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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

Differential gene expression in Prop1df/df and Pou1f1dw/dw pituitary stem-cell-derived colonies at P13.(A) A Venn diagram illustrates the total number of genes with significantly different expression in Prop1 mutants relative to their wild-type littermates (blue circle) and Pou1f1 mutants relative to their wild-type counterparts (red circle). The table shows examples of genes up regulated (green) or down regulated (red) (p value ≤ 0.05, /log2FCΙ ≥ 1) uniquely in the Prop1 mutant colonies relative to Pou1f1 mutants. (B) RT-qPCR validation of RNA-Seq data. GAPDH was used as an internal control (N = 4) OWA and * indicates p<0.05 relative to control. Immunostaining for CDH1 shows an increased in protein expression on colonies from Prop1df/df pituitaries compare to control. CYCLIN E immunostaining reveals that Prop1+/+ colonies express this protein but is absent on Prop1df/df colonies. Cell nuclei were stained with DAPI (blue). Scale bar 100 µm. Plots denote the mean ± SEM. qPCRs were done using at least three technical replicates.DOI:http://dx.doi.org/10.7554/eLife.14470.009
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fig5: Differential gene expression in Prop1df/df and Pou1f1dw/dw pituitary stem-cell-derived colonies at P13.(A) A Venn diagram illustrates the total number of genes with significantly different expression in Prop1 mutants relative to their wild-type littermates (blue circle) and Pou1f1 mutants relative to their wild-type counterparts (red circle). The table shows examples of genes up regulated (green) or down regulated (red) (p value ≤ 0.05, /log2FCΙ ≥ 1) uniquely in the Prop1 mutant colonies relative to Pou1f1 mutants. (B) RT-qPCR validation of RNA-Seq data. GAPDH was used as an internal control (N = 4) OWA and * indicates p<0.05 relative to control. Immunostaining for CDH1 shows an increased in protein expression on colonies from Prop1df/df pituitaries compare to control. CYCLIN E immunostaining reveals that Prop1+/+ colonies express this protein but is absent on Prop1df/df colonies. Cell nuclei were stained with DAPI (blue). Scale bar 100 µm. Plots denote the mean ± SEM. qPCRs were done using at least three technical replicates.DOI:http://dx.doi.org/10.7554/eLife.14470.009

Mentions: In order to discover the mechanism whereby Prop1 deficiency uniquely affects the population of stem cells in the postnatal pituitaries, RNA-Seq experiments were performed on stem cell colonies originating from P13 pituitaries from Prop1df/df, Pou1f1dw/dw and their respective wild-type littermates. Three independent samples of total RNA for each experimental group were collected and analyzed using RNA-Seq technology. A comparison of the gene expression differences in wild types and mutants revealed that the Prop1 mutation had the greatest effect on gene expression in stem-cell-derived colonies. A total of 2035 genes were differentially expressed (p value ≤ 0.05, /log2FCΙ ≥ 1) in colonies from Prop1 mutants compared with their wild-type littermates, while only about 1/3 that number were differentially expressed in Pou1f1 mutants compared to their wild-type littermates (752 genes; p value ≤ 0.05, /log2FCΙ ≥ 1). The two mutants share a total of 386 genes that are differentially expressed relative to their wild-type littermates (p value ≤ 0.05, /log2FCΙ ≥ 1), revealing a set of common effects on colony-forming properties of postnatal pituitary progenitors (Figure 5A).10.7554/eLife.14470.009Figure 5.Differential gene expression in Prop1df/df and Pou1f1dw/dw pituitary stem-cell-derived colonies at P13.


PROP1 triggers epithelial-mesenchymal transition-like process in pituitary stem cells
Differential gene expression in Prop1df/df and Pou1f1dw/dw pituitary stem-cell-derived colonies at P13.(A) A Venn diagram illustrates the total number of genes with significantly different expression in Prop1 mutants relative to their wild-type littermates (blue circle) and Pou1f1 mutants relative to their wild-type counterparts (red circle). The table shows examples of genes up regulated (green) or down regulated (red) (p value ≤ 0.05, /log2FCΙ ≥ 1) uniquely in the Prop1 mutant colonies relative to Pou1f1 mutants. (B) RT-qPCR validation of RNA-Seq data. GAPDH was used as an internal control (N = 4) OWA and * indicates p<0.05 relative to control. Immunostaining for CDH1 shows an increased in protein expression on colonies from Prop1df/df pituitaries compare to control. CYCLIN E immunostaining reveals that Prop1+/+ colonies express this protein but is absent on Prop1df/df colonies. Cell nuclei were stained with DAPI (blue). Scale bar 100 µm. Plots denote the mean ± SEM. qPCRs were done using at least three technical replicates.DOI:http://dx.doi.org/10.7554/eLife.14470.009
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fig5: Differential gene expression in Prop1df/df and Pou1f1dw/dw pituitary stem-cell-derived colonies at P13.(A) A Venn diagram illustrates the total number of genes with significantly different expression in Prop1 mutants relative to their wild-type littermates (blue circle) and Pou1f1 mutants relative to their wild-type counterparts (red circle). The table shows examples of genes up regulated (green) or down regulated (red) (p value ≤ 0.05, /log2FCΙ ≥ 1) uniquely in the Prop1 mutant colonies relative to Pou1f1 mutants. (B) RT-qPCR validation of RNA-Seq data. GAPDH was used as an internal control (N = 4) OWA and * indicates p<0.05 relative to control. Immunostaining for CDH1 shows an increased in protein expression on colonies from Prop1df/df pituitaries compare to control. CYCLIN E immunostaining reveals that Prop1+/+ colonies express this protein but is absent on Prop1df/df colonies. Cell nuclei were stained with DAPI (blue). Scale bar 100 µm. Plots denote the mean ± SEM. qPCRs were done using at least three technical replicates.DOI:http://dx.doi.org/10.7554/eLife.14470.009
Mentions: In order to discover the mechanism whereby Prop1 deficiency uniquely affects the population of stem cells in the postnatal pituitaries, RNA-Seq experiments were performed on stem cell colonies originating from P13 pituitaries from Prop1df/df, Pou1f1dw/dw and their respective wild-type littermates. Three independent samples of total RNA for each experimental group were collected and analyzed using RNA-Seq technology. A comparison of the gene expression differences in wild types and mutants revealed that the Prop1 mutation had the greatest effect on gene expression in stem-cell-derived colonies. A total of 2035 genes were differentially expressed (p value ≤ 0.05, /log2FCΙ ≥ 1) in colonies from Prop1 mutants compared with their wild-type littermates, while only about 1/3 that number were differentially expressed in Pou1f1 mutants compared to their wild-type littermates (752 genes; p value ≤ 0.05, /log2FCΙ ≥ 1). The two mutants share a total of 386 genes that are differentially expressed relative to their wild-type littermates (p value ≤ 0.05, /log2FCΙ ≥ 1), revealing a set of common effects on colony-forming properties of postnatal pituitary progenitors (Figure 5A).10.7554/eLife.14470.009Figure 5.Differential gene expression in Prop1df/df and Pou1f1dw/dw pituitary stem-cell-derived colonies at P13.

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