doi: 10.1242/10.1242/dev.00478
Elevated SMAD1/ß-catenin molecular complexes and renal medullary cystic dysplasia in ALK3 transgenic mice
Ming Chang Hu1,
Tino D. Piscione1,2 and
Norman D. Rosenblum1,2,*
1 Program in Developmental Biology, Research Institute, The Hospital for Sick
Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada
2 Division of Nephrology, Department of Paediatrics, University of Toronto, 555
University Avenue, Toronto, Ontario M5G 1X8, Canada
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Fig. 1. Transgenic mice overexpressing ALK3QD. (A) Structure of the
transgene. The positions of the Hoxb7 promoter, Q233D mutation, the
HA tag, and the human ß-globin sequence are shown. (B) Western blot
analysis of proteins isolated from mouse kidney at P10 showing expression of
HA-tagged ALK3 in TgALK3QD mice and a marked increase in
ALK3-HA levels in TgALK3QD/QD mice. (C)
ALK3QD-HA expression in TgALK3QD mice at P10.
Expression of the HA epitope was detected by immunohistochemistry using an
anti-HA antibody (brown stain). HA was undetectable in the cortex and medulla
of control kidneys. In hemizygous (TgALK3QD) mice, low
levels of HA (arrow) were detected in cortical collecting ducts (T) and in
medullary collecting ducts but not in glomeruli (G). In homozygous
(TgALK3QD/QD) mice, much higher levels of HA were detected
in a small subset of cortical tubules (T) and in medullary collecting ducts
(T).
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Fig. 2. Renal phenotype of TgALK3QD and
TgALK3QD/QD mice at E18.5 (A-F) and P10 (G-L) in
representative 4 µm tissue sections. Sections in A-J are stained with
Haematoxylin and Eosin. (A-F) E18.5. (A) The kidney of control mice is
characterized by a well-differentiated cortex (C) and medulla (M). The box
highlights the outer cortex. (B) Higher magnification view of the cortex in a
control kidney. It is characterized by glomerular progenitors in the outer
cortex and a mature glomerulus (G) in the deep cortex. (C) Higher
magnification view of the medulla in a control kidney. It consists of a
densely packed linear array of tubules. Note the lack of intervening
mesenchyme. (D) The kidney of TgALK3QD/QD mice is
characterized by decreased density of immature glomeruli in the outer cortex,
cortical cysts, a paucity of medullary collecting ducts in the medulla, a
marked increase in mesenchyme in the medulla and medullary cysts. (E) Higher
magnification view of the kidney cortex of a TgALK3QD/QD
mouse. Note the decreased density of glomerular progenitors in the outer
cortex (compared with B) and the presence of a cyst (Cy). (F) Higher
magnification view of the kidney medulla of a TgALK3QD/QD
mouse. Note the cystic dilatation of tubules, the decreased density of tubules
and the increase in intervening mesenchyme. (G-L) P10. (G) The kidney of a
control mouse is characterized by a well-developed cortex, medulla and papilla
(P). (H) The kidney of a TgALK3QD mouse is smaller in
area. It exhibits fewer medullary tubules, has cysts in the cortex and medulla
and has a smaller papilla. (I) The kidney of a TgALK3QD/QD
mouse is malformed by large cysts in the cortex and medulla. The outer cortex
is compressed. The medulla contains few tubules and shows a large increase in
intervening mesenchyme. (J) Higher power view of the renal medulla of a
TgALK3QD/QD mouse. Note the cystic tubules. Some are
characterized by a cuboidal epithelium (Cy), whereas others have a squamous
epithelium. The intertubular mesenchyme is increased and contains fibroblastic
cells. (K,L) A medullary cyst in the kidney of a
TgALK3QD/QD mouse stained with DBA. (L) Positive staining
indicates that the cyst epithelium is of ureteric bud origin.
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Fig. 3. Cellular changes in the kidneys of TgALK3QD/QD mice.
