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 May 1;22(9):1433-42.
doi: 10.1089/scd.2012.0620. Epub 2013 Feb 19.

NKX2-1 activation by SMAD2 signaling after definitive endoderm differentiation in human embryonic stem cell

Yong Li  1 , Kristel Eggermont, Veerle Vanslembrouck, Catherine M Verfaillie
Affiliations

NKX2-1 activation by SMAD2 signaling after definitive endoderm differentiation in human embryonic stem cell

Yong Li et al. Stem Cells Dev. .

Erratum in

Abstract

Expression of NKX2-1 is required to specify definitive endoderm to respiratory endoderm. However, the transcriptional regulation of NKX2-1 is not fully understood. Here we demonstrate that aside from specifying undifferentiated human embryonic stem cell (hESC) to definitive endoderm, high concentrations of Activin-A are also necessary and sufficient to induce hESC-derived definitive endodermal progeny to a FOXA2/NKX2-1/GATA6/PAX9 positive respiratory epithelial fate. Activin-A directly mediates the induction of NKX2-1 by interacting with ALK4, leading to phosphorylation of SMAD2, which binds directly to the NKX2-1 promoter and activates its expression. Activin-A can be replaced by GDF11 but not transforming growth factor β1. Addition of Wnt3a, SHH, FGF2, or BMP4 failed to induce NKX2-1. These results suggest that direct binding of Activin-A-responsive SMAD2 to the NKX2-1 promoter plays essential role during respiratory endoderm specification.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
NKX2-1 expression can be induced by Activin-A. (A) Differentiation scheme used. (B) Reverse transcription quantitative polymerase chain reaction (RT-qPCR) measurement of FOXA2 and NKX2-1 mRNA levels in human embryonic stem cell (hESC)-H9 cultured under different conditions. Data are represented as mean±SEM from 3 independent experiments (*P<0.05; **P<0.01). (C) Immunostaining for NKX2-1 and FOXA2 in hESC on day 0 (ES) and day 6 for Medium (M), A, W, AW, AWA, and AWW. Scale bar, 100 μm. Percentage FOXA2 positive cells: M=5.8%±5.4%, A=16.3%±8.2%, W=7.2%±3.9%, AW=16.4%±11.3%, AWA=18%±14.7%, AWW=8.7%±4.3%; Percentage NKX2-1 positive cells: 0%–2% in A, AW and AWA conditions, all other conditions 0%. Color images available online at www.liebertpub.com/scd
FIG. 2.
FIG. 2.
Expression of NKX2-1, PAX9 and GATA6 is induced by Activin-A. (A) Differentiation scheme. (B) RT-qPCR measurement of FOXA2, GATA6, PAX9, and NKX2-1 mRNA in cells cultured in the different culture conditions. Data are represented as mean±SEM from 6 independent experiments (*P<0.05). (C) Immunostaining for NKX2-1, FOXA2, GATA6 and PAX9 in day hESC-H9 progeny cultured from d6 with medium alone or with Activin-A. Scale bar, 100 μm. Percentage FOXA2 positive cells: M=65.6%±8.5%, A=89%±2.8%; Percentage NKX2-1 positive cells: M=0%; A=6.1%±4.2% (n=3). Color images available online at www.liebertpub.com/scd
FIG. 3.
FIG. 3.
Induction of NKX2-1 expression is Activin-A specific. (A) RT-qPCR measurement and immunostaining for NKX2-1, FOXA2, GATA6, and PAX9 of hESC-H9 cells treated with transforming growth factor (TGF)β1 and TGFβ1+Activin-A between d6 and d8. Data are represented as mean±SEM from 5 independent experiments (*P<0.05; **P<0.01). Scale bar, 100 μm. Percentage FOXA2 positive cells: T5=72.3%±8.4%, AT5=79.5%±11.8%; Percentage NKX2-1 positive cells: T5=0%; AT5=17%±11.6% (n=3). (B) RT-qPCR measurement and immunostaining for NKX2-1, FOXA2, GATA6 and PAX9 of hESC-H9 cells treated with SB431542 and SB431542+Activin-A between d6 and d8. Data are represented as mean±SEM from 5 independent experiments. Scale bar, 100 μm. Percentage FOXA2 positive cells: SB=78.1%±5%, ASB=86.9%±7.9%; Percentage NKX2-1 positive cells=0% (n=2). Color images available online at www.liebertpub.com/scd
