β-Catenin is necessary to keep cells of ureteric bud/Wolffian duct epithelium in a precursor state

AbstractDifferentiation is the process by which tissues/organs take on their final, physiologically functional form. This process is mediated in part by the silencing of embryonic genes and the activation of terminal, differentiation gene products. Mammalian kidney development is initiated when the Wolffian duct branches and invades the overlying metanephric mesenchyme. The newly formed epithelial bud, known as the ureteric bud, will continue to branch ultimately differentiating into the collecting duct system and ureter. Here, we show that Hoxb7-Cre mediated removal of β-catenin from the mouse Wolffian duct epithelium leads to the premature expression of gene products normally associated with the differentiated kidney collecting duct system including the water channel protein, Aquaporin-3 and the tight junction protein isoform, ZO-1α+. Mutant cells fail to maintain expression of some genes associated with embryonic development, including several mediators of branching morphogenesis, which subsequently leads to kidney aplasia or hypoplasia. Reciprocally, expression of a stabilized form of β-catenin appears to block differentiation of the collecting ducts. All of these defects occur in the absence of any effects on the adherens junctions. These data indicate a role for β-catenin in maintaining cells of the Wolffian ducts and the duct derived ureteric bud/collecting duct system in an undifferentiated or precursor state

Gata3 Acts Downstream of β-Catenin Signaling to Prevent Ectopic Metanephric Kidney Induction

Metanephric kidney induction critically depends on mesenchymal–epithelial interactions in the caudal region of the nephric (or Wolffian) duct. Central to this process, GDNF secreted from the metanephric mesenchyme induces ureter budding by activating the Ret receptor expressed in the nephric duct epithelium. A failure to regulate this pathway is believed to be responsible for a large proportion of the developmental anomalies affecting the urogenital system. Here, we show that the nephric duct-specific inactivation of the transcription factor gene Gata3 leads to massive ectopic ureter budding. This results in a spectrum of urogenital malformations including kidney adysplasia, duplex systems, and hydroureter, as well as vas deferens hyperplasia and uterine agenesis. The variability of developmental defects is reminiscent of the congenital anomalies of the kidney and urinary tract (CAKUT) observed in human. We show that Gata3 inactivation causes premature nephric duct cell differentiation and loss of Ret receptor gene expression. These changes ultimately affect nephric duct epithelium homeostasis, leading to ectopic budding of interspersed cells still expressing the Ret receptor. Importantly, the formation of these ectopic buds requires both GDNF/Ret and Fgf signaling activities. We further identify Gata3 as a central mediator of β-catenin function in the nephric duct and demonstrate that the β-catenin/Gata3 pathway prevents premature cell differentiation independently of its role in regulating Ret expression. Together, these results establish a genetic cascade in which Gata3 acts downstream of β-catenin, but upstream of Ret, to prevent ectopic ureter budding and premature cell differentiation in the nephric duct

In vitro analysis of the <i>β-catenin</i>-<i>Gata3</i>-<i>Ret</i> molecular cascade.

<p>(A, B) Bioinformatic analysis of <i>Gata3</i> and <i>Ret</i> genomic loci 50 kb upstream of the translational start site. Conserved regions between mouse and human sequences are depicted as thick black lines. Conserved TCF/Lef binding sites (consensus sequence (C/G)TTG(A/T)(A/T)) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000316#pgen.1000316-Waterman1" target="_blank">[69]</a> (A) and Gata3 binding sites (consensus sequence (A/T)GATA(A/T)) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000316#pgen.1000316-Orkin1" target="_blank">[70]</a> (B), are represented as asterisks. (B) A putative Gata3 binding site located in a previously reported <i>RET</i> regulatory element <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000316#pgen.1000316-Fisher1" target="_blank">[50]</a> and the mutated version used in (D) are indicated in brackets. (C) Quantitative RT-PCR analysis of <i>Gata3</i> expression in mIMCD3 reveals a marked increase in endogenous <i>Gata3</i> expression upon treatment with 3 µM BIO versus DMSO control. Results are expressed as ratios relative to control (n = 3, ** P<0.01, Student's t-test). (D) β-Galactosidase reporter activity is decreased in Gata3-expressing mIMCD3 cells transfected with an expression construct containing the mutated Gata3 binding sequence (as indicated in B), when compared to the wild-type control. Results are expressed as ratios relative to control. (n = 3, * P<0.05, Student's t-test).</p

Gata3 is necessary for maintenance of <i>Ret</i> expression.

