; TTF, thyroid transcription factor; WB, Western blot; ZO, zonula occludens.

This work was supported in part by grants from Ministero per l’Istruzione, l’Universita’ e la Ricerca, Biotechnology and Molecular Genetics in Southern Italy, Associazione Italiana per la Ricerca sul Cancro (to L.N. and M.Z.) and the Telethon Foundation (to L.N.).

Disclosure Statement: The authors have nothing to disclose.

Received October 4, 2006.

Accepted for publication February 26, 2007.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Takeichi M 1995 Morphogenetic roles of classic cadherins. Curr Opin Cell Biol 7:619–627[CrossRef][Medline]
2. Gumbiner BM 1996 Cell adhesion: the molecular basis of tissue architecture and morphogenesis. Cell 84:345–357[CrossRef][Medline]
3. Steinberg MS, McNutt PM 1999 Cadherins and their connections: adhesion junctions have broader functions. Curr Opin Cell Biol 11:554–560[CrossRef][Medline]
4. Yap AS, Brieher WM, Gumbiner BM 1997 Molecular and functional analysis of cadherin-based adherens junctions. Annu Rev Cell Dev Biol 13:119–146[CrossRef][Medline]
5. Aberle H, Schwartz H, Kemler R 1996 Cadherin-catenin complex: protein interactions and their implications for cadherin function. J Cell Biochem 61:514–523[CrossRef][Medline]
6. Rimm DL, Koslov ER, Kebriaei P, Cianci CD, Morrow JS 1995 1(E)-catenin is an actin-binding and -bundling protein mediating the attachment of F-actin to the membrane adhesion complex. Proc Natl Acad Sci USA 92:8813–8817[Abstract/Free Full Text]
7. Gumbiner B, Stevenson B, Grimaldi A 1988 The role of the cell adhesion molecule uvomorulin in the formation and maintenance of the epithelial junctional complex. J Cell Biol 107:1575–1587[Abstract/Free Full Text]
8. Gumbiner B, Simons K 1986 A functional assay for proteins involved in establishing an epithelial occluding barrier: identification of a uvomorulin-like polypeptide. J Cell Biol 102:457–468[Abstract/Free Full Text]
9. Behrens J 1999 Cadherins and catenins: role in signal transduction and tumor progression. Cancer Metastasis Rev 18:15–30[CrossRef][Medline]
10. Larue L, Ohsugi M, Hirchenhain J, Kemler R 1994 E-cadherin null mutant embryos fail to form a trophectoderm epithelium. Proc Natl Acad Sci USA 91:8263–8267[Abstract/Free Full Text]
11. Gu H, Marth JD, Orban PC, Mossmann H, Rajewsky K 1994 Deletion of a DNA polymerase ß gene segment in T cells using cell type-specific gene targeting. Science 265:103–106[Abstract/Free Full Text]
12. Boussadia O, Kutsch S, Hierholzer A, Delmas V, Kemler R 2002 E-cadherin is a survival factor for the lactating mouse mammary gland. Mech Dev 115:53–62[CrossRef][Medline]
13. Young P, Boussadia O, Halfter H, Grose R, Berger P, Leone DP, Robenek H, Charnay P, Kemler R, Suter U 2003 E-cadherin controls adherens junctions in the epidermis and the renewal of hair follicles. EMBO J 22:5723–5733[CrossRef][Medline]
14. Tinkle CL, Lechler T, Pasolli HA, Fuchs E 2004 Conditional targeting of E-cadherin in skin: insights into hyperproliferative and degenerative responses. Proc Natl Acad Sci USA 101:552–557[Abstract/Free Full Text]
15. Tunggal JA, Helfrich I, Schmitz A, Schwarz H, Gunzel D, Fromm M, Kemler R, Krieg T, Niessen CM 2005 E-cadherin is essential for in vivo epidermal barrier function by regulating tight junctions. EMBO J 24:1146–1156[CrossRef][Medline]
16. Brabant G, Hoang-Vu C, Behrends J, Cetin Y, Potter E, Dumont JE, Maenhaut C 1995 Regulation of the cell-cell adhesion protein, E-cadherin, in dog and human thyrocytes in vitro. Endocrinology 136:3113–3119[Abstract]
17. Nilsson M, Fagman H, Ericson LE 1996 Ca2+-dependent and Ca2+-independent regulation of the thyroid epithelial junction complex by protein kinases. Exp Cell Res 225:1–11[CrossRef][Medline]
18. Brabant G, Hoang-Vu C, Cetin Y, Dralle H, Scheumann G, Molne J, Hansson G, Jansson S, Ericson LE, Nilsson M 1993 E-cadherin: a differentiation marker in thyroid malignancies. Cancer Res 53:4987–4993[Abstract/Free Full Text]
19. von Wasielewski R, Rhein A, Werner M, Scheumann GF, Dralle H, Potter E, Brabant G, Georgii A 1997 Immunohistochemical detection of E-cadherin in differentiated thyroid carcinomas correlates with clinical outcome. Cancer Res 57:2501–2507[Abstract/Free Full Text]
