Limits...
All-trans retinoic acid directs urothelial specification of murine embryonic stem cells via GATA4/6 signaling mechanisms.

Mauney JR, Ramachandran A, Yu RN, Daley GQ, Adam RM, Estrada CR - PLoS ONE (2010)

Bottom Line: The urinary bladder and associated tract are lined by the urothelium, a transitional epithelium that acts as a specialized permeability barrier that protects the underlying tissue from urine via expression of a highly specific group of proteins known as the uroplakins (UP).Analysis of endodermal transcription factors such as GATA4 and GATA6 revealed significant upregulation and nuclear enrichment in RA-treated UP2-GFP+ populations.Collectively, these data suggest that RA mediates ESC specification toward a urothelial lineage via GATA4/6-dependent processes.

View Article: PubMed Central - PubMed

Affiliation: Urological Diseases Research Center, Children's Hospital Boston, Boston, Massachusetts, USA.

ABSTRACT
The urinary bladder and associated tract are lined by the urothelium, a transitional epithelium that acts as a specialized permeability barrier that protects the underlying tissue from urine via expression of a highly specific group of proteins known as the uroplakins (UP). To date, our understanding of the developmental processes responsible for urothelial differentiation has been hampered due to the lack of suitable models. In this study, we describe a novel in vitro cell culture system for derivation of urothelial cells from murine embryonic stem cells (ESCs) following cultivation on collagen matrices in the presence all trans retinoic acid (RA). Upon stimulation with micromolar concentrations of RA, ESCs significantly downregulated the pluripotency factor OCT-4 but markedly upregulated UP1A, UP1B, UP2, UP3A, and UP3B mRNA levels in comparison to naïve ESCs and spontaneously differentiating controls. Pan-UP protein expression was associated with both p63- and cytokeratin 20-positive cells in discrete aggregating populations of ESCs following 9 and 14 days of RA stimulation. Analysis of endodermal transcription factors such as GATA4 and GATA6 revealed significant upregulation and nuclear enrichment in RA-treated UP2-GFP+ populations. GATA4-/- and GATA6-/- transgenic ESC lines revealed substantial attenuation of RA-mediated UP expression in comparison to wild type controls. In addition, EMSA analysis revealed that RA treatment induced formation of transcriptional complexes containing GATA4/6 on both UP1B and UP2 promoter fragments containing putative GATA factor binding sites. Collectively, these data suggest that RA mediates ESC specification toward a urothelial lineage via GATA4/6-dependent processes.

Show MeSH

Related in: MedlinePlus

Murine UP1B and UP2 promoters contain GATA-DNA binding sites which recruit complexes containing GATA4/6 in response to RA stimulation.Nuclear extracts from RA-treated ESCs following 9 d show distinct GATA-DNA complexes with 32P-labeled consensus oligos specific for GATA binding to 2 kb UP1B [A, C] or UP2 [B, D] promoter fragments. Complexes were absent in spontaneously differentiating controls (C). Both complexes were inhibited by addition of respective excess unlabeled wild type (wt) but not mutant GATA oligos (m1, m2). [C, D] The presence of GATA4 (G4) and GATA6 (G6) in both the GATA-UP complexes was determined by supershift or immunodepletion (ID) following antibody-specific incubation (G4, G6), but not species-matched isotype control antibodies (IgG). NS represents non specific band. FP denotes excess free probe.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2903484&req=5

pone-0011513-g006: Murine UP1B and UP2 promoters contain GATA-DNA binding sites which recruit complexes containing GATA4/6 in response to RA stimulation.Nuclear extracts from RA-treated ESCs following 9 d show distinct GATA-DNA complexes with 32P-labeled consensus oligos specific for GATA binding to 2 kb UP1B [A, C] or UP2 [B, D] promoter fragments. Complexes were absent in spontaneously differentiating controls (C). Both complexes were inhibited by addition of respective excess unlabeled wild type (wt) but not mutant GATA oligos (m1, m2). [C, D] The presence of GATA4 (G4) and GATA6 (G6) in both the GATA-UP complexes was determined by supershift or immunodepletion (ID) following antibody-specific incubation (G4, G6), but not species-matched isotype control antibodies (IgG). NS represents non specific band. FP denotes excess free probe.

