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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.


Immunofluorescence for PROP1 on e12.5 sagittal sections.Immunostaining with specific antibody against PROP1 in wild-type and Prop1-/- embryos. Cell nuclei were stained with DAPI (blue).DOI:http://dx.doi.org/10.7554/eLife.14470.006
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fig3s1: Immunofluorescence for PROP1 on e12.5 sagittal sections.Immunostaining with specific antibody against PROP1 in wild-type and Prop1-/- embryos. Cell nuclei were stained with DAPI (blue).DOI:http://dx.doi.org/10.7554/eLife.14470.006

Mentions: To further analyze the expression of Prop1 and stem cell markers postnatally, we performed double immunohistochemistry using antibodies against PROP1, SOX2 and SOX9. The missense mutation in Prop1 mutant mice does not lead to enhanced protein degradation (Gage et al., 1996), and the mutated protein can be detect using the PROP1 antibody directed against the N terminus. We characterized the specificity of the PROP1 antibody by performing immunofluorescence for PROP1 on e12.5 embryos from wild-type and Prop1-/- mice, which carry a deletion in the Prop1 gene and are not expected to produce a stable protein (Figure 3—figure supplement 1) (Nasonkin et al., 2004). As expected, strong, nuclear PROP1 immunoreactivity is detected throughout the middle zone of Rathke’s pouch in wild-type embryos. No PROP1 staining was detected in the pituitaries of Prop1-/- mice, confirming the specificity of the antibody (Figure 3—figure supplement 1). The expansion of SOX2-positive cells in Prop1df/df pituitaries correlates with an expansion of Prop1-expressing cells in the marginal zone (Figure 3A and C). The expanded population of SOX2-expressing cells in Prop1df/df dwarf pituitaries exhibit less co-localization with SOX9 relative to controls (Figure 3B and D). Overall, these results suggest that Prop1 is crucial to promote stem cell engagement in the postnatal pituitary.10.7554/eLife.14470.005Figure 3.Prop1 regulates Sox2+ stem cell population.


PROP1 triggers epithelial-mesenchymal transition-like process in pituitary stem cells
Immunofluorescence for PROP1 on e12.5 sagittal sections.Immunostaining with specific antibody against PROP1 in wild-type and Prop1-/- embryos. Cell nuclei were stained with DAPI (blue).DOI:http://dx.doi.org/10.7554/eLife.14470.006
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4940164&req=5

fig3s1: Immunofluorescence for PROP1 on e12.5 sagittal sections.Immunostaining with specific antibody against PROP1 in wild-type and Prop1-/- embryos. Cell nuclei were stained with DAPI (blue).DOI:http://dx.doi.org/10.7554/eLife.14470.006
Mentions: To further analyze the expression of Prop1 and stem cell markers postnatally, we performed double immunohistochemistry using antibodies against PROP1, SOX2 and SOX9. The missense mutation in Prop1 mutant mice does not lead to enhanced protein degradation (Gage et al., 1996), and the mutated protein can be detect using the PROP1 antibody directed against the N terminus. We characterized the specificity of the PROP1 antibody by performing immunofluorescence for PROP1 on e12.5 embryos from wild-type and Prop1-/- mice, which carry a deletion in the Prop1 gene and are not expected to produce a stable protein (Figure 3—figure supplement 1) (Nasonkin et al., 2004). As expected, strong, nuclear PROP1 immunoreactivity is detected throughout the middle zone of Rathke’s pouch in wild-type embryos. No PROP1 staining was detected in the pituitaries of Prop1-/- mice, confirming the specificity of the antibody (Figure 3—figure supplement 1). The expansion of SOX2-positive cells in Prop1df/df pituitaries correlates with an expansion of Prop1-expressing cells in the marginal zone (Figure 3A and C). The expanded population of SOX2-expressing cells in Prop1df/df dwarf pituitaries exhibit less co-localization with SOX9 relative to controls (Figure 3B and D). Overall, these results suggest that Prop1 is crucial to promote stem cell engagement in the postnatal pituitary.10.7554/eLife.14470.005Figure 3.Prop1 regulates Sox2+ stem cell population.

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.