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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 enrichment of UP+ populations coincides with markers of stratified urothelium.[A] Photomicrographs of adult murine bladder (C57BL/6) showing native urothelium organization and architecture. Left panel: immunofluorescence of pan-UP expression (red, Cy3) localized in urothelium. Scale bar = 500 µm. Right panel: immunofluorescence of nuclear p63 expression (red, Cy3) localized to the basal and intermediate urothelial layers (denoted with white arrow). Absence of nuclear p63 staining was noted in superficial urothelial cells (denoted with yellow arrow). Scale bar = 100 µm. For both panels, images were merged with DAPI nuclear counterstain (blue). [B] Photomicrographs of spontaneously differentiating control and RA-treated ESCs following 9 d of cultivation showing acquisition of epithelial morphology and co-localization of UP (green, FITC) and nuclear p63 expression (red, Cy3). Pan-UP+ populations merged with DAPI were observed with (denoted with white arrow) and without (denoted with yellow arrow) p63+ nuclear staining. Scale bar = 30 µm. [C] Photomicrographs of phase and immunofluorescence fields of RA-treated ESCs following 14 d of cultivation. Samples demonstrated 3-D cell aggregates coupled with pan-UP expression (red, Cy3). DAPI nuclear counterstain (blue). Scale bar = 100 µm. [D] Real time RT-PCR analysis of CK18 and CK20 expression in cultures described in [B]. Levels normalized to GAPDH expression. Mean ± SD per data point. [E] Photomicrographs of immunofluorescence fields of RA-treated UP2-GFP ESCs following 14 d of cultivation demonstrated GFP expression colocalized with CK20 expression (red, Cy3). Cells exhibiting co-staining of GFP and Cy3 are denoted with orange arrows. DAPI nuclear counterstain (blue). Scale bar = 60 µm.
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pone-0011513-g002: RA enrichment of UP+ populations coincides with markers of stratified urothelium.[A] Photomicrographs of adult murine bladder (C57BL/6) showing native urothelium organization and architecture. Left panel: immunofluorescence of pan-UP expression (red, Cy3) localized in urothelium. Scale bar = 500 µm. Right panel: immunofluorescence of nuclear p63 expression (red, Cy3) localized to the basal and intermediate urothelial layers (denoted with white arrow). Absence of nuclear p63 staining was noted in superficial urothelial cells (denoted with yellow arrow). Scale bar = 100 µm. For both panels, images were merged with DAPI nuclear counterstain (blue). [B] Photomicrographs of spontaneously differentiating control and RA-treated ESCs following 9 d of cultivation showing acquisition of epithelial morphology and co-localization of UP (green, FITC) and nuclear p63 expression (red, Cy3). Pan-UP+ populations merged with DAPI were observed with (denoted with white arrow) and without (denoted with yellow arrow) p63+ nuclear staining. Scale bar = 30 µm. [C] Photomicrographs of phase and immunofluorescence fields of RA-treated ESCs following 14 d of cultivation. Samples demonstrated 3-D cell aggregates coupled with pan-UP expression (red, Cy3). DAPI nuclear counterstain (blue). Scale bar = 100 µm. [D] Real time RT-PCR analysis of CK18 and CK20 expression in cultures described in [B]. Levels normalized to GAPDH expression. Mean ± SD per data point. [E] Photomicrographs of immunofluorescence fields of RA-treated UP2-GFP ESCs following 14 d of cultivation demonstrated GFP expression colocalized with CK20 expression (red, Cy3). Cells exhibiting co-staining of GFP and Cy3 are denoted with orange arrows. DAPI nuclear counterstain (blue). Scale bar = 60 µm.

Mentions: IHC analysis of UP protein expression in the adult murine bladder demonstrated specific localization throughout each layer of the urothelium with maximal expression observed in the apical layer of the superficial umbrella cells lining the lumen (Figure 2A). Previous studies have reported the expression of UPs and their assembly into AUM is essential for the maintenance of urothelial barrier function [15]–[18]. Transgenic mice lacking the basal cell marker p63 present severe epithelial defects, including epidermis and prostate bud agenesis and loss of basal and intermediate urothelial cell layers [42]. Our results demonstrated nuclear p63 expression localized to the urothelium of the murine bladder with detection restricted to basal and intermediate cell layers. These data were consistent with previous observations of urothelial marker distribution in the murine bladder [15]–[16], [42].


