Limits...
Gata3 acts downstream of beta-catenin signaling to prevent ectopic metanephric kidney induction.

Grote D, Boualia SK, Souabni A, Merkel C, Chi X, Costantini F, Carroll T, Bouchard M - PLoS Genet. (2008)

Bottom Line: This results in a spectrum of urogenital malformations including kidney adysplasia, duplex systems, and hydroureter, as