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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

Loss of Prop1 affects EMT pathway genes in pituitary tissues.(A) Upper panel:In situ hybridization using a Zeb2 antisense riboprobe at e14.5 detected expression in the control pituitary. Most of the positive cells are in the ventral area, around the cleft (between the lines). In Prop1df/df pituitaries very few cells are expressing Zeb2. Lower panel: Sagittal sections of 14.5 embryos were stained for CDH1 (green) and CDH2 (red) by immunohistochemistry. In the wild-type pituitary, CDH1 expression is dispersed throughout the Rathke’s Pouch, while CDH2 expression is more concentrated in the rostral area and in the forming anterior lobe. In Prop1 mutant pituitaries, CDH1 expression is increased and concentrated in the area ventral to the lumen, where Zeb2 expression was absent. White boxes indicate where higher magnification photos were taken. (B) Immunohistochemestry for CDH1 at P13 shows that Prop1df/df pituitaries have an expanded expression of this endothelial protein in the anterior lobe compared to control pituitaries. White boxes indicate where higher magnification photos were taken. Cell nuclei were stained with DAPI (blue). Scale bar 100 µm.DOI:http://dx.doi.org/10.7554/eLife.14470.011
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fig7: Loss of Prop1 affects EMT pathway genes in pituitary tissues.(A) Upper panel:In situ hybridization using a Zeb2 antisense riboprobe at e14.5 detected expression in the control pituitary. Most of the positive cells are in the ventral area, around the cleft (between the lines). In Prop1df/df pituitaries very few cells are expressing Zeb2. Lower panel: Sagittal sections of 14.5 embryos were stained for CDH1 (green) and CDH2 (red) by immunohistochemistry. In the wild-type pituitary, CDH1 expression is dispersed throughout the Rathke’s Pouch, while CDH2 expression is more concentrated in the rostral area and in the forming anterior lobe. In Prop1 mutant pituitaries, CDH1 expression is increased and concentrated in the area ventral to the lumen, where Zeb2 expression was absent. White boxes indicate where higher magnification photos were taken. (B) Immunohistochemestry for CDH1 at P13 shows that Prop1df/df pituitaries have an expanded expression of this endothelial protein in the anterior lobe compared to control pituitaries. White boxes indicate where higher magnification photos were taken. Cell nuclei were stained with DAPI (blue). Scale bar 100 µm.DOI:http://dx.doi.org/10.7554/eLife.14470.011

Mentions: We examined expression of the EMT inducer Zeb2 and cadherins during pituitary development in control and Prop1df/df mice (Figure 7A). At e14.5, Zeb2 is normally expressed in cells in the ventral part of the Rathke’s Pouch (Figure 7A, between the two lines), and this zone of enriched expression is almost absent in Prop1 mutant pituitaries. CDH1 is normally expressed throughout the Rathke’s pouch, but CDH1 staining is stronger in the ventral part of the Prop1 mutant pouch, particularly along the marginal zone (Figure 7A, box and higher magnification image), although there was no obvious difference in expression of the mesenchymal marker, N-CADHERIN or CDH2. Next, we explored CDH1 expression at P13, the time point at which the CFCs were collected for subsequent RNA-Seq analysis. We found that Prop1df/df mutants have abnormal expression of CDH1 in cells distant from the marginal zone. Taken together, these results show that Prop1 is necessary for suppressing SOX2 expression in stem cells and for driving them to undergo an EMT-like process critical for populating the anterior lobe with progenitors capable of differentiating into the PIT1 lineage.10.7554/eLife.14470.011Figure 7.Loss of Prop1 affects EMT pathway genes in pituitary tissues.


PROP1 triggers epithelial-mesenchymal transition-like process in pituitary stem cells
Loss of Prop1 affects EMT pathway genes in pituitary tissues.(A) Upper panel:In situ hybridization using a Zeb2 antisense riboprobe at e14.5 detected expression in the control pituitary. Most of the positive cells are in the ventral area, around the cleft (between the lines). In Prop1df/df pituitaries very few cells are expressing Zeb2. Lower panel: Sagittal sections of 14.5 embryos were stained for CDH1 (green) and CDH2 (red) by immunohistochemistry. In the wild-type pituitary, CDH1 expression is dispersed throughout the Rathke’s Pouch, while CDH2 expression is more concentrated in the rostral area and in the forming anterior lobe. In Prop1 mutant pituitaries, CDH1 expression is increased and concentrated in the area ventral to the lumen, where Zeb2 expression was absent. White boxes indicate where higher magnification photos were taken. (B) Immunohistochemestry for CDH1 at P13 shows that Prop1df/df pituitaries have an expanded expression of this endothelial protein in the anterior lobe compared to control pituitaries. White boxes indicate where higher magnification photos were taken. Cell nuclei were stained with DAPI (blue). Scale bar 100 µm.DOI:http://dx.doi.org/10.7554/eLife.14470.011
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fig7: Loss of Prop1 affects EMT pathway genes in pituitary tissues.(A) Upper panel:In situ hybridization using a Zeb2 antisense riboprobe at e14.5 detected expression in the control pituitary. Most of the positive cells are in the ventral area, around the cleft (between the lines). In Prop1df/df pituitaries very few cells are expressing Zeb2. Lower panel: Sagittal sections of 14.5 embryos were stained for CDH1 (green) and CDH2 (red) by immunohistochemistry. In the wild-type pituitary, CDH1 expression is dispersed throughout the Rathke’s Pouch, while CDH2 expression is more concentrated in the rostral area and in the forming anterior lobe. In Prop1 mutant pituitaries, CDH1 expression is increased and concentrated in the area ventral to the lumen, where Zeb2 expression was absent. White boxes indicate where higher magnification photos were taken. (B) Immunohistochemestry for CDH1 at P13 shows that Prop1df/df pituitaries have an expanded expression of this endothelial protein in the anterior lobe compared to control pituitaries. White boxes indicate where higher magnification photos were taken. Cell nuclei were stained with DAPI (blue). Scale bar 100 µm.DOI:http://dx.doi.org/10.7554/eLife.14470.011
Mentions: We examined expression of the EMT inducer Zeb2 and cadherins during pituitary development in control and Prop1df/df mice (Figure 7A). At e14.5, Zeb2 is normally expressed in cells in the ventral part of the Rathke’s Pouch (Figure 7A, between the two lines), and this zone of enriched expression is almost absent in Prop1 mutant pituitaries. CDH1 is normally expressed throughout the Rathke’s pouch, but CDH1 staining is stronger in the ventral part of the Prop1 mutant pouch, particularly along the marginal zone (Figure 7A, box and higher magnification image), although there was no obvious difference in expression of the mesenchymal marker, N-CADHERIN or CDH2. Next, we explored CDH1 expression at P13, the time point at which the CFCs were collected for subsequent RNA-Seq analysis. We found that Prop1df/df mutants have abnormal expression of CDH1 in cells distant from the marginal zone. Taken together, these results show that Prop1 is necessary for suppressing SOX2 expression in stem cells and for driving them to undergo an EMT-like process critical for populating the anterior lobe with progenitors capable of differentiating into the PIT1 lineage.10.7554/eLife.14470.011Figure 7.Loss of Prop1 affects EMT pathway genes in pituitary tissues.

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