20. Shimshek DR, Kim J, Hubner MR, Spergel DJ, Buchholz F, Casanova E, Stewart AF, Seeburg PH, Sprengel R 2002 Codon-improved Cre recombinase (iCre) expression in the mouse. Genesis 32:19–26[CrossRef][Medline]
21. Wintermantel TM, Mayer AK, Schutz G, Greiner EF 2002 Targeting mammary epithelial cells using a bacterial artificial chromosome. Genesis 33:125–130[CrossRef][Medline]
22. Buchholz F, Angrand PO, Stewart AF 1998 Improved properties of FLP recombinase evolved by cycling mutagenesis. Nat Biotechnol 16:657–662[CrossRef][Medline]
23. Soriano P 1999 Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet 21:70–71[CrossRef][Medline]
24. Young P, Boussadia O, Berger P, Leone DP, Charnay P, Kemler R, Suter U 2002 E-cadherin is required for the correct formation of autotypic adherens junctions of the outer mesaxon but not for the integrity of myelinated fibers of peripheral nerves. Mol Cell Neurosci 21:341–351[CrossRef][Medline]
25. Tacchetti C, Favre A, Moresco L, Meszaros P, Luzzi P, Truini M, Rizzo F, Grossi CE, Ciccone E 1997 HIV is trapped and masked in the cytoplasm of lymph node follicular dendritic cells. Am J Pathol 150:533–542[Abstract]
26. De Felice M, Di Lauro R 2004 Thyroid development and its disorders: genetics and molecular mechanisms. Endocr Rev 25:722–746[Abstract/Free Full Text]
27. Thomson RB, Igarashi P, Biemesderfer D, Kim R, Abu-Alfa A, Soleimani M, Aronson PS 1995 Isolation and cDNA cloning of Ksp-cadherin, a novel kidney-specific member of the cadherin multigene family. J Biol Chem 270:17594–17601[Abstract/Free Full Text]
28. Thomson RB, Ward DC, Quaggin SE, Igarashi P, Muckler ZE, Aronson PS 1998 cDNA cloning and chromosomal localization of the human and mouse isoforms of Ksp-cadherin. Genomics 51:445–451[CrossRef][Medline]
29. Fagman H, Grande M, Edsbagge J, Semb H, Nilsson M 2003 Expression of classical cadherins in thyroid development: maintenance of an epithelial phenotype throughout organogenesis. Endocrinology 144:3618–3624[Abstract/Free Full Text]
30. McNeill H, Ozawa M, Kemler R, Nelson WJ 1990 Novel function of the cell adhesion molecule uvomorulin as an inducer of cell surface polarity. Cell 62:309–316[CrossRef][Medline]
31. Nitta T, Hata M, Gotoh S, Seo Y, Sasaki H, Hashimoto N, Furuse M, Tsukita S 2003 Size-selective loosening of the blood-brain barrier in claudin-5-deficient mice. J Cell Biol 161:653–660[Abstract/Free Full Text]
32. Wetzel BK, Wollman SH 1969 Fine structure of a second kind of thyroid follicle in the C3H mouse. Endocrinology 84:563–578[Abstract/Free Full Text]
33. Helmbrecht K, Kispert A, von Wasielewski R, Brabant G 2001 Identification of a Wnt/ß-catenin signaling pathway in human thyroid cells. Endocrinology 142:5261–5266[Abstract/Free Full Text]
34. Aberle H, Bauer A, Stappert J, Kispert A, Kemler R 1997 ß-Catenin is a target for the ubiquitin-proteasome pathway. EMBO J 16:3797–3804[CrossRef][Medline]
35. Hinck L, Nathke IS, Papkoff J, Nelson WJ 1994 Dynamics of cadherin/catenin complex formation: novel protein interactions and pathways of complex assembly. J Cell Biol 125:1327–1340[Abstract/Free Full Text]
36. Green KJ, Jones JC 1996 Desmosomes and hemidesmosomes: structure and function of molecular components. FASEB J 10:871–881[Abstract]
37. Itoh M, Nagafuchi A, Moroi S, Tsukita S 1997 Involvement of ZO-1 in cadherin-based cell adhesion through its direct binding to catenin and actin filaments. J Cell Biol 138:181–192[Abstract/Free Full Text]
38. Wendeler MW, Praus M, Jung R, Hecking M, Metzig C, Gessner R 2004 Ksp-cadherin is a functional cell-cell adhesion molecule related to LI-cadherin. Exp Cell Res 294:345–355[CrossRef][Medline]
39. Nelson WJ 2003 Adaptation of core mechanisms to generate cell polarity. Nature 422:766–774[CrossRef][Medline]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Calì, G. Articles by Nitsch, L. Search for Related Content PubMed PubMed Citation Articles by Calì, G. Articles by Nitsch, L. Pubmed/NCBI databases Gene GEO Profiles HomoloGene UniGene Substance via MeSH