(A) Cell proliferation in kidney tissue of P10 mice detected by anti-Ki67 and
HRP-conjugated secondary antibodies. Left: Hematoxylin-stained tissue
demonstrates a marked increase in the number of cells expressing Ki-67 (red)
in non-cystic and cystic tissue elements of Tg mouse kidneys compared with
control kidneys. Right: quantitation of cell proliferation showed a 6.4-fold
and 17.1-fold increase in non-cystic and cystic tissue elements, respectively,
of TgALK3QD/QD versus control mice. (B) E-cadherin
expression is decreased in TgALK3QD/QD kidney tissue.
Left: quantitation of E-cadherin protein in whole kidney lysate demonstrated a
64% decrease in TgALK3QD/QD versus control mice. Right:
immunohistochemistry using anti-E-cadherin and HRP-conjugated secondary
antibodies. E-cadherin is expressed in all epithelial tubules in control
kidneys. By contrast, in TgALK3QD/QD kidney tissue,
expression is reduced in a subset of tubules with cuboidal (*) or squamous (#)
epithelium. (C) MYC expression is increased in TgALK3QD/QD
kidney tissue. Left: quantitation of MYC protein in whole kidney lysate
demonstrated a 542% increase in TgALK3QD/QD versus control
mice. Right: immunohistochemistry using anti-MYC and HRP-conjugated secondary
antibodies. MYC is almost undetectable in control kidneys. By contrast, in
TgALK3QD/QD kidney tissue, expression is markedly
increased in cystic epithelium (red color marking cell nuclei). Data are
mean±s.d. Number of independent experiments were: E-cadherin,
n=4; MYC, n=6.
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Fig. 4. Renal branching morphogenesis is reduced in kidneys from E13.5
TgALK3QD/QD embryos. (A) Ureteric bud branching in
TgALK3QD/QD versus control kidneys. Left: DBA-stained
whole-mount preparations of E13.5 kidneys. Ureteric bud branches are
highlighted. Right: quantitation of branch points demonstrated 36% fewer
branches in TgALK3QD/QD versus control kidneys. (B)
Ureteric bud cell proliferation in TgALK3QD/QD versus
control kidneys. Left: Hematoxylin-stained E13.5 kidney tissue. BrdU
incorporation is identified by HRP-conjugated mouse anti-BrdU antibody
yielding red colored cell nuclei. Note the marked reduction of BrdU uptake in
the ureteric bud (UB) of TgALK3QD/QD mice. By contrast,
BrdU in glomerular progenitors and mesenchymal cells is similar in
TgALK3QD/QD and control kidney sections. Right:
quantitation of the percentage of BrdU-positive ureteric bud cells
demonstrated a 60% reduction in TgALK3QD/QD versus control
mice. Data are mean±s.d from three separate experiments.
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Fig. 5. Phospho-SMAD1 expression is increased in the P10
TgALK3QD/QD kidney. (A) Phospho-SMAD1 expression in
TgALK3QD/QD kidney. Left: Hematoxylin-stained control
kidney tissue (medulla) stained with anti-phospho-SMAD1 antibody demonstrates
very weak expression of phospho-SMAD1. Middle: Hematoxylin-stained tissue from
a TgALK3QD/QD mouse demonstrating a marked increase in
phospho-SMAD1 expression. Right: Hematoxylin-stained tissue containing an
epithelial cyst showing phospho-SMAD1 expression in cells lining the cyst. (B)
Quantification of SMAD1 and phospho-SMAD1 protein expression in whole kidney
lysate. Left: expression of total SMAD1 (unphosphorylated and phosphorylated)
is similar in kidneys from control (white), TgALK3QD (QD;
gray) and TgALK3QD/QD (QD/QD, black) mice. Right:
phospho-SMAD1 expression is increased by 51% in TgALK3QD
versus control kidney, and by 86% in TgALK3QD/QD kidneys.
