Lineage specification of parietal epithelial cells requires β-catenin/Wnt signaling

Abstract

β-Catenin/Wnt signaling is essential during early inductive stages of kidney development, but its role during postinductive stages of nephron development and maturation is not well understood. In this study, we used Pax8Cre mice to target β-catenin deficiency to renal epithelial cells at the late S-shaped body stage and the developing collecting ducts. The conditional β-catenin knockout mice formed abnormal kidneys and had reduced renal function. The kidneys were hypoplastic with a thin cortex; a superficial layer of tubules was missing. A high proportion of glomeruli had small, underdeveloped capillary tufts. In these glomeruli, well differentiated podocytes replaced parietal epithelial cells in Bowman's capsule; capillaries toward the outer aspect of these podocytes mimicked the formation of glomerular capillaries. Tracing nephrogenesis in embryonic conditional β-catenin knockout mice revealed that these "parietal podocytes" derived from precursor cells in the parietal layer of the S-shaped body by direct lineage switch. Taken together, these findings demonstrate that β-catenin/Wnt signaling is important during the late stages of nephrogenesis and for the lineage specification of parietal epithelial cells.

Figure 1.

TCF and Pax8Cre activity are found in presumptive parietal epithelial cells during glomerulogenesis. (A) Enzymatic β-galactosidase staining of embryonic kidneys (embryonic day 15.5 [E15.5]) derived from TCF/lacZ reporter mice. Specific β-galactosidase expression is seen in parietal epithelial cells of maturing glomeruli. (B) Enzymatic β-galactosidase staining of cryosections of embryonic kidneys (E15.5) derived from Pax8Cre/Rosa26R mice. β-Galactosidase positivity is seen in all epithelial cells of the kidney including cells of the S-shaped body (SS), collecting duct (CD), visceral epithelial cells (VECs), and parietal epithelial cells (PECs) of a maturing glomerulus and tubules (T). Cells of the metanephric blastema (MB), interstitial cells, and blood vessels are negative. (C and D) Immunostaining for β-catenin in control (lox/lox) and β-catenin–deficient (lox/lox Pax8Cre) mice at 3 months of age. (C) Control kidneys show β-catenin expression in all tubular epithelial cells; podocytes are negative. (D) In kidneys of β-catenin–deficient mice, β-catenin is efficiently removed in all cells. Only a few single cells show persistent positive staining for β-catenin (arrows). (E–H) Double staining for aquaporin 2 (red) and β-catenin (green) in control (lox/lox) and β-catenin–deficient (lox/lox Pax8Cre) mice at 3 months of age. (E) Control kidneys show β-catenin in all tubular epithelial cells and parietal epithelial cells (arrow) at the basolateral surface. Aquaporin 2 is detected at the apical surface of collecting duct cells. UP, urinary pole. (F) In kidneys of β-catenin–deficient mice, β-catenin is efficiently removed in nearly all epithelial cells. (arrow = parietal epithelial cells). A few epithelial cells show persistent β-catenin expression (arrowhead). (G) Aquaporin 2 positive tubules in control kidneys show strong β-catenin expression. (H) In β-catenin–deficient mice, aquaporin 2 expression is similar in distribution and intensity compared with those in control mice. A few epithelial cells show persistent β-catenin expression (arrowhead). Original magnifications: ×500 in A; ×125 in B; ×500 in C and D; ×750 in E–H.

Figure 2.

β-Catenin–deficient mice show abnormal glomeruli featuring parietal podocytes, parietal capillaries, and underdeveloped capillary tufts. Semithin sections (A and B) and electron microscopy (C–H) of adult β-catenin–deficient (lox/lox Pax8Cre) kidneys. (A) The outermost cortex is missing. Glomeruli show underdeveloped capillary tufts and are cystically widened. Juxtamedullary glomeruli mostly appeared normal. (B) The glomeruli have a normal tubular pole (TP). (C) Electron microscopy reveals that the parietal epithelial cells are parietal podocytes (PP) with foot processes extending and covering Bowman’s capsule. Capillaries (asterisks) are arranged on the outer aspect of Bowman's capsule adjacent to parietal podocytes. VP, visceral podocytes. (D) Parietal podocytes (PP) and foot processes at higher magnification. Large parietal capillaries (PC) have formed adjacent to the parietal podocytes. (E) Parietal podocytes form a filtration-like barrier consisting of three layers: fenestrated endothelial cells (FE), a parietal basement membrane (BM), and foot processes (FP). The filtration slits between the foot processes are bridged by a slit membrane (arrows), and the endothelial fenestrae are bridged by diaphragms (arrowheads). (F) Parietal capillaries (asterisks) associated with parietal podocytes; one capillary bulges into Bowman's space (BS). (G) Bulging capillary with a well developed capillary neck (boxed area enlarged in I). (H) A schematic draft of the image of (G). Parietal podocytes (blue; PP) are covering the surface of a bulging parietal capillary (red; PC). Parietal basement membrane is dark blue. BS, Bowman’s space. (I) At the neck, the peripheral endothelial basement membrane reflects into the parietal basement membrane (white arrows); the bridging portion of the endothelium is underlain only by a thin and fragmented basement membrane (arrowheads). The cells within the neck region have many actin-filled processes (black arrows) that are connected to the basement membrane at the turning points. (J and K) Both visceral and parietal podocytes (arrows) stain positive for WT1. (L and M) Both visceral and parietal podocytes (arrows) stain positive for VEGF. (N and O) Double staining for synaptopodin (red) and WT1 (green). Synaptopodin in control kidneys is exclusively expressed in visceral podocytes. Parietal epithelial cells are negative for synaptopodin. In β-catenin–deficient mice, synaptopodin is expressed in both visceral and parietal podocytes (arrow). Original magnifications: ×125 in A; ×500 in B, J, K, N, and O; ×570 in C; ×3400 in D; ×40,000 in E; ×2000 in F; ×6300 in G; ×12,500 in I; ×750 in L and M.

