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PROP1 triggers epithelial-mesenchymal transition-like process in pituitary stem cells

View Article: PubMed Central - PubMed

ABSTRACT

Mutations in PROP1 are the most common cause of hypopituitarism in humans; therefore, unraveling its mechanism of action is highly relevant from a therapeutic perspective. Our current understanding of the role of PROP1 in the pituitary gland is limited to the repression and activation of the pituitary transcription factor genes Hesx1 and Pou1f1, respectively. To elucidate the comprehensive PROP1-dependent gene regulatory network, we conducted genome-wide analysis of PROP1 DNA binding and effects on gene expression in mutant mice, mouse isolated stem cells and engineered mouse cell lines. We determined that PROP1 is essential for stimulating stem cells to undergo an epithelial to mesenchymal transition-like process necessary for cell migration and differentiation. Genomic profiling reveals that PROP1 binds to genes expressed in epithelial cells like Claudin 23, and to EMT inducer genes like Zeb2, Notch2 and Gli2. Zeb2 activation appears to be a key step in the EMT process. Our findings identify PROP1 as a central transcriptional component of pituitary stem cell differentiation.

Doi:: http://dx.doi.org/10.7554/eLife.14470.001

No MeSH data available.


Related in: MedlinePlus

Pituitary stem-cell-derived colonies from Prop1 and Pou1f1 mutant pituitaries.Immunostaining with specific antibodies performed on fixed colonies show expression of stem cell markers, SOX2, SOX9 and GFRa2 and CYCLIN D2. No differences were found between genotypes. Cell nuclei were stained with DAPI (blue).DOI:http://dx.doi.org/10.7554/eLife.14470.008
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fig4s1: Pituitary stem-cell-derived colonies from Prop1 and Pou1f1 mutant pituitaries.Immunostaining with specific antibodies performed on fixed colonies show expression of stem cell markers, SOX2, SOX9 and GFRa2 and CYCLIN D2. No differences were found between genotypes. Cell nuclei were stained with DAPI (blue).DOI:http://dx.doi.org/10.7554/eLife.14470.008

Mentions: Adherent colonies arising from single stem cells can be cultured from postnatal and adult pituitaries in vitro. These colonies represent a mixture of stem cells and cells that have engaged in the transitioning process to differentiation (Andoniadou et al., 2013). We analyzed the number of colony-forming cells (CFCs) per pituitary in normal and mutant mice postnatally, as a measure of the stem cell reserve and engagement (Gaston-Massuet et al., 2011). We demonstrated that both mutants, Prop1df/df and Pou1f1dw/dw, can generate progenitor CFCs in culture that express stem cell markers, such as SOX2, SOX9, GFRA2 and CYCLIN D2 by immunostaining (Figure 4—figure supplement 1). The number of CFC per pituitary at postnatal days 7, 13 and 38 is reduced in Prop1 mutant relative to wild-type pituitaries (Figure 4A, left panel). Colonies from Prop1df/df mice differ qualitatively from those of normal mice. Wild-type mice produce colonies that have two visually distinct types. About half of the colonies are comprised of cells that are compact and piled up, which is typical for undifferentiated progenitors (Gaston-Massuet et al., 2011). The rest of the wild-type colonies are comprised of large, flat cells, but 100% of Prop1 mutant colonies are the flat cell type (Figure 4B and C, left panel). After 4 days in culture, the difference in morphology in the Prop1 mutant colonies is already apparent (Figure 4D). In contrast to Prop1df/df, the pituitaries from Pou1f1dw/dw mice produced similar number of colonies per pituitary gland at all ages analyzed relative to wild types, and their morphology was representative of wild type (Figure 4A–C right panels). Together, these studies demonstrate that Prop1 is necessary to maintain normal stem cell pools during postnatal pituitary maturation and adulthood.10.7554/eLife.14470.007Figure 4.Prop1 regulates pituitary stem cells after birth.


