Stromally Expressed β-Catenin Modulates Wnt9b Signaling in the Ureteric Epithelium

<div><p>The mammalian kidney undergoes cell interactions between the epithelium and mesenchyme to form the essential filtration unit of the kidney, termed the nephron. A third cell type, the kidney stroma, is a population of fibroblasts located in the kidney capsule, cortex and medulla and is ideally located to affect kidney formation. We found β-catenin, a transcriptional co-activator, is strongly expressed in distinctive intracellular patterns in the capsular, cortical, and medullary renal stroma. We investigated β-catenin function in the renal stroma using a conditional knockout strategy that genetically deleted β-catenin specifically in the renal stroma cell lineage (β-cat^s-/-). <i>β-cat^s-/-</i> mutant mice demonstrate marked kidney abnormalities, and surprisingly we show β-catenin in the renal stroma is essential for regulating the condensing mesenchyme cell population. We show that the population of induced mesenchyme cells is significantly reduced in <i>β-cat^s-/-</i> mutants and exhibited decreased cell proliferation and a specific loss of Cited 1, while maintaining the expression of other essential nephron progenitor proteins. <i>Wnt9b</i>, the key signal for the induction of nephron progenitors, was markedly reduced in adjacent ureteric epithelial cells in <i>β-cat^s-/-</i>. Analysis of Wnt9b-dependent genes in the neighboring nephron progenitors was significantly reduced while Wnt9b-independent genes remained unchanged. In contrast mice overexpressing β-catenin exclusively in the renal stroma demonstrated massive increases in the condensing mesenchyme population and <i>Wnt9b</i> was markedly elevated. We propose that β-catenin in the renal stroma modulates a genetic program in ureteric epithelium that is required for the induction of nephron progenitors.</p></div

β-catenin in the renal stroma modulates Wnt9b expression in ureteric epithelial cells.

<p>(A-C) When compared to <i>WT</i>, In situ hybridization and real-time quantitative PCR for <i>Wnt9b</i> demonstrates <i>Wnt9b</i> mRNA expression is significantly reduced (1.00 versus 0.29, p=0.008) in E14.5 <i>β-cat^S-/-</i> kidneys. (D-I) In situ hybridization and Real-time quantitative PCR for <i>Ret</i> and <i>Wnt11</i> demonstrated no differences in mRNA expression between <i>WT</i> and <i>β-cat^S-/-</i> at E14.5. (J-K) Histological analysis of <i>β-cat^GOF-S</i> mutant kidneys demonstrate a marked increase in condensing mesenchyme population when compared to <i>WT</i> at E14.5. (L-N) In situ hybridization and quantitative PCR demonstrate <i>Wnt9b</i> expression in <i>β-cat^GOF-S</i> kidneys was significantly increased (1.02 versus 3.102, p=0.021) as compared to <i>WT</i> at E14.5. (scale bar = 50 μm, rc-renal capsule, cm= condensing mesenchyme, ub = ureteric epithelium).</p

<i>β-cat</i>^<i>S-/</i>- mutants demonstrate multiple kidney abnormalities.

<p>(A,B) Gross anatomy of PN0 <i>WT</i> and <i>β-cat</i>^<i>S-/</i>- kidneys show comparable size and shape. (C-J) In contrast to <i>WT</i>, histological analysis of <i>β-cat</i>^<i>S-/</i>- mutant kidneys demonstrate numerous kidney abnormalities. (C,D) As compared to <i>WT</i>, <i>β-cat</i>^<i>S-/</i>- mutant kidneys were lobular, lacked a distinct boarder, contained numerous cysts in the medulla and cortex (star), with an ill-defined cortical medullary axis and misplaced glomeruli (arrow). (E-J) In contrast to <i>WT</i>, high magnification of <i>β-cat</i>^<i>S-/</i>- kidneys at PN0 revealed a non-adherent sporadic renal capsule (F and J), misplaced tubules just under the renal capsule (F and J), glomeruli in the medulla (H) and a marked reduction in medullary stroma (H). (A, B scale bar = 1mm, C, D scale bar = 100μm, ad = adrenal gland, k = kidney, b = bladder, rc = renal capsule, cs = cortical stroma, ms = medullary stroma, g = glomerulus).</p

β-catenin is expressed in distinctive patterns in the renal stroma.