Figure 3.

Parietal capillaries are in continuity with peritubular capillaries in β-catenin–deficient mice. (A) Superficial parietal capillary in continuity with a peritubular capillary (arrow). T, tubule; G, glomerulus. (B) Glomerular profile with an efferent arteriole running alongside the outer glomerular circumference (arrows). (C) Cross section through an efferent arteriole associated with parietal podocytes and Bowman's capsule. Note the continuous endothelium and the many profiles of smooth muscle cells (asterisks). G, glomerulus. (D) Glomerular profile with a misshapen vascular pole. The afferent (a) and efferent (e) arterioles are widely separated; the vascular pole in between appears to be unorganized. (E) Cells of the proximal tubule (left) appear cuboidal with microvilli at the apical side forming the brush border. They are densely packed with mitochondria. Parietal podocytes (right) are flat and characterized by foot processes. Between those cell types, there is always a morphologically different epithelial cell (EC), which has none of the morphological characteristics of the neighboring cell types. Original magnifications: ×500 in A, B, and D; ×5000 in C; ×2500 in E.

Figure 4.

Parietal podocytes develop directly from the parietal layer of S-shaped bodies in β-catenin–deficient mice. (A) Immunostaining for β-catenin reveals β-catenin expression in all cells of the comma-shaped body in both control (lox/lox) and β-catenin–deficient (lox/lox Pax8Cre) mice. (B and C) At the S-shaped body stage, β-catenin is expressed in precursor cells of the tubules and the parietal epithelial cells (PECs) (arrowheads) but only weakly in visceral epithelial cells (VECs) (asterisks) (boxed areas enlarged in C). There is no difference between control and β-catenin–deficient mice at this stage. (D and E) In maturing glomeruli, β-catenin staining is only observed in PECs (arrowheads) of control mice. VECs (asterisks) are neither positive in control nor in β-catenin–deficient mice (boxed areas enlarged in E). (F) At the S-shaped body stage, WT1 stains positive in both VECs (asterisks) and PECs (arrowheads). (G) In maturing glomeruli of control mice, WT1 expression is still observed in VECs (asterisks) but not in PECs (arrowheads). In β-catenin–deficient mice, both VECs and PECs remain positive for WT1 expression. (H) At the S-shaped body stage, VECs (asterisks) are strongly positive for VEGF, whereas PECs (arrowheads) are only weakly positive. (I) In maturing glomeruli, positive VEGF staining is observed in VECs (asterisks) but not in PECs (arrowheads) in control mice. In β-catenin–deficient mice, both VECs and PECs remain positive for VEGF. (J through N) Transmission electron micrographs of developing glomeruli. (J–L) S-shaped body stage. (M and N) Capillary loop stage. At the S-shaped body stage, presumptive PECs (asterisks) of control mice are flat (J), whereas the presumptive PECs from β-catenin–deficient mice (K and L) are columnar in shape (asterisks) and associated with capillaries (arrows). At the maturing glomerulus stage from a β-catenin–deficient mouse (M) (accompanied by a schematic draft in N), PECs have developed foot processes lining up along the parietal basement membrane (blue in the scheme) as have the VECs on the glomerular basement membrane (red in the scheme). Large capillaries (lumina are orange in the scheme) are found at the outer side of the parietal basement membrane. E18.5. Original magnifications: ×750 in A–I; ×5000 in J; ×2000 in K and L; ×1500 in M.

Figure 5.

The cell fate decision of parietal epithelial cells of the glomerulus is dependent on β-catenin/Wnt signaling. β-Catenin/Wnt signaling directs proper differentiation of prospective parietal epithelial cells. In the absence of β-catenin/Wnt signaling, parietal epithelial precursor cells switch to the visceral, podocyte-specific cell fate and become parietal podocytes (PPs). DT, distal tubule; PT, proximal tubule; VECs, visceral epithelial cells; PECs, parietal epithelial cells.