PROP1 triggers epithelial-mesenchymal transition-like process in pituitary stem cells
Pituitary stem-cell-derived colonies from Prop1 and Pou1f1 mutant pituitaries.Immunostaining with specific antibodies performed on fixed colonies show expression of stem cell markers, SOX2, SOX9 and GFRa2 and CYCLIN D2. No differences were found between genotypes. Cell nuclei were stained with DAPI (blue).DOI:http://dx.doi.org/10.7554/eLife.14470.008
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4940164&req=5

fig4s1: Pituitary stem-cell-derived colonies from Prop1 and Pou1f1 mutant pituitaries.Immunostaining with specific antibodies performed on fixed colonies show expression of stem cell markers, SOX2, SOX9 and GFRa2 and CYCLIN D2. No differences were found between genotypes. Cell nuclei were stained with DAPI (blue).DOI:http://dx.doi.org/10.7554/eLife.14470.008
Mentions: Adherent colonies arising from single stem cells can be cultured from postnatal and adult pituitaries in vitro. These colonies represent a mixture of stem cells and cells that have engaged in the transitioning process to differentiation (Andoniadou et al., 2013). We analyzed the number of colony-forming cells (CFCs) per pituitary in normal and mutant mice postnatally, as a measure of the stem cell reserve and engagement (Gaston-Massuet et al., 2011). We demonstrated that both mutants, Prop1df/df and Pou1f1dw/dw, can generate progenitor CFCs in culture that express stem cell markers, such as SOX2, SOX9, GFRA2 and CYCLIN D2 by immunostaining (Figure 4—figure supplement 1). The number of CFC per pituitary at postnatal days 7, 13 and 38 is reduced in Prop1 mutant relative to wild-type pituitaries (Figure 4A, left panel). Colonies from Prop1df/df mice differ qualitatively from those of normal mice. Wild-type mice produce colonies that have two visually distinct types. About half of the colonies are comprised of cells that are compact and piled up, which is typical for undifferentiated progenitors (Gaston-Massuet et al., 2011). The rest of the wild-type colonies are comprised of large, flat cells, but 100% of Prop1 mutant colonies are the flat cell type (Figure 4B and C, left panel). After 4 days in culture, the difference in morphology in the Prop1 mutant colonies is already apparent (Figure 4D). In contrast to Prop1df/df, the pituitaries from Pou1f1dw/dw mice produced similar number of colonies per pituitary gland at all ages analyzed relative to wild types, and their morphology was representative of wild type (Figure 4A–C right panels). Together, these studies demonstrate that Prop1 is necessary to maintain normal stem cell pools during postnatal pituitary maturation and adulthood.10.7554/eLife.14470.007Figure 4.Prop1 regulates pituitary stem cells after birth.

View Article: PubMed Central - PubMed

ABSTRACT

Mutations in PROP1 are the most common cause of hypopituitarism in humans; therefore, unraveling its mechanism of action is highly relevant from a therapeutic perspective. Our current understanding of the role of PROP1 in the pituitary gland is limited to the repression and activation of the pituitary transcription factor genes Hesx1 and Pou1f1, respectively. To elucidate the comprehensive PROP1-dependent gene regulatory network, we conducted genome-wide analysis of PROP1 DNA binding and effects on gene expression in mutant mice, mouse isolated stem cells and engineered mouse cell lines. We determined that PROP1 is essential for stimulating stem cells to undergo an epithelial to mesenchymal transition-like process necessary for cell migration and differentiation. Genomic profiling reveals that PROP1 binds to genes expressed in epithelial cells like Claudin 23, and to EMT inducer genes like Zeb2, Notch2 and Gli2. Zeb2 activation appears to be a key step in the EMT process. Our findings identify PROP1 as a central transcriptional component of pituitary stem cell differentiation.

Doi:: http://dx.doi.org/10.7554/eLife.14470.001

No MeSH data available.


Related in: MedlinePlus