Ectopic ureter budding in <i>Gata3^ND−/</i>⁻ embryos depends on GDNF/Ret and Fgf signaling.

<p>(A, B) E10.5 UGS organ cultures recapitulate the phenotype of both control and <i>Gata3^ND−/</i>⁻ embryos observed <i>in vivo</i>. (C, D) Treatment of cultures with the Ret kinase inhibitor SU5416 efficiently inhibits both primary (*) and ectopic budding in control and <i>Gata3^ND−/</i>⁻ UGSs. (E, F) Supplementing the organ cultures with recombinant GDNF induces ectopic ureteric budding in control UGS cultures (arrows in E) and enhances ectopic bud growth in <i>Gata3^ND−/</i>⁻ UGS cultures (arrows in F). (G, H) Organ culture of control or <i>Gata3</i>-deficient UGSs treated with a GDNF blocking antibody suppresses primary and ectopic bud formation, respectively. (I, J) Soluble FgfR2-Fc fusion protein has no major effect on primary budding in control UGSs, but successfully inhibits ectopic budding in <i>Gata3^ND−/</i>⁻ UGSs. ub, ureteric bud; *, primary bud agenesis.</p

<i>Gata3</i> regulates cell differentiation in a Ret independent manner.

<p>(A,C) Co-immunostainings for β-catenin and Zo1+ reveal an increase of Zo1+ expression in <i>Gata3</i>-deficient nephric ducts at E11.5. (B, D) Similarly, E-cadherin/DBA co-labeling shows stronger DBA staining in <i>Gata3^ND−/−</i> embryos. (E,F and A,B) No change in Zo1+ or DBA expression was observed between <i>Ret^−/−</i> and control embryos at E11.5. The <i>HoxB7</i>-driven Venus signal outlines the nephric duct. (G) Quantification of the DBA positive cells reveal a tenfold increase of DBA+ cells in <i>Gata3^ND−/−</i> embryos when compared to control and <i>Ret^−/−</i> embryos (n = 4, P<0.001, Student's t-test).</p

Ectopic ureteric budding in <i>Gata3^ND−/−</i> embryos.

<p>(A–F) Dissected urogenital systems of control (<i>HoxB7-Cre</i>; <i>Gata3^flox/+</i>; <i>Rosa26^STOPlacZ/+</i>) and <i>Gata3^ND−/−</i> (<i>HoxB7-Cre</i>; <i>Gata3^flox/flox</i>; <i>Rosa26^STOPlacZ/+</i>) embryos were stained for β-Galactosidase activity at the indicated embryonic stages. (A, B) Swelling of the caudal portion of the nephric duct (nd) at E10.5 marks the ureteric bud induction site (ub) in control and <i>Gata3</i> mutant embryos. (C, D) At E11.5, the ureteric bud of control embryos invades the metanephric mesenchyme to form a T-stage kidney. In <i>Gata3^ND−/</i>⁻ embryos, ectopic buds form along the entire length of the nephric duct (arrowheads). <i>Gata3</i>-deficient embryos frequently fail to induce the primary bud (*). (E, F) At E12.5 the metanephric kidney (k) undergoes its third round of branching morphogenesis in control embryos. In <i>Gata3^ND−/</i>⁻ embryos, some ectopic buds give rise to ectopic kidneys (ek) showing deficient branching morphogenesis, whereas the most rostral buds start to regress. Note the aberrant branch point of the ectopic kidneys (red arrowhead).</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

<i>Gata3^ND−/−</i> embryos display multiple urogenital system abnormalities.

<p>Urogenital systems were dissected at E18.5 and either processed as whole mount (A–G) or subjected to H&E analysis on tissue sections (H–M). (A) Wild-type male UGS showing normal kidneys (k) and vas deferens (vd). (B) UGS of a <i>Gata3^ND−/</i>⁻ male embryo, showing unilateral kidney agenesis (*), an aplastic kidney (ak) and bilateral vas deferens dilations (white arrowheads). (C) Wild-type female UGS harboring a normal uterus (ut). (D) Female <i>Gata3^ND−/−</i> UGS, with dysplastic kidneys (dk) and bilateral agenesis of the uterus (white arrowheads point to remaining connective tissue). (E) Wild-type kidney and ureter. (F) Duplex kidneys and ureters (white arrowheads) and (G) hydroureter, hydronephrosis and kidney dysplasia are frequently observed in conditional <i>Gata3^ND−/</i>⁻ embryos. (H, I) H&E histological analysis of wild-type (H) and <i>Gata3^ND−/−</i> (I) kidneys reveals severe kidney dysplasia and hydronephrosis affecting the collecting duct system and all nephron segments. (J, K) Histological analysis of wild-type (J) and <i>Gata3^ND−/−</i> (K) male genital tracts highlighting the dilated and fluid filled vas deferens in mutant embryos. (L, M) H&E staining of wild-type (L) and <i>Gata3^ND−/−</i> (M) female genital tract, confirming uterus agenesis (*) and showing normal morphology of the ovaries (ov) and oviducts (od) in mutant embryos. cd, collecting duct; dt, distal tubule; g, glomerulus; pt, proximal tubule.</p

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