Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Apr;23(4):385-91.
doi: 10.1089/thy.2012.0644. Epub 2013 Mar 18.

Thyroid follicle formation and thyroglobulin expression in multipotent endodermal stem cells

Risheng Ma  1 , Rauf Latif, Terry F Davies
Affiliations

Thyroid follicle formation and thyroglobulin expression in multipotent endodermal stem cells

Risheng Ma et al. Thyroid. 2013 Apr.

Abstract

Objective: The aim of this study was to assess the impact of transcriptional induction on thyroid follicular cell (TFC) differentiation from endodermally matured embryonic stem (ES) cells. The thyroid transcription factors-NKx2 homeobox 1 (NKx2-1, formerly called TTF-1) and Paired box gene 8 (Pax8)-are known to associate biochemically and synergistically in the activation of thyroid functional genes including the sodium/iodide symporter (NIS), thyrotropin (TSH) receptor (TSHR), thyroglobulin (Tg), and thyroid peroxidase (TPO) genes. In this study, we investigated the ability of ectopically expressed Pax8 and NKx2-1 to further the induction and differentiation of murine ES cells into potential TFCs.

Methods: ES cells were stably transfected with either the Pax8 gene, the NKx2-1 gene, or both genes to study the induction of NIS, TSHR, Tg, and TPO genes as assessed using both quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and protein expression. The derived cells were cultured with or without the presence of activin A to allow their differentiation into multipotent endodermal cells.

Results: The four thyroid-specific genes NIS, TSHR, Tg, and TPO were all significantly activated by expressing both transcription factors within the same ES cell. In contrast, significant but much lower transcriptional activity of the TSHR, Tg, and TPO genes was detected in cells expressing just NKx2-1, and only the NIS and TSHR genes responded to Pax8 alone. No Tg protein expression could be detected prior to their development into endodermal derivatives. However, after further differentiation of postembryoid body ES cells with activin A and TSH into endodermal cell lines, those cells with dual transfection of Pax8 and NKx2-1 demonstrated greatly enhanced expression of the NIS, TSHR, Tg, and TPO genes to such a degree that it was similar to that found in control thyroid cells. Furthermore, these same cells formed three-dimensional neofollicles in vitro and expressed Tg protein, but these phenomena were absent from lines expressing only Pax8 or NKx2-1.