<p>(A–F) Gata3 maintains <i>Ret</i> expression in the nephric duct at E11.5. (A) Whole mount in situ hybridization of control UGS with a <i>Ret</i> cRNA probe shows the smooth gradient of <i>Ret</i> expression along the entire length of the nephric duct. (B) In <i>Gata3^ND−/−</i> embryos, <i>Ret</i> expression is lost in most nephric duct cells except for some cells located in the ectopic ureteric bud tips (arrows). (C,D) In situ hybridizations with a <i>Gata3</i> cRNA probe specific for exon 4 confirm that the <i>Ret</i> expressing cells in the ectopic buds still express <i>Gata3</i>. In these embryos, <i>Gata3</i>+ cells segregate from <i>Gata3</i>− cells. A similar tendency is observed in <i>Gata3^ND−/−</i> embryos stained for β-Galactosidase activity (D insert). (E, F) The caudal nephric duct efficiently deleted <i>Gata3</i> already at this stage. (G–L) Deficient branching morphogenesis and nephron differentiation in <i>Gata3^ND−/−</i> hypodysplastic kidneys. (G,H) In situ hybridization with the <i>Gata3-exon 4</i> cRNA probe confirms <i>Gata3</i> inactivation in <i>Gata3^ND−/−</i> metanephric kidneys. (I, J) <i>Ret</i> expression becomes strongly downregulated in highly dysplastic <i>Gata3</i>-mutant kidneys, in contrast to control kidneys. (J insert) Milder kidney dysplasia showing a pattern of <i>Ret</i> expression intermediate between wild-type and highly dysplastic kidneys. (K,L) <i>Fgf8</i> in situ hybridizations reveal impaired nephron induction in <i>Gata3^ND−/−</i> kidneys. Tissue sections in D (insert) and I–L are counterstained with nuclear fast red. nd, nephric duct; cl, cloaca; ut, ureter tip; rv, renal vesicle.</p

Genital tract anomalies in <i>Gata3^ND−/−</i> embryos.

<p>(A, B) β-Galactosidase staining of E14.5 genital ridges marks the beginning of Wolffian duct hyperplasia in male <i>Gata3^ND−/</i>⁻ embryos (red arrowhead in B). (C, D) Immunostainings for the mitosis marker phosphorylated Histone H3 (p-H3) show an increase in cell proliferation (n = 4, P = 0.09, Student's t-test) in <i>Gata3^ND−/−</i> nephric ducts at E13.5. E-cadherin labeling marks the genital tracts. (E–H) Whole-mount in situ hybridizations for <i>Emx2</i> (E, F) or <i>Wnt4</i> (G, H) of control and <i>Gata3^ND−/−</i> E13.5 genital ridges reveal an arrest of Müllerian duct elongation (red arrowhead in F, H) in <i>Gata3^ND−/−</i> embryos. Notably, the elongation arrest occurs at the level of persisting ectopic ureteric buds (black arrows in B). nd, nephric duct; md, Müllerian duct; k, kidney; g, gonad.</p

Molecular characterization of ectopic ureteric bud formation in <i>Gata3^ND−/−</i> embryos.

