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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

Characterization of Prop1-biotag transgenic mice.(A) Pituitary paraffin sections were prepared from e12.5 embryos of the genotype indicated and immunohistochemistry was performed. A polyclonal antibody against PROP1 detects the PROP1 protein in the pituitary primordium - Rathke’s Pouch (upper panel). The transgenic PROP1-biotag protein can be detected with fluorescein-conjugated streptavidin only when is co-expressed with BirA (lower panel). (B) Body weight at P21. Prop1-/- mice are dwarfed, but the presence of the Prop1-biotag transgene alone or co-expressed with BirA rescues the dwarf phenotype. The mean of body weight of mice of each genotype is graphed. Error bars represent the standard deviation. (n = 25–40) ANOVA *p<0.05. (C) Dysmorphology characteristic of the Prop1 mutant pituitary gland is rescued by the biotin-tagged Prop1 transgene. Upper panel: Hematoxylin and eosin staining of e16.5 embryos. Lower panel: Immunostaining with POU1F1-1 antibody at e16.5 paraffin sections. Scale bars, 100 µm.DOI:http://dx.doi.org/10.7554/eLife.14470.014
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fig8s1: Characterization of Prop1-biotag transgenic mice.(A) Pituitary paraffin sections were prepared from e12.5 embryos of the genotype indicated and immunohistochemistry was performed. A polyclonal antibody against PROP1 detects the PROP1 protein in the pituitary primordium - Rathke’s Pouch (upper panel). The transgenic PROP1-biotag protein can be detected with fluorescein-conjugated streptavidin only when is co-expressed with BirA (lower panel). (B) Body weight at P21. Prop1-/- mice are dwarfed, but the presence of the Prop1-biotag transgene alone or co-expressed with BirA rescues the dwarf phenotype. The mean of body weight of mice of each genotype is graphed. Error bars represent the standard deviation. (n = 25–40) ANOVA *p<0.05. (C) Dysmorphology characteristic of the Prop1 mutant pituitary gland is rescued by the biotin-tagged Prop1 transgene. Upper panel: Hematoxylin and eosin staining of e16.5 embryos. Lower panel: Immunostaining with POU1F1-1 antibody at e16.5 paraffin sections. Scale bars, 100 µm.DOI:http://dx.doi.org/10.7554/eLife.14470.014

Mentions: To confirm that the Prop1Tag protein has normal function, we generated transgenic mice expressing the Prop1Tag construct, and carried out the appropriate crosses with Rosa26BirA/BirA transgenics and Prop1+/- mice to generate Prop1Tag, Rosa26BirA, Prop1-/- mice. Mice with the latter genotype grew normally, confirming that biotinylated PROP1 is active and functional and able to correct the dwarfism characteristic of Prop1-/- mutants (Figure 8—figure supplement 1). The expression of Pit1 and pituitary dysmorphology were also rescued during development by the Prop1Tag transgene (Figure 8—figure supplement 1).


PROP1 triggers epithelial-mesenchymal transition-like process in pituitary stem cells
Characterization of Prop1-biotag transgenic mice.(A) Pituitary paraffin sections were prepared from e12.5 embryos of the genotype indicated and immunohistochemistry was performed. A polyclonal antibody against PROP1 detects the PROP1 protein in the pituitary primordium - Rathke’s Pouch (upper panel). The transgenic PROP1-biotag protein can be detected with fluorescein-conjugated streptavidin only when is co-expressed with BirA (lower panel). (B) Body weight at P21. Prop1-/- mice are dwarfed, but the presence of the Prop1-biotag transgene alone or co-expressed with BirA rescues the dwarf phenotype. The mean of body weight of mice of each genotype is graphed. Error bars represent the standard deviation. (n = 25–40) ANOVA *p<0.05. (C) Dysmorphology characteristic of the Prop1 mutant pituitary gland is rescued by the biotin-tagged Prop1 transgene. Upper panel: Hematoxylin and eosin staining of e16.5 embryos. Lower panel: Immunostaining with POU1F1-1 antibody at e16.5 paraffin sections. Scale bars, 100 µm.DOI:http://dx.doi.org/10.7554/eLife.14470.014
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Related In: Results  -  Collection

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fig8s1: Characterization of Prop1-biotag transgenic mice.(A) Pituitary paraffin sections were prepared from e12.5 embryos of the genotype indicated and immunohistochemistry was performed. A polyclonal antibody against PROP1 detects the PROP1 protein in the pituitary primordium - Rathke’s Pouch (upper panel). The transgenic PROP1-biotag protein can be detected with fluorescein-conjugated streptavidin only when is co-expressed with BirA (lower panel). (B) Body weight at P21. Prop1-/- mice are dwarfed, but the presence of the Prop1-biotag transgene alone or co-expressed with BirA rescues the dwarf phenotype. The mean of body weight of mice of each genotype is graphed. Error bars represent the standard deviation. (n = 25–40) ANOVA *p<0.05. (C) Dysmorphology characteristic of the Prop1 mutant pituitary gland is rescued by the biotin-tagged Prop1 transgene. Upper panel: Hematoxylin and eosin staining of e16.5 embryos. Lower panel: Immunostaining with POU1F1-1 antibody at e16.5 paraffin sections. Scale bars, 100 µm.DOI:http://dx.doi.org/10.7554/eLife.14470.014
Mentions: To confirm that the Prop1Tag protein has normal function, we generated transgenic mice expressing the Prop1Tag construct, and carried out the appropriate crosses with Rosa26BirA/BirA transgenics and Prop1+/- mice to generate Prop1Tag, Rosa26BirA, Prop1-/- mice. Mice with the latter genotype grew normally, confirming that biotinylated PROP1 is active and functional and able to correct the dwarfism characteristic of Prop1-/- mutants (Figure 8—figure supplement 1). The expression of Pit1 and pituitary dysmorphology were also rescued during development by the Prop1Tag transgene (Figure 8—figure supplement 1).

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