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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.

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RA stimulation of ESCs induces UP expression in a time and concentration-dependent manner.[A] Real time RT-PCR analysis of UP and OCT-4 expression by ESCs cultured on collagen matrices in the presence (+RA) or absence (C) of 10 µM RA for up to 9 d. (*) = p<0.05, in comparison to spontaneously differentiating controls and undifferentiated ESCs. [B] Effect of RA concentration on UP expression following 9 d. For [A–B], levels normalized to GAPDH expression. Mean ± SD per data point. [C] Photomicrographs of merged phase and GFP expression in spontaneously differentiating controls and RA-stimulated UP2-GFP ESC line following 14 d of cultivation. Scale bar = 80 µm. [D] Flow cytometry histograms comparing the extent of GFP expression in untransfected (WT) and UP2-GFP ESC lines either as spontaneously differentiating controls (left panel) or RA-treated cultures following 14 d (right panel). Extent of fluorescence intensity per amount of population events is displayed with shaded plots representing WT cultures and open plots representing UP2-GFP ESC line.
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pone-0011513-g001: RA stimulation of ESCs induces UP expression in a time and concentration-dependent manner.[A] Real time RT-PCR analysis of UP and OCT-4 expression by ESCs cultured on collagen matrices in the presence (+RA) or absence (C) of 10 µM RA for up to 9 d. (*) = p<0.05, in comparison to spontaneously differentiating controls and undifferentiated ESCs. [B] Effect of RA concentration on UP expression following 9 d. For [A–B], levels normalized to GAPDH expression. Mean ± SD per data point. [C] Photomicrographs of merged phase and GFP expression in spontaneously differentiating controls and RA-stimulated UP2-GFP ESC line following 14 d of cultivation. Scale bar = 80 µm. [D] Flow cytometry histograms comparing the extent of GFP expression in untransfected (WT) and UP2-GFP ESC lines either as spontaneously differentiating controls (left panel) or RA-treated cultures following 14 d (right panel). Extent of fluorescence intensity per amount of population events is displayed with shaded plots representing WT cultures and open plots representing UP2-GFP ESC line.

Mentions: RA is known to function in both a time- and concentration-dependent manner to selectively regulate lineage specification during ESC differentiation [27], [40], [41]. We first investigated the ability of continuous cultivation of ESCs in the presence of RA (0.01–10 µM) to stimulate urothelial marker expression. Real time RT-PCR analysis demonstrated that withdrawal of LIF as well as the addition of micromolar concentrations of RA led to the down-regulation of OCT-4 mRNA transcript levels in ESC cultures, indicating a progression toward differentiation (Figure 1A). In addition, RA acted synergistically with LIF withdrawal to promote pluripotency marker decline. RA stimulation increased mRNA transcript levels of all five uroplakins (UPs) over those seen in spontaneously differentiating controls and naïve ESCs by day 6 of cultivation with significant elevation occurring by 9 d and continuing to rise through 14 d of culture (Figure S1). The ability of RA to stimulate UP expression was concentration-dependent, with micromolar concentrations stimulating maximal extents of mRNA transcription over the 9 d time course (Figure 1B).


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)

RA stimulation of ESCs induces UP expression in a time and concentration-dependent manner.[A] Real time RT-PCR analysis of UP and OCT-4 expression by ESCs cultured on collagen matrices in the presence (+RA) or absence (C) of 10 µM RA for up to 9 d. (*) = p<0.05, in comparison to spontaneously differentiating controls and undifferentiated ESCs. [B] Effect of RA concentration on UP expression following 9 d. For [A–B], levels normalized to GAPDH expression. Mean ± SD per data point. [C] Photomicrographs of merged phase and GFP expression in spontaneously differentiating controls and RA-stimulated UP2-GFP ESC line following 14 d of cultivation. Scale bar = 80 µm. [D] Flow cytometry histograms comparing the extent of GFP expression in untransfected (WT) and UP2-GFP ESC lines either as spontaneously differentiating controls (left panel) or RA-treated cultures following 14 d (right panel). Extent of fluorescence intensity per amount of population events is displayed with shaded plots representing WT cultures and open plots representing UP2-GFP ESC line.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2903484&req=5

pone-0011513-g001: RA stimulation of ESCs induces UP expression in a time and concentration-dependent manner.[A] Real time RT-PCR analysis of UP and OCT-4 expression by ESCs cultured on collagen matrices in the presence (+RA) or absence (C) of 10 µM RA for up to 9 d. (*) = p<0.05, in comparison to spontaneously differentiating controls and undifferentiated ESCs. [B] Effect of RA concentration on UP expression following 9 d. For [A–B], levels normalized to GAPDH expression. Mean ± SD per data point. [C] Photomicrographs of merged phase and GFP expression in spontaneously differentiating controls and RA-stimulated UP2-GFP ESC line following 14 d of cultivation. Scale bar = 80 µm. [D] Flow cytometry histograms comparing the extent of GFP expression in untransfected (WT) and UP2-GFP ESC lines either as spontaneously differentiating controls (left panel) or RA-treated cultures following 14 d (right panel). Extent of fluorescence intensity per amount of population events is displayed with shaded plots representing WT cultures and open plots representing UP2-GFP ESC line.
Mentions: RA is known to function in both a time- and concentration-dependent manner to selectively regulate lineage specification during ESC differentiation [27], [40], [41]. We first investigated the ability of continuous cultivation of ESCs in the presence of RA (0.01–10 µM) to stimulate urothelial marker expression. Real time RT-PCR analysis demonstrated that withdrawal of LIF as well as the addition of micromolar concentrations of RA led to the down-regulation of OCT-4 mRNA transcript levels in ESC cultures, indicating a progression toward differentiation (Figure 1A). In addition, RA acted synergistically with LIF withdrawal to promote pluripotency marker decline. RA stimulation increased mRNA transcript levels of all five uroplakins (UPs) over those seen in spontaneously differentiating controls and naïve ESCs by day 6 of cultivation with significant elevation occurring by 9 d and continuing to rise through 14 d of culture (Figure S1). The ability of RA to stimulate UP expression was concentration-dependent, with micromolar concentrations stimulating maximal extents of mRNA transcription over the 9 d time course (Figure 1B).

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