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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 induction of UP expression is correlated with markers of hindgut definitive endoderm (DE), in contrast to markers of the extraembryonic endoderm (ExE).[A, B] Real time RT-PCR analysis of endoderm marker expression by ESCs cultured on collagen matrices in the presence (+RA) or absence (C) of 10 µM RA for up to 9 d. [C] Effect of RA concentration on ESC differentiation markers of ExE or hindgut DE, as well as various RA-responsive lineages following 9 d of stimulation. For all panels, levels were normalized to GAPDH expression. N = 3–4 per data point.
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pone-0011513-g003: RA induction of UP expression is correlated with markers of hindgut definitive endoderm (DE), in contrast to markers of the extraembryonic endoderm (ExE).[A, B] Real time RT-PCR analysis of endoderm marker expression by ESCs cultured on collagen matrices in the presence (+RA) or absence (C) of 10 µM RA for up to 9 d. [C] Effect of RA concentration on ESC differentiation markers of ExE or hindgut DE, as well as various RA-responsive lineages following 9 d of stimulation. For all panels, levels were normalized to GAPDH expression. N = 3–4 per data point.

Mentions: RA is known to promote differentiation of murine ESCs toward particular endodermal phenotypes via stimulation of transcriptional networks that mediate embryonic patterning in vivo. In order to evaluate potential pathways and transcriptional mediators responsible for urothelial specification, we first assessed the temporal expression of early endoderm transcription factors, SOX17 [44] and its associated downstream targets, FOXA1 [45] and FOXA2 [46] (Figure 3A), as well as GATA 4/5/6 (Figure 3B) in relation to the onset (6–9 d) of RA-mediated UP expression.


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 induction of UP expression is correlated with markers of hindgut definitive endoderm (DE), in contrast to markers of the extraembryonic endoderm (ExE).[A, B] Real time RT-PCR analysis of endoderm marker expression by ESCs cultured on collagen matrices in the presence (+RA) or absence (C) of 10 µM RA for up to 9 d. [C] Effect of RA concentration on ESC differentiation markers of ExE or hindgut DE, as well as various RA-responsive lineages following 9 d of stimulation. For all panels, levels were normalized to GAPDH expression. N = 3–4 per data point.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0011513-g003: RA induction of UP expression is correlated with markers of hindgut definitive endoderm (DE), in contrast to markers of the extraembryonic endoderm (ExE).[A, B] Real time RT-PCR analysis of endoderm marker expression by ESCs cultured on collagen matrices in the presence (+RA) or absence (C) of 10 µM RA for up to 9 d. [C] Effect of RA concentration on ESC differentiation markers of ExE or hindgut DE, as well as various RA-responsive lineages following 9 d of stimulation. For all panels, levels were normalized to GAPDH expression. N = 3–4 per data point.
Mentions: RA is known to promote differentiation of murine ESCs toward particular endodermal phenotypes via stimulation of transcriptional networks that mediate embryonic patterning in vivo. In order to evaluate potential pathways and transcriptional mediators responsible for urothelial specification, we first assessed the temporal expression of early endoderm transcription factors, SOX17 [44] and its associated downstream targets, FOXA1 [45] and FOXA2 [46] (Figure 3A), as well as GATA 4/5/6 (Figure 3B) in relation to the onset (6–9 d) of RA-mediated UP expression.

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