Conclusion: These findings provide further evidence that co-expression of Pax8 and NKx2-1 in murine ES cells may induce the differentiation of thyroid-specific gene expression within endodermally differentiated ES cells and commit them to form three-dimensional neofollicular structures.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
Protocol summary.
FIG. 2.
FIG. 2.
Generation and characterization of stable Pax8+, NKx2-1+, and Pax8+/NKx2-1+ mouse ES cell lines. (A) Reverse transcription polymerase chain reaction (RT-PCR) analysis of selected Pax8-, NKx2-1–, or Pax8/NKx2-1–expressing mouse embryonic stem (ES) cell lines for thyroid transcription factors Pax8 and NKx2-1 and pluripotent markers Rex1, Sox2, and Oct4 after the cells were cultured in selection medium for up to four weeks. (B) Immunofluorescence analysis of Pax8 and NKx2-1 in the selected double transfected cell line. Scale=20 μm. (C) Representative immunodetection analysis of pluripotent markers SSEA-1 and Oct4 expression in Pax8+/NKx2-1+ cells. Scale=100 μm.
FIG. 3.
FIG. 3.
(A) qRT-PCR analysis for thyroid-specific gene activity and Foxe1 expression in Pax8+, NKx2-1+, and Pax8+/NKx2-1+ ES cell lines. Inset: The quantitative reverse-transcription polymerase chain reaction (qRT-PCR) data for rat thyroid (FRTL5) cells. Relative expression of each transcript is presented as fold change compared to ES cells (mean±SEM, n=3), and represent one of three separate experiments. (B) RT-PCR analysis for thyroid-specific gene expression in untreated Pax8+/NKx2-1+ embryoid bodies (EB) cells. The gel images represent one of three separate experiments.
FIG. 4.
FIG. 4.
(A) qRT-PCR analysis of NIS, TSHR, Pax8, Tg, and TPO genes in EB cells and in EB cells treated for five days with activin A. Although there was significant expression of NIS, TSHR, and Pax8 after activin treatment, there was no induction of Tg or TPO in these cells. (B) qRT-PCR analysis for TSHR and endoderm markers (AFP, Sox17, and Fox2) in EB cells formed from activin A treated and untreated Pax8+/NKx2-1+ ES cells. Data were expressed as mean±SEM, and represent one of three separate experiments. All the data were significantly different from –LIF cells (p<0.01). LIF, leukemia inhibitory factor; AA, activin A.
FIG. 5.
FIG. 5.
Differentiation of Pax8-, NKx2-1–, or Pax8/NKx2-1–expressing mouse ES cell lines into thyroid cells. (A) qRT-PCR analysis for thyroid transcriptional factors (Pax8, NKx2-1, and Foxe1) in differentiated Pax8+, NKx2-1+, and Pax8+/NKx2-1+ ES cells at day 21. (B) qRT-PCR analysis for thyroid functional genes (NIS, TSHR, Tg, and TPO) in differentiated Pax8+, NKx2-1+, and Pax8+/NKx2-1+ ES cells at day 21. The relative expression of each transcript is presented as fold change compared to undifferentiated ES cells (mean±SEM) and represent one out of three separate experiments. All data were significant (p<0.01) when compared to control ES cells.
FIG. 6.
FIG. 6.
Immunodetection of NIS (red) in the differentiated Pax8+, NKx2-1+, and Pax8+/NKx2-1+ EB cells at day 21 with DAPI (blue) nuclear staining. Immunodetection of Tg (green) expression was only seen in the Pax8+/NKx2-1+ cells. Scale=20 μm.
FIG. 7.
FIG. 7.
Immunostaining of endoderm-derived thyroid neofollicles. (A) Immunodetection of Pax8 (green) and NIS (red) expression in a thyroid neofollicle derived from differentiated Pax8+/NKx2-1+ EB cells at day 21. Note that the NIS expression is seen on the cell surface, and the staining for transcription factor Pax8 is seen within the nucleus. Scale bar=20 μm. (B) Immunodetection of Tg (green) and NKx2-1 (red) expression in a thyroid neofollicle derived from differentiated Pax8+/NKx2-1+ EB cells at day 21. Note that the Tg is seen in the intrafollicluar lumen and the staining for transcription factor NKx2-1 is seen within the nucleus. Extravasation of Tg is seen around this three-dimensional structure within the extracellular matrix support. Scale bar=20 μm.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Damante G. Di Lauro R. Thyroid-specific gene expression. Biochim Biophys Acta. 1994;1218:255–266. - PubMed
    1. Damante G. Tell G. Di Lauro R. A unique combination of transcription factors controls differentiation of thyroid cells. Prog Nucleic Acid Res Mol Biol. 2001;66:307–356. - PubMed
    1. Di Palma T. Nitsch R. Mascia A. Nitsch L. Di Lauro R. Zannini M. The paired domain-containing factor Pax8 and the homeodomain-containing factor TTF-1 directly interact and synergistically activate transcription. J Biol Chem. 2003;278:3395–3402. - PubMed
    1. Espinoza CR. Schmitt TL. Loos U. Thyroid transcription factor 1 and Pax8 synergistically activate the promoter of the human thyroglobulin gene. J Mol Endocrinol. 2001;27:59–67. - PubMed
    1. Lin RY. Kubo A. Keller GM. Davies TF. Committing embryonic stem cells to differentiate into thyrocyte-like cells in vitro. Endocrinology. 2003;144:2644–2649. - PubMed

Publication types

MeSH terms

LinkOut - more resources