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 enrichment of UP+ populations coincides with markers of stratified urothelium.[A] Photomicrographs of adult murine bladder (C57BL/6) showing native urothelium organization and architecture. Left panel: immunofluorescence of pan-UP expression (red, Cy3) localized in urothelium. Scale bar = 500 µm. Right panel: immunofluorescence of nuclear p63 expression (red, Cy3) localized to the basal and intermediate urothelial layers (denoted with white arrow). Absence of nuclear p63 staining was noted in superficial urothelial cells (denoted with yellow arrow). Scale bar = 100 µm. For both panels, images were merged with DAPI nuclear counterstain (blue). [B] Photomicrographs of spontaneously differentiating control and RA-treated ESCs following 9 d of cultivation showing acquisition of epithelial morphology and co-localization of UP (green, FITC) and nuclear p63 expression (red, Cy3). Pan-UP+ populations merged with DAPI were observed with (denoted with white arrow) and without (denoted with yellow arrow) p63+ nuclear staining. Scale bar = 30 µm. [C] Photomicrographs of phase and immunofluorescence fields of RA-treated ESCs following 14 d of cultivation. Samples demonstrated 3-D cell aggregates coupled with pan-UP expression (red, Cy3). DAPI nuclear counterstain (blue). Scale bar = 100 µm. [D] Real time RT-PCR analysis of CK18 and CK20 expression in cultures described in [B]. Levels normalized to GAPDH expression. Mean ± SD per data point. [E] Photomicrographs of immunofluorescence fields of RA-treated UP2-GFP ESCs following 14 d of cultivation demonstrated GFP expression colocalized with CK20 expression (red, Cy3). Cells exhibiting co-staining of GFP and Cy3 are denoted with orange arrows. DAPI nuclear counterstain (blue). Scale bar = 60 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2903484&req=5

pone-0011513-g002: RA enrichment of UP+ populations coincides with markers of stratified urothelium.[A] Photomicrographs of adult murine bladder (C57BL/6) showing native urothelium organization and architecture. Left panel: immunofluorescence of pan-UP expression (red, Cy3) localized in urothelium. Scale bar = 500 µm. Right panel: immunofluorescence of nuclear p63 expression (red, Cy3) localized to the basal and intermediate urothelial layers (denoted with white arrow). Absence of nuclear p63 staining was noted in superficial urothelial cells (denoted with yellow arrow). Scale bar = 100 µm. For both panels, images were merged with DAPI nuclear counterstain (blue). [B] Photomicrographs of spontaneously differentiating control and RA-treated ESCs following 9 d of cultivation showing acquisition of epithelial morphology and co-localization of UP (green, FITC) and nuclear p63 expression (red, Cy3). Pan-UP+ populations merged with DAPI were observed with (denoted with white arrow) and without (denoted with yellow arrow) p63+ nuclear staining. Scale bar = 30 µm. [C] Photomicrographs of phase and immunofluorescence fields of RA-treated ESCs following 14 d of cultivation. Samples demonstrated 3-D cell aggregates coupled with pan-UP expression (red, Cy3). DAPI nuclear counterstain (blue). Scale bar = 100 µm. [D] Real time RT-PCR analysis of CK18 and CK20 expression in cultures described in [B]. Levels normalized to GAPDH expression. Mean ± SD per data point. [E] Photomicrographs of immunofluorescence fields of RA-treated UP2-GFP ESCs following 14 d of cultivation demonstrated GFP expression colocalized with CK20 expression (red, Cy3). Cells exhibiting co-staining of GFP and Cy3 are denoted with orange arrows. DAPI nuclear counterstain (blue). Scale bar = 60 µm.
Mentions: IHC analysis of UP protein expression in the adult murine bladder demonstrated specific localization throughout each layer of the urothelium with maximal expression observed in the apical layer of the superficial umbrella cells lining the lumen (Figure 2A). Previous studies have reported the expression of UPs and their assembly into AUM is essential for the maintenance of urothelial barrier function [15]–[18]. Transgenic mice lacking the basal cell marker p63 present severe epithelial defects, including epidermis and prostate bud agenesis and loss of basal and intermediate urothelial cell layers [42]. Our results demonstrated nuclear p63 expression localized to the urothelium of the murine bladder with detection restricted to basal and intermediate cell layers. These data were consistent with previous observations of urothelial marker distribution in the murine bladder [15]–[16], [42].

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