Mentions: To determine the presence of putative GATA binding elements within the UP promoters, we analyzed a 2.0 kb fragment upstream from the transcriptional start site of each murine UP promoter sequence in silico using commercially available MatInspector software (Genomatix, Ann Arbor, MI) as previously described [63]. We identified one putative GATA-binding site within both the UP1B and UP2 promoter sequences at the following positions relative to the transcriptional start site: UP1B (−698 to −710) and UP2 (−1872 to −1885). In addition, promoter regions of the UP1A, UP3A, UP3B genes contained multiple putative GATA-binding sites. To determine whether GATA4/6 were recruited to the UP1B and UP2 promoters, we performed EMSA using nuclear extracts of RA-treated ESCs and spontaneously differentiating controls. As shown in Figure 6A and 6B, we observed robust complex formation with labeled probes corresponding to the GATA binding sites in both the UP1B and UP2 promoter sequences of RA-treated samples, while control nuclear extracts showed negligible binding. The complexes were competed out with the corresponding unlabeled UP1B or UP2 consensus GATA oligonucleotides, but not with mutant versions of these sequences. In addition, we observed supershift of the UP1B complex in the presence of GATA4 and GATA6 antibodies (Figure 6C), consistent with the presence of GATA4/6 in the complex. Supershift was also noted with the UP2 complex in the presence of GATA4 antibody, but not with IgG control; however the presence of GATA6 antibody resulted in substantial immunodepletion of the UP2 complex (Figure 6D) relative to IgG control. Together, these data suggest that RA-mediated signals promote recruitment of GATA4/6 to GATA binding sites within the UP1B/2 promoters during ESC specification.


All-trans retinoic acid directs urothelial specification of murine embryonic stem cells via GATA4/6 signaling mechanisms.

Mauney JR, Ramachandran A, Yu RN, Daley GQ, Adam RM, Estrada CR - PLoS ONE (2010)

Murine UP1B and UP2 promoters contain GATA-DNA binding sites which recruit complexes containing GATA4/6 in response to RA stimulation.Nuclear extracts from RA-treated ESCs following 9 d show distinct GATA-DNA complexes with 32P-labeled consensus oligos specific for GATA binding to 2 kb UP1B [A, C] or UP2 [B, D] promoter fragments. Complexes were absent in spontaneously differentiating controls (C). Both complexes were inhibited by addition of respective excess unlabeled wild type (wt) but not mutant GATA oligos (m1, m2). [C, D] The presence of GATA4 (G4) and GATA6 (G6) in both the GATA-UP complexes was determined by supershift or immunodepletion (ID) following antibody-specific incubation (G4, G6), but not species-matched isotype control antibodies (IgG). NS represents non specific band. FP denotes excess free probe.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2903484&req=5