<p>(A-I) Immunofluorescence demonstrating β-catenin spatial and temporal expression in stromal cells. (A) At E11.5 Pbx1 is expressed in the nucleus of stromal cells (arrow head-inset) surrounding the condensing mesenchyme. Some Pbx1 positive stromal cells locate within the condensing mesenchyme, directly adjacent to epithelial cells (arrows). (B,C) At E11.5 β-catenin is expressed in the condensing mesenchyme and ureteric epithelium, and co-localizes with Pbx1 demonstrating expression in the renal stroma. At E11.5, some Pbx1 cells co-localize with β-catenin in the nuclear compartment of stromal cells (arrowhead-inset). (D) At E13.5, Pbx1 is expressed in capsular and cortical stroma surrounding the condensing mesenchyme. (E, F) At E13.5, β-catenin co-localizes in the cytoplasm of capsular stromal cells. The stromal cells located between developing nephrons express β-catenin in the cytoplasmic (arrow-inset) and nuclear compartment (arrowhead-inset). (G) At E17.5, Pbx1 marks the capsular, cortical, and medullary stroma. (H-I) β-catenin is expressed in the medullary stroma and co-localizes strongly with Pbx1 in the nuclear compartment (arrowhead-inset). (scale bar = 100μm, s = stroma, cm = condensing mesenchyme, ub = ureteric epithelium, rc = renal capsule, ms = medullary stroma, rp = renal pelvis).</p

The condensing mesenchyme cell population is reduced in <i>β-cat</i>^<i>S-/</i>- mutant kidneys.

<p>(A,B) As compared to <i>WT</i>, which demonstrates 3–4 cell layers of aggregated condensing mesenchyme, <i>β-cat</i>^<i>S-/</i>- kidneys display a reduced, single cell layer of loosely aggregated condensing mesenchyme. (C-H) Analysis of cell proliferation in the condensing mesenchyme was performed using Brdu cell proliferation assay. (C-E) As compared to <i>WT</i>, <i>β-cat</i>^<i>S-/</i>- mutants demonstrated a 6.46% reduction in condensing mesenchyme cell proliferation at E14.5 (<i>WT</i>, 34.56%±1.45, n = 28 versus <i>β-cat</i>^<i>S-/</i>-, 28.02%±1.05, n = 27, p = 0.0006). (F-G) At E15.5 <i>β-cat</i>^<i>S-/</i>- mutants demonstrated a 7.35% reduction in condensing mesenchyme cell proliferation when compared to <i>WT</i> (<i>WT</i>, 34.09%±1.65, n = 17 versus <i>β-cat</i>^<i>S-/</i>-, 26.73%±2.21, n = 15, p = 0.01). (I,J) A TUNEL assay at E15.5 did not reveal any changes in apoptosis in the condensing mesenchyme between <i>WT</i> and <i>β-cat</i>^<i>S-/</i>-. Scale Bar = 50μm</p

Investigation of the renal stroma in <i>β-cat</i>^<i>S-/</i>- mutant kidneys.

<p>(A-L) Analysis of the renal stroma using stromal markers; Meis1/2, Pbx1, Foxd1, and TN-C at E15.5. As compared to <i>WT</i>, no overt changes were observed in the cortical stroma with respect to Meis1/2 (A, B), Pbx1 (E, F), Foxd1 (I, J), and TN-C (K, L). However, a reduction of Meis1/2 (C, D) and Pbx1 (G, H) was observed in the medullary region in <i>β-cat</i>^<i>S-/</i>- kidneys. (M, N) TUNEL assay at E15.5 reveals an increase in apoptosis in the medullary stroma of <i>β-cat</i>^<i>S-/</i>- kidneys compared to <i>WT</i>. (rc = renal capsule, cs = cortical stroma, ms = medullary stroma, A = apoptosis). Scale Bar = 50μm</p

<i>β-cat</i>^<i>S-/</i>- mutants demonstrate altered Wnt9b signaling to the condensing mesenchyme.