Representative blots are shown above. Data are mean±s.d. from four
separate experiments.
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Fig. 6. ß-catenin is increased in postnatal TgALK3QD/QD
mouse kidney tissue. (A) Immunohistochemistry of kidney tissue from control
and TgALK3QD/QD mice at P10, using rabbit anti-human
ß-catenin and HRP-conjugated anti-rabbit antibodies. Top left panels:
Hematoxylin-stained kidney tissue from control mouse demonstrates absence of
detectable ß-catenin protein in the medulla. Hematoxylin-stained kidney
tissue from TgALK3QD/QD mouse demonstrates marked
upregulation of ß-catenin expression in a subset of tubules and
epithelial cysts in the medulla. Bottom left panels: higher power view of
kidney cortex of a TgALK3QD/QD mouse. Low levels of
ß-catenin were detected. By contrast, ß-catenin levels in the
medulla of a TgALK3QD/QD mouse kidney are greatly
increased in dilated tubules (cysts). Right panel: ß-catenin expression
in whole kidney lysate was increased by 46% in TgALK3QD
(gray) versus control (white) mice, and by a further 60% in
TgALK3QD/QD (black) mice. A representative blot is shown
above. (B) Activation of ALK3 signaling increases the activity of a
ß-catenin/TCF reporter gene in vivo. ß-galactosidase activity was
assayed in the kidneys of newborn QD/QD, Tcf-gal and QD/QD;
Tcf-gal mice. Top panels demonstrate ß-gal expression in
Eosin-stained cross-sections of the entire kidney (composite image). Lower
panels demonstrate ß-gal expression in representative corresponding
tissue sections. No ß-galexpression was detected in QD/QD
kidneys. Weak expression was detected in Tcf-gal mice. By contrast,
ß-gal expression was markedly increased in QD/QD; Tcf-gal mouse
kidney, with localization in tubular epithelium. Data are mean±s.d.
from three independent experiments.
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Fig. 7. SMAD1 and ß-catenin are associated in molecular complexes in
TgALK3QD/QD (QD/QD) mice. Molecular associations
in postnatal kidney tissue (P10) isolated from different mice were assayed by
immunoprecipitation-immunoblot assays using specific antisera. Immunoblots and
quantitation are shown in the left and right panels, respectively. (A) The
amount of ß-catenin associated with SMAD1 was increased ninefold in Tg
mice versus controls. (B) The amount of phospho-SMAD1 associated with
ß-catenin was increased ninefold in Tg mice versus controls. (C) The
amount of SMAD4 associated with ß-catenin was increased twofold in Tg
mice versus controls. Data are mean±s.d. from three independent
experiments.
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Fig. 8. Phospho-SMAD1 and ß-catenin expression is increased in human
dysplastic fetal kidney. (A-C) Phospho-SMAD1 expression detected by
immunohistochemistry using anti-phospho-SMAD1 and HRP-conjugated secondary
antibodies. (A) Phospho-SMAD1 is undectable in normal Hematoxylin-stained
tissue. (B) Phospho-SMAD1 is markedly unregulated in Hematoxylin-stained
dysplastic tissue (red cells), including a branched ureteric bud (arrowhead)
and in cysts (C). (D-I) ß-catenin expression detected by
immunohistochemistry using anti-ß-catenin and HRP-conjugated secondary
antibodies. (D,G) ß-catenin is weakly expressed in epithelial tubules
(arrows) of Hematoxylin-stained normal kidney tissue. (E,F) ß-catenin is
highly expressed in epithelial tubules (arrows) of Hematoxylin-stained
dysplastic kidney tissue. (H,I) ß-catenin is highly expressed in the
epithelium of ureteric bud-derived cysts. (H) ß-catenin is highly
expressed in cyst epithelium (arrow). (I) Fluoresence image of cyst in H
stained with fluorescence-conjugated DBA. Positive staining demonstrates that
the cyst is derived from the ureteric bud.
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