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The PI3K pathway balances self-renewal and differentiation of nephron progenitor cells through β-catenin signaling.

Lindström NO, Carragher NO, Hohenstein P - Stem Cell Reports (2015)

Bottom Line: Nephron progenitor cells differentiate to form nephrons during embryonic kidney development.In contrast, self-renewal maintains progenitor numbers and premature depletion leads to impaired kidney function.Here we analyze the PI3K pathway as a point of convergence for the multiple pathways that are known to control self-renewal in the kidney.

View Article: PubMed Central - PubMed

Affiliation: The Roslin Institute, University of Edinburgh, Easter Bush Campus, Midlothian EH25 9RG, UK; Edinburgh Cancer Research Centre, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XR, UK. Electronic address: nils.lindstrom@roslin.ed.ac.uk.

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Nephron Progenitors Differentiate into Nephrons when PI3K Is Inhibited(A–C) qRT-PCR analyses on FACS-sorted cells from dissociated E12.5 Six2+/GCiP;Rosa26tdRFP kidneys cultured for 24 hr. Cells from three kidneys were grouped to form each mRNA isolate replicate. Experiments were performed in triplicate with nine kidneys per treatment. All error bars indicate SEM. P values calculated using Student’s t test.(D and E) E12.5 kidneys cultured for 24 hr.(F–H) E12.5 kidneys cultured for 48 hr. Blue dashed line surrounds the nephron progenitor cells; white dashed line surrounds the ureteric bud; white arrowheads indicate points of ectopic expression. Cm, cap mesenchyme; ub, ureteric bud; ec, ectopic nephron. Culture conditions and labeling are as indicated in figures. See also Figure S2.
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fig2: Nephron Progenitors Differentiate into Nephrons when PI3K Is Inhibited(A–C) qRT-PCR analyses on FACS-sorted cells from dissociated E12.5 Six2+/GCiP;Rosa26tdRFP kidneys cultured for 24 hr. Cells from three kidneys were grouped to form each mRNA isolate replicate. Experiments were performed in triplicate with nine kidneys per treatment. All error bars indicate SEM. P values calculated using Student’s t test.(D and E) E12.5 kidneys cultured for 24 hr.(F–H) E12.5 kidneys cultured for 48 hr. Blue dashed line surrounds the nephron progenitor cells; white dashed line surrounds the ureteric bud; white arrowheads indicate points of ectopic expression. Cm, cap mesenchyme; ub, ureteric bud; ec, ectopic nephron. Culture conditions and labeling are as indicated in figures. See also Figure S2.

Mentions: Current models for nephron development assume that mesenchymal ENPs undergo a MET before segment-specific expression programs are activated (Costantini and Kopan, 2010). We noted, however, that the ectopic nephrons that form under conditions of PI3K inhibition show signs of differentiation, for instance, expression of JAG1 and not all JAG1+ cells being fully epithelialized, as shown by the lack of CDH1 expression (Figure 1D). We analyzed this further at the 24 hr time point before the ENP population differentiated fully using qRT-PCR on RNA from Six2+/GCiPRosa26+/tdRFP kidneys (Figures S2A–S2C). This confirmed that, in ENPs (GFP+/RFP+), cell expression of the ENP markers Six2 and Cited1 did not change after PI3K inhibition, though expression of Osr1, a marker of intermediate mesoderm that is maintained in the ENP stage, was reduced (Figure 2A). In contrast, in the same cells, Ly294002 treatment resulted in an upregulation of induction markers Wnt4, Lhx1, and Cdh1 (Figure 2B) and segment markers Jag1, Dll1, and HeyL (Figure 2C). Note that, although we detected a modest upregulation of Cdh1 mRNA 24 hr after PI3K inhibition (Figure 2B), at the same time point there was no sign of CDH1 protein expression in SIX2+ cells (Figure 1C). After 24 hr of PI3K inhibition, ectopic nephrons showed expression of JAG1 protein, while the tight junction marker ZO-1 and adherence junction protein β-catenin were increased in expression, but no CDH1 protein (MET marker, Figures 2D and 2E). After 48 hr in Ly2094002 expression of LEF1, PAX2 (induction markers), JAG1, and ZO-1 as well as CDH1 (Figures 2F–2H) confirmed that full MET eventually takes place in these structures. Expression of SIX2 was almost completely gone from cells that expressed CDH1 protein (data not shown).