FIG. 4.
FIG. 4.
NKX2-1 expression by the Activin-A is dependent on SMAD2 activation. (A–C) Western blot analysis of phospho-SMAD2 and total SMAD2 in whole-cell lysates. (A) Undifferentiated hESC-H9=ES; hESC-H9 differentiated with Activin-A+Wnt3a d0–d4 and Activin-A between d4–6=AWA6; hESC-H9 differentiated between d6–d8 with Activin-A+SB431542 (ASB10), SB431542 alone (SB10), medium (M), 10 ng/mL Activin-A (A10), 50 ng/mL Activin-A (A50), 5 ng/mL TGFβ1 (T5), Activin-A+TGFβ1 (AT5). (B) Undifferentiated hESC-H9=ES; hESC-H9 differentiated with 250 ng/mL GDF11+Wnt3a between d0–d4 and GDF11 between d4–6 (GWG6); between d6–d8 with medium (M), 50 ng/mL Activin-A (A) or 250 ng/mL GDF11 (G). (C) Schematic representation of NKX2-1 distal promoter region (not to scale). TSS: transcription start site; SBE: SMAD binding element. (D) NCI-H441 cells were transfected with wild-type pNKX2-1-luc(WT), pNKX2-1(mSBE1)-Luc(Mut-1), pNKX2-1(mSBE2)-Luc(Mut-2), pNKX2-1(mSBE3)-Luc(Mut-3), pNKX2-1(mSBEctr)-Luc(Mut-ctr), pNKX2-1(dSBE1)-Luc(Del-1), pNKX2-1(dSBE2)-Luc(Del-2), pNKX2-1(dSBEctr)-Luc(Del-ctr), on day 1 (all with pGV). Cells were harvested after 2 days of treatment and luciferase activity was analyzed. (*: P<0.01 vs. WT; #: P<0.01 vs. Mut-1; Δ: P<0.05 vs. Mut-2; □: P<0.05 vs. Del-ctr). (E) chromatin immunoprecipitation (ChIP) assay for SMAD binding to the NKX2-1 distal promoter. Differentiated hESC-H9 cells were cultured in M, 10 ng/mL Activin-A, 100 ng/mL Activin-A, or 100 ng/mL Activin-A+10 μM SB431542 for another 2 days. Cells were harvested and ChIP performed with an isogenic or anti-SMAD2 antibodies. Enrichment for the NKX2-1 promoter in the precipitated DNA was analyzed by qPCR using primers that flank the distal promoter region. Results from triplicate experiments are shown as fold change of DNA enrichment. *P<0.05.
FIG. 5.
FIG. 5.
Model of SMAD Regulation of NKX2-1 transcription in Human pluripotent stem cells. Arrows represent induction, and hammer-ended lines represent inhibition.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Kimura S. Hara Y. Pineau T. Fernandez-Salguero P. Fox CH. Ward JM. Gonzalez FJ. The T/ebp null mouse: thyroid-specific enhancer-binding protein is essential for the organogenesis of the thyroid, lung, ventral forebrain, and pituitary. Genes Dev. 1996;1:60–69. - PubMed
    1. Bohinski RJ. Di Lauro R. Whitsett JA. The lung-specific surfactant protein B gene promoter is a target for thyroid transcription factor 1 and hepatocyte nuclear factor 3, indicating common factors for organ-specific gene expression along the foregut axis. Mol Cell Biol. 1994;14:5671–5681. - PMC - PubMed
    1. Ikeda K. Clark JC. Shaw-White JR. Stahlman MT. Boutell CJ. Whitsett JA. Gene structure and expression of human thyroid transcription factor-1 in respiratory epithelial cells. J Biol Chem. 1995;270:8108–8114. - PubMed
    1. Lazzaro D. Price M. de Felice M. Di Lauro R. The transcription factor TTF-1 is expressed at the onset of thyroid and lung. Development. 1991;113:1093–1104. - PubMed
    1. Liu D. Yi M. Smith M. Mendelson CR. TTF-1 response element is critical for temporal and spatial regulation and necessary for hormonal regulation of human surfactant protein-A2 promoter activity. Am J Physiol Lung Cell Mol Physiol. 2008;2:L264–L271. - PMC - PubMed

Publication types

MeSH terms