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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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GATA4 and GATA6 are crucial signaling molecules in RA-mediated upregulation of UP expression in ESCs.Real time RT-PCR analysis of uroplakin expression in WT, GATA4−/− [A] or GATA6−/− [B] ESCs cultured in the presence (+RA) or absence (C) of 10 µM RA for up to 9 d. ESC(0)  =  undifferentiated ESCs. Levels normalized to GAPDH expression. Mean ± SD per data point. N = 3–4 per data point. (*) = p<0.05, in comparison to levels observed with RA-treated WT cultures.
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pone-0011513-g005: GATA4 and GATA6 are crucial signaling molecules in RA-mediated upregulation of UP expression in ESCs.Real time RT-PCR analysis of uroplakin expression in WT, GATA4−/− [A] or GATA6−/− [B] ESCs cultured in the presence (+RA) or absence (C) of 10 µM RA for up to 9 d. ESC(0)  =  undifferentiated ESCs. Levels normalized to GAPDH expression. Mean ± SD per data point. N = 3–4 per data point. (*) = p<0.05, in comparison to levels observed with RA-treated WT cultures.

Mentions: Previous reports have demonstrated that knockout mice deficient in either GATA4 or 6 are embryonic lethal at E6.5–8, prior to the onset of bladder specification due to deficiencies in extraembryonic tissue formation [28], [34]. To determine the role of GATA4/6 in the regulation of markers associated with urothelial differentiation, GATA4−/− and GATA6−/− homozygous ESCs were subjected to RA stimulation (10 µM) for 9 d and compared to wild type (WT) controls. Real time RT-PCR analysis revealed that mutant ESC lines were capable of expressing low baseline levels of certain UPs similar to WT controls. However, RA-treated GATA4−/− ESCs failed to enhance UP1B and UP2 expression in response to RA treatment as compared to unstimulated controls (Figure 5A). In addition, UP1A and UP3A expression were significantly attenuated compared to wild type cultures stimulated with RA in parallel. GATA6−/− ESCs failed to upregulate mRNA transcript levels of any of the major UPs (Figure 5B). These results demonstrate that GATA4/6 transcription factors play essential roles in regulating UP expression in RA-stimulated ESCs.


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)

GATA4 and GATA6 are crucial signaling molecules in RA-mediated upregulation of UP expression in ESCs.Real time RT-PCR analysis of uroplakin expression in WT, GATA4−/− [A] or GATA6−/− [B] ESCs cultured in the presence (+RA) or absence (C) of 10 µM RA for up to 9 d. ESC(0)  =  undifferentiated ESCs. Levels normalized to GAPDH expression. Mean ± SD per data point. N = 3–4 per data point. (*) = p<0.05, in comparison to levels observed with RA-treated WT cultures.
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Related In: Results  -  Collection

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

pone-0011513-g005: GATA4 and GATA6 are crucial signaling molecules in RA-mediated upregulation of UP expression in ESCs.Real time RT-PCR analysis of uroplakin expression in WT, GATA4−/− [A] or GATA6−/− [B] ESCs cultured in the presence (+RA) or absence (C) of 10 µM RA for up to 9 d. ESC(0)  =  undifferentiated ESCs. Levels normalized to GAPDH expression. Mean ± SD per data point. N = 3–4 per data point. (*) = p<0.05, in comparison to levels observed with RA-treated WT cultures.
Mentions: Previous reports have demonstrated that knockout mice deficient in either GATA4 or 6 are embryonic lethal at E6.5–8, prior to the onset of bladder specification due to deficiencies in extraembryonic tissue formation [28], [34]. To determine the role of GATA4/6 in the regulation of markers associated with urothelial differentiation, GATA4−/− and GATA6−/− homozygous ESCs were subjected to RA stimulation (10 µM) for 9 d and compared to wild type (WT) controls. Real time RT-PCR analysis revealed that mutant ESC lines were capable of expressing low baseline levels of certain UPs similar to WT controls. However, RA-treated GATA4−/− ESCs failed to enhance UP1B and UP2 expression in response to RA treatment as compared to unstimulated controls (Figure 5A). In addition, UP1A and UP3A expression were significantly attenuated compared to wild type cultures stimulated with RA in parallel. GATA6−/− ESCs failed to upregulate mRNA transcript levels of any of the major UPs (Figure 5B). These results demonstrate that GATA4/6 transcription factors play essential roles in regulating UP expression in RA-stimulated ESCs.

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