<p>(A, B) At E10.5 in <i>Gata3^ND−/−</i> embryos, the Ret+ nephric duct cells (nd) segregate from Ret− cells prior to budding. (C,D) Adjacent sections show that Ret+ cells are localized next to <i>GDNF</i> expressing cells in the metanephric mesenchyme (mm). (E, F) Ectopic kidneys (ek) maintain mesenchymal <i>GDNF</i> expression in the metanephric mesenchyme at E11.5. (G, H) Induced ectopic ureteric buds in <i>Gata3</i>-deficient embryos strongly upregulate the expression of <i>Wnt11</i> at E10.5. (I–L) <i>Slit2</i> and <i>Spry1</i> are slightly downregulated in <i>Gata3</i> mutant (nd) cells at E10.5. (M,N) FgfR2-Fc staining shows a strong but unaffected expression of Fgf ligands in both control and <i>Gata3^ND−/−</i> embryos at E11.5. (O,P) <i>Fgf10</i> is expressed in the mesenchymal cells adjacent to the nephric duct in both wild-type and <i>Gata3^ND−/−</i> embryos. nd k: kidney, ur ureter.</p

β-catenin regulates <i>Gata3</i> expression in the nephric duct.

<p>(A, B) In situ hybridizations with a <i>Gata3</i> cRNA probe reveal a strong downregulation of <i>Gata3</i> expression in the nephric duct of E11.5 <i>Ctnnb1^ND−/−</i> embryos. (C, D) Likewise, in situ hybridizations with a <i>Ret</i> probe show a strong decrease of <i>Ret</i> expression in <i>Ctnnb1</i>-deficient nephric ducts. (E, F) Adjacent sections stained for the β-catenin transcriptional target <i>Axin2</i>, confirm loss of β-catenin signaling in <i>Ctnnb1^ND−/−</i> embryos. (G,H) Phosphorylated β-catenin levels are not significantly perturbed in <i>Gata3^ND−/−</i> embryos. E-cadherin was used as a nephric duct marker. (I–L) In situ hybridizations for <i>Axin2</i> (I, J) and <i>Daple</i> (K, L) show a normal β-catenin signaling response in <i>Gata3^ND−/−</i> nephric ducts.</p

Generation of a conditional <i>Gata3</i> knockout allele.

<p>(A) The conditional <i>Gata3</i> knockout allele was generated by crossing <i>Gata3^ex4GFP</i> mice with <i>FLPe</i> germline deleter mice. The FLPe recombinase removed the <i>Ires-GFP-neo</i> cassette, thereby generating a <i>Gata3</i> allele with <i>loxP</i> sites flanking exon 4 (<i>Gata3^flox</i>). Subsequent crosses of <i>Gata3^flox</i> mice with <i>More-Cre</i> germline deleter mice resulted in <i>Gata3</i>-exon 4 excision and the creation of a null allele (<i>Gata3^D</i>). (B) Representative genotyping by PCR for wild-type (+/+), <i>Gata3^flox/flox</i> (flox/flox) and <i>Gata3^Δ/+</i> (Δ/+) mice prepared from tail DNA.</p

Summary of Gata3 function in progenitor cell state maintenance in the nephric duct.

<p>(A) Genetic regulation experiments with <i>Gata3</i>, <i>β-catenin</i> and <i>Ret</i> mutant embryos identify a cascade whereby <i>Gata3</i> acts downstream of the <i>β-catenin</i> developmental program to independently maintain <i>Ret</i> expression and to prevent premature differentiation of nephric duct cells. (B–D) Panel B- In wild-type nephric duct cells, high levels of Gata3 expression maintain nephric duct homeostasis. Panel C- A critical downregulation in Gata3 expression levels (dark blue cells) in <i>Gata3^ND−/−</i> mice (and possibly also in HDR-patients) causes an imbalance in the response to local growth factors such as GDNF and Fgfs (green circles). This may come as a result of premature cell differentiation and differential cell adhesion properties of <i>Gata3</i>-deficient cells. Panel D- Consequently, cells with sufficient levels of Gata3 (and Ret) segregate from <i>Gata3</i>-mutant cells and expand, thereby forming ectopic buds and kidneys.</p