CXCR7, CXCR4, and CXCL12 expression in mature glomeruli.

<p>(A–C) Brightfield micrographs of cresyl violet stained mature glomeruli (E16.5) after hybridization with ³⁵S- labeled probes for CXCR7, CXCR4, and CXCL12. (<b>A,B</b>) Signals for CXCR7 are restricted to the podocyte layer whereas CXCR4 is restricted to the center of the glomerulus. (<b>C</b>) CXCL12 is detected both in the podocytes and in the area of the vascular pole (vp, arrowhead). (D–E) Mature glomeruli after dual in situ hybridization with ³⁵S labeled probes for CXCR4 (D), CXCL12 (E), and a DIG-labeled probe for podocyte marker WT1 (D,E). (<b>D</b>) The darkfield micrograph reveals CXCR4 labeling (white signals) close to the vascular pole but not in WT1 stained podocytes of the visceral blade of Bowman's capsule. (<b>E</b>) The brightfield image shows labeling for CXCL12 mRNA (black grains) in the WT1 positive podocyte layer and in the WT1 negative area of the vascular pole (vp, arrowhead). (F–G) Confocal images of dual immunofluorescent stainings for GFP/CXCR4 (F) and GFP/podocin (G) in E16.5 BAC transgenic mice expressing EGFP under the control of the CXCR7 promoter. (<b>F</b>) CXCR4 immunoreactivity is present in the glomerular tuft (arrow), presumptive arterioles (arrowheads), and at the luminar membrane of tubular epithelial cells (asteriscs). Some tubules are co-positive for CXCR7-GFP and CXCR4 (filled asteristics), others display exclusively CXCR4 protein signals (open asteristics). In the glomerulus, signals for CXCR4 and CXCR7-GFP do not overlap. (<b>G</b>) Podocytes labeled by the selective marker podocin (red) are CXCR7-GFP positive. Scale bars represent 10 µm (C,G′″) and 20 µm (F).</p

Transmission electron microscopic analysis of the glomeruli.

<p>Glomerular capillaries at 3.300× magnification (<b>A</b>,<b>B</b>) and details of the filtration barrier at 21.600× magnification (<b>C</b>,<b>D</b>; framing in A,B). CXCR7^+/+ (C) and CXCR7^−/− (D) showed no differences in general morphology and glomerular basement membrane (gbm) attachment of podocytes (pod). However endothelial cells (en) seemed to be detached from the gbm (arrows). Scale bars correspond to 2 µm (A,B) and 0.3 µm (C,D).</p

Differential expression of CXCR7 and CXCR4 in nephrogenic mesenchyme, ureteric bud, and forming glomeruli.

<p>(A–D) A digoxigenin (DIG) labeled WT1 antisense probe was used as a marker for mesenchymal and mesenchyme derived nephrogenic structures at E14.5 and co-hybridized with ³⁵S labeled probes for CXCR7 or CXCR4. (<b>A</b>) Strong CXCR7 signals (black grains) are present in a T-shaped WT1 negative early ureteric bud tip (eub) which is associated with a CXCR7/WT1 co-positive pretubular aggregate (pa; black grains/brown staining). Weaker CXCR7 expression is found in a late ureteric bud tip (lub) which is associated with CXCR7/WT1 co-positive renal vesicles (rv). Note weak CXCR7 mRNA expression in the cap mesenchyme (cm) and strong CXCR7 gene activity above the cm (brown staining) at the renal capsule (rc). (<b>B,B′,C,C′</b>) Bright- and darkfield views of a comma-shaped body (cb in B) and S-shaped body (sb in C) after hybridization with a DIG labeled WT1 probe and a ³⁵S labeled CXCR7 riboprobe (black grains in B,C; white grains in B′,C′). Both WT1 positive structures exhibit clear CXCR7 antisense mRNA signals. (<b>D,D′</b>) Bright- and darkfield micrographs showing WT1-positive renal tissue (D) and radiosignals of CXCR4 riboprobe (D′). Strong CXCR4 gene expression is detected in a WT1 negative early ureteric bud tip (eub in D, dotted line in D′). Weak CXCR4 labeling is seen in a late ureteric bud tip (lub) which is associated with a WT1 positive/CXCR4 negative renal vesicle (rv). Note that CXCR4 mRNA is also present in WT1 positive cap mesenchyme (cm). WT1 positive epithelial cells of S-Shaped bodies display no CXCR4 mRNA expression. The vascular cleft of S-shaped bodies (arrows in D′) as well as putative arterioles (arrowhead in D′) are CXCR4 positive. (<b>E</b>,<b>F</b>) GFP immunostaining in sections from BAC transgenic mice expressing EGFP under the control of the CXCR7 promotor (G) or CXCR4 promotor (H). Calbindin was co-stained as an ureteric bud marker. (<b>G</b>) CXCR7-GFP is highly expressed in the renal capsule (rc) as well as in comma- and S-shaped bodies (cb, sb) associated with a calbindin positive late ureteric bud (lub). CXCR7-GFP is weak expressed in cap mesenchyme (cm) and not present in the late ureteric bud. (<b>H</b>) Strong CXCR4-GFP signals are detected only in cap mesenchyme (cm). All scale bars equal 20 µm.</p

Expression of CXCL12, CXCR7 and CXCR7 mRNA during nephrogenesis.