The PI3K pathway balances self-renewal and differentiation of nephron progenitor cells through β-catenin signaling.

Lindström NO, Carragher NO, Hohenstein P - Stem Cell Reports (2015)

Nephron Progenitors Differentiate into Nephrons when PI3K Is Inhibited(A–C) qRT-PCR analyses on FACS-sorted cells from dissociated E12.5 Six2+/GCiP;Rosa26tdRFP kidneys cultured for 24 hr. Cells from three kidneys were grouped to form each mRNA isolate replicate. Experiments were performed in triplicate with nine kidneys per treatment. All error bars indicate SEM. P values calculated using Student’s t test.(D and E) E12.5 kidneys cultured for 24 hr.(F–H) E12.5 kidneys cultured for 48 hr. Blue dashed line surrounds the nephron progenitor cells; white dashed line surrounds the ureteric bud; white arrowheads indicate points of ectopic expression. Cm, cap mesenchyme; ub, ureteric bud; ec, ectopic nephron. Culture conditions and labeling are as indicated in figures. See also Figure S2.
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fig2: Nephron Progenitors Differentiate into Nephrons when PI3K Is Inhibited(A–C) qRT-PCR analyses on FACS-sorted cells from dissociated E12.5 Six2+/GCiP;Rosa26tdRFP kidneys cultured for 24 hr. Cells from three kidneys were grouped to form each mRNA isolate replicate. Experiments were performed in triplicate with nine kidneys per treatment. All error bars indicate SEM. P values calculated using Student’s t test.(D and E) E12.5 kidneys cultured for 24 hr.(F–H) E12.5 kidneys cultured for 48 hr. Blue dashed line surrounds the nephron progenitor cells; white dashed line surrounds the ureteric bud; white arrowheads indicate points of ectopic expression. Cm, cap mesenchyme; ub, ureteric bud; ec, ectopic nephron. Culture conditions and labeling are as indicated in figures. See also Figure S2.
Mentions: Current models for nephron development assume that mesenchymal ENPs undergo a MET before segment-specific expression programs are activated (Costantini and Kopan, 2010). We noted, however, that the ectopic nephrons that form under conditions of PI3K inhibition show signs of differentiation, for instance, expression of JAG1 and not all JAG1+ cells being fully epithelialized, as shown by the lack of CDH1 expression (Figure 1D). We analyzed this further at the 24 hr time point before the ENP population differentiated fully using qRT-PCR on RNA from Six2+/GCiPRosa26+/tdRFP kidneys (Figures S2A–S2C). This confirmed that, in ENPs (GFP+/RFP+), cell expression of the ENP markers Six2 and Cited1 did not change after PI3K inhibition, though expression of Osr1, a marker of intermediate mesoderm that is maintained in the ENP stage, was reduced (Figure 2A). In contrast, in the same cells, Ly294002 treatment resulted in an upregulation of induction markers Wnt4, Lhx1, and Cdh1 (Figure 2B) and segment markers Jag1, Dll1, and HeyL (Figure 2C). Note that, although we detected a modest upregulation of Cdh1 mRNA 24 hr after PI3K inhibition (Figure 2B), at the same time point there was no sign of CDH1 protein expression in SIX2+ cells (Figure 1C). After 24 hr of PI3K inhibition, ectopic nephrons showed expression of JAG1 protein, while the tight junction marker ZO-1 and adherence junction protein β-catenin were increased in expression, but no CDH1 protein (MET marker, Figures 2D and 2E). After 48 hr in Ly2094002 expression of LEF1, PAX2 (induction markers), JAG1, and ZO-1 as well as CDH1 (Figures 2F–2H) confirmed that full MET eventually takes place in these structures. Expression of SIX2 was almost completely gone from cells that expressed CDH1 protein (data not shown).

Bottom Line: Nephron progenitor cells differentiate to form nephrons during embryonic kidney development.In contrast, self-renewal maintains progenitor numbers and premature depletion leads to impaired kidney function.Here we analyze the PI3K pathway as a point of convergence for the multiple pathways that are known to control self-renewal in the kidney.

View Article: PubMed Central - PubMed

Affiliation: The Roslin Institute, University of Edinburgh, Easter Bush Campus, Midlothian EH25 9RG, UK; Edinburgh Cancer Research Centre, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XR, UK. Electronic address: nils.lindstrom@roslin.ed.ac.uk.

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Related in: MedlinePlus