<p>(A-P) Analysis of Wnt9b dependent and independent gene targets by immunofluorescence and real-time quantitative PCR at E15.5 and E14.5 respectively. (A-F) In contrast to <i>WT</i> at E15.5, the number of Pax2 and Six2 positive cells in the condensing mesenchyme was reduced in <i>β-cat</i>^<i>S-/</i>- kidneys. No changes were observed in the <i>Pax2</i> and <i>Six2</i> mRNA expression levels at E14.5 by qRT-PCR. (G-I) Both the number of Cited 1 positive cells and <i>Cited 1</i> mRNA expression levels were reduced (1.00 versus 0.62, p = 0.003) in <i>β-cat</i>^<i>S-/</i>- kidneys. (J-L) The levels of Amphiphysin were significantly reduced in the condensing mesenchyme at both the protein and mRNA levels in <i>β-cat</i>^<i>S-/</i>- kidneys (1.01 versus 0.48, p = 0.025) (M-O) In situ hybridization and qRT-PCR analysis of <i>Wnt4</i> demonstrates a reduction in <i>Wnt4</i> mRNA levels in E14.5 <i>β-cat</i>^<i>S-/</i>- kidneys (1.00 versus 0.30, p = 0.0007). (P) QRT-PCR of Wnt9b-independent gene <i>Eya1</i> demonstrates no changes in mRNA expression levels in <i>β-cat</i>^<i>S-/</i>- kidneys. In contrast, Wnt9b-dependent gene <i>Tafa5</i> (1.00 versus 0.42, p = 0.003) was significantly decreased in <i>β-cat</i>^<i>S-/</i>- kidneys (scale bar = 50μm) (rv = renal vesicle, ub = ureteric bud).</p

Intracellular localization of β-catenin in capsular, cortical, and medullary stroma.

<p>(A-L) Immunofluorescence showing β-catenin intracellular distribution in the capsular, cortical, and medullary stroma. (A-C) In the capsular stroma, β-catenin localizes in a membrane and cytoplasmic pattern and does not co-localize with nuclear stromal factor Pbx1. (D) Schematic diagram of the intracellular β-catenin localization and a suggested role in cell-cell adhesion via adherens junctions. (E-G) β-catenin weakly co-localizes with Pbx1 in the nuclear compartment but is primarily cytoplasmic. (H) Schematic diagram of the intracellular β-catenin localization showing possible roles in the cytoplasm and nucleus. (I-K) In the medulla, β-catenin co-localizes with Pbx1 primarily to the nuclear compartment but some cytoplasmic β-catenin expression is observed. (L) Schematic diagram of the intracellular β-catenin localization showing a more prominent role in the nucleus. (scale bar = 5μm, M = membrane, C = cytoplasm, N = nuclear, AJ = Adherens junctions).</p

Temporal analysis of the embryonic kidney phenotype in <i>β-cat</i>^<i>S-/</i>- mutants.

<p>(A, B) Histological analysis of <i>WT</i> and <i>β-cat</i>^<i>S-/</i>- embryonic kidneys at E13.5 demonstrates no abnormalities in the stromal population, developing nephrons, or kidney patterning. (C, D) In contrast to <i>WT</i> at E14.5, <i>β-cat</i>^<i>S-/</i>- kidneys demonstrate abnormally located glomeruli, and a non-adherent irregular patterned renal capsule. (E-J) In contrast to <i>WT</i> at E15.5, the non-adherent capsular phenotype persists in <i>β-cat</i>^<i>S-/</i>- kidneys (H) and the cortical stroma is reduced and loosely packed (H). Similarly, the medullary stroma in <i>β-cat</i>^<i>S-/</i>- kidneys is markedly reduced compared to <i>WT</i> (J) and glomeruli are also abnormally located within the medulla (rc = renal capsule, cs = cortical stroma, ms = medullary stroma, g = glomerulus, arrowhead = ureter). Scale Bar C-F = 100μm, G-J = 50μm</p