<p>Mapping of CXCL12, CXCR7, and CXCR4 expression was performed using the markers WT1 (dual in situ hybridization) and Podocin as well as VEGFR-2 (dual immunofluorescence). +, strong expression; (+) weak expression; (−) very weak expression; − no expression. ep, epithelial cells; mes, mesenchymal cells; mes→ep, mesenchymal-to-epithelial transition.</p

Histological analysis of CXCR7-deficient kidneys.

<p>Kidney sections of E16.5 wildtype (A–C) and CXCR7 deficient littermates (D–F) were stained with periodic acid-Schiff (PAS) reagents. (<b>A,D</b>) Macroscopic images demonstrate that the overall kidney morphology is normal in the CXCR7 knockout embryo. (<b>B,C,E,F</b>) Microscopic views of pretubular aggregates (pa), renal vesicles (rv), ureteric buds (ub) (B,E), and S-shaped bodies (C,F) do not reveal any abnormalities in the CXCR7 knockout kidney. Scale bars equal 200 µm (D) and 20 µm (B,C).</p

Patterns of CXCL12, CXCR7, and CXCR4 mRNAs in the early developing kidney.

<p>Adjacent kidney sections were hybridized with radiolabeled antisense riboprobes for CXCL12, CXCR7, and CXCR4 at E12.5 and E14.5. (<b>A</b>, <b>D</b>) CXCL12 expression pattern changed remarkably from E12.5 to E14.5 as no CXCL12 mRNA could be detected within the E12.5 kidney. At E14.5, CXCL12 signals are present in the kidney in stromal cells (asteristics in D), around the ureter (u) as well as in glomeruli (arrowheads in D). (<b>B</b>, <b>E</b>) CXCR7 mRNA is expressed in the region of the renal capsule (rc) and ureter at both indicated embryonic stages. The gene is also active in some ureteric buds (open arrows in B, E), immature glomeruli (closed arrows in E), and mature glomeruli (arrowheads in E). (<b>C</b>, <b>F</b>) CXCR4 mRNA is highly expressed in mesenchymal cells below the rc region at E12.5 (arrowheads in C) as well as in the cap mesenchyme at E14.5 (cm in F). CXCR4 is also expressed in some ureteric buds at E12.5 (open arrows in C). At E14.5, CXCR4 expression is found in presumptive blood vessels (closed arrows in F) and glomerular tufts (arrowheads in F). Allocation of detection signals to renal structures was performed using counterstaining with hematoxylin &amp; eosin. ag, adrenal gland; ov, ovarium; Scale bars correspond to 100 µm.</p

CXCR4 immunoreactivity in CXCR7 deficient kidneys.

<p>(<b>A,B</b>) In situ hybridizations with a radiolabeled CXCR4 probe show similar CXCR4 expression patterns and labeling intensities in the E14.5 kidney of a wildtype and a CXCR7 deficient mutant. (<b>C,D</b>) Immunostaining of the CXCR4 protein in E16.5 kidney sections of a wildtype and a CXCR7 mutant littermate show severely reduced immunoreactivity in the nephrogenic zone (nz), in glomerular tufts (arrowheads) but not in tubular structures (arrows) of the mutant. (<b>E–H</b>) Higher magnification pictures showing CXCR4 protein signals in wildtype and CXCR7 deficient nephrogenic zone (E,F) and tubular structures (G,H). In the cap mesenchyme (cm) of the CXCR7^−/− embryo (F), CXCR4 immunoreactivity is heavily reduced to single protein signals allocated to the cytoplasm of the mesenchymal cells (arrows). CXCR4 protein signals at the apical site of tubular epithelial cells (ep) are similar in a CXCR7 wildtype (G) and a mutant littermate (H). (<b>I</b>) Measurement of CXCR4 mRNA-radiosignal positive area in the nephrogenic zone (see A,B: nz). (<b>J</b>) Measurement of CXCR4 immunoreactivity positive area in the nephrogenic zone (see C,D: nz) reveals severe downregulation of the CXCR4 protein in CXCR7^−/− kidney sections (Mann-Whitney U test, p&lt;0.001). Data represent mean values ±SEM as percentage of the wildtype group (I,J). (<b>K</b>) Western Blot showing CXCR4 protein expression from 22 pooled E14.5 kidneys of control and CXCR7 knockout embryos. Endogenous transferring receptor (TFR) was used as loading control. lum, lumen; rp, renal pelvis: ub, ureteric bud. Scale bars equal 100 µm (B), 200 µm (D) and 10 µm (F,H).</p