pone-0011513-g006: Murine UP1B and UP2 promoters contain GATA-DNA binding sites which recruit complexes containing GATA4/6 in response to RA stimulation.Nuclear extracts from RA-treated ESCs following 9 d show distinct GATA-DNA complexes with 32P-labeled consensus oligos specific for GATA binding to 2 kb UP1B [A, C] or UP2 [B, D] promoter fragments. Complexes were absent in spontaneously differentiating controls (C). Both complexes were inhibited by addition of respective excess unlabeled wild type (wt) but not mutant GATA oligos (m1, m2). [C, D] The presence of GATA4 (G4) and GATA6 (G6) in both the GATA-UP complexes was determined by supershift or immunodepletion (ID) following antibody-specific incubation (G4, G6), but not species-matched isotype control antibodies (IgG). NS represents non specific band. FP denotes excess free probe.
Mentions: To determine the presence of putative GATA binding elements within the UP promoters, we analyzed a 2.0 kb fragment upstream from the transcriptional start site of each murine UP promoter sequence in silico using commercially available MatInspector software (Genomatix, Ann Arbor, MI) as previously described [63]. We identified one putative GATA-binding site within both the UP1B and UP2 promoter sequences at the following positions relative to the transcriptional start site: UP1B (−698 to −710) and UP2 (−1872 to −1885). In addition, promoter regions of the UP1A, UP3A, UP3B genes contained multiple putative GATA-binding sites. To determine whether GATA4/6 were recruited to the UP1B and UP2 promoters, we performed EMSA using nuclear extracts of RA-treated ESCs and spontaneously differentiating controls. As shown in Figure 6A and 6B, we observed robust complex formation with labeled probes corresponding to the GATA binding sites in both the UP1B and UP2 promoter sequences of RA-treated samples, while control nuclear extracts showed negligible binding. The complexes were competed out with the corresponding unlabeled UP1B or UP2 consensus GATA oligonucleotides, but not with mutant versions of these sequences. In addition, we observed supershift of the UP1B complex in the presence of GATA4 and GATA6 antibodies (Figure 6C), consistent with the presence of GATA4/6 in the complex. Supershift was also noted with the UP2 complex in the presence of GATA4 antibody, but not with IgG control; however the presence of GATA6 antibody resulted in substantial immunodepletion of the UP2 complex (Figure 6D) relative to IgG control. Together, these data suggest that RA-mediated signals promote recruitment of GATA4/6 to GATA binding sites within the UP1B/2 promoters during ESC specification.

Bottom Line: The urinary bladder and associated tract are lined by the urothelium, a transitional epithelium that acts as a specialized permeability barrier that protects the underlying tissue from urine via expression of a highly specific group of proteins known as the uroplakins (UP).Analysis of endodermal transcription factors such as GATA4 and GATA6 revealed significant upregulation and nuclear enrichment in RA-treated UP2-GFP+ populations.Collectively, these data suggest that RA mediates ESC specification toward a urothelial lineage via GATA4/6-dependent processes.

View Article: PubMed Central - PubMed

Affiliation: Urological Diseases Research Center, Children's Hospital Boston, Boston, Massachusetts, USA.

ABSTRACT
The urinary bladder and associated tract are lined by the urothelium, a transitional epithelium that acts as a specialized permeability barrier that protects the underlying tissue from urine via expression of a highly specific group of proteins known as the uroplakins (UP). To date, our understanding of the developmental processes responsible for urothelial differentiation has been hampered due to the lack of suitable models. In this study, we describe a novel in vitro cell culture system for derivation of urothelial cells from murine embryonic stem cells (ESCs) following cultivation on collagen matrices in the presence all trans retinoic acid (RA). Upon stimulation with micromolar concentrations of RA, ESCs significantly downregulated the pluripotency factor OCT-4 but markedly upregulated UP1A, UP1B, UP2, UP3A, and UP3B mRNA levels in comparison to naïve ESCs and spontaneously differentiating controls. Pan-UP protein expression was associated with both p63- and cytokeratin 20-positive cells in discrete aggregating populations of ESCs following 9 and 14 days of RA stimulation. Analysis of endodermal transcription factors such as GATA4 and GATA6 revealed significant upregulation and nuclear enrichment in RA-treated UP2-GFP+ populations. GATA4-/- and GATA6-/- transgenic ESC lines revealed substantial attenuation of RA-mediated UP expression in comparison to wild type controls. In addition, EMSA analysis revealed that RA treatment induced formation of transcriptional complexes containing GATA4/6 on both UP1B and UP2 promoter fragments containing putative GATA factor binding sites. Collectively, these data suggest that RA mediates ESC specification toward a urothelial lineage via GATA4/6-dependent processes.

Show MeSH
Related in: MedlinePlus