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. 2015 Oct 5;3(10):e12537. doi: 10.14814/phy2.12537

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© 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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Loss of stromal miRNAs resulted in expansion of nephron progenitors and an intrinsic stromal defect. (A, B) Whole-mount immunofluorescence staining for Six2 (nephron progenitor), FoxD1 (renal stroma), and Calbindin (ureteric tips) revealed disorganization and expansion of nephron progenitors surrounding the ureteric tips, with decreased FoxD1⁺ cells, in E18.5 FoxD1^GC;Dicer^fl/fl kidneys. (C–H) Section immunofluorescence also reaffirmed the aberrant expansion of Six2⁺ (red) nephron progenitors, and loss of Anxa2⁺ (green) stroma in the interdigitating renal cortical stroma of E18.5 FoxD1^GC;Dicer^fl/fl kidneys (white arrows). (I) Quantitative real-time PCR performed on control and FoxD1^GC;Dicer^fl/fl E18.5 kidneys confirmed increased levels of Six2, Cited1, and Eya1 expression in nephron progenitor and reduction of FoxD1 transcripts. n = 6, *P < 0.05, ***P < 0.001. (J–O) Lineage tracing of the renal stroma performed with tdTomato reporter mice (red) demonstrated that while lineage tagged FoxD1^GC;Dicer^fl/fl stroma (FoxD1^GC;tdTomato;Dicer^fl/fl) adopts the expression pattern observed in E18.5 FoxD1^GC;tdTomato;Dicer^fl/+ kidneys (white arrows), the interdigitating stroma lacked Anxa2 expression (green). (P–U) Lineage tracing and Six2 immunostaining at E18.5 revealed that expansion of FoxD1^GC;Dicer^fl/fl Six2⁺ nephron progenitors is not due to transdifferentiation of the FoxD1⁺ renal stroma into nephron progenitors. Immunofluorescent studies were performed on at least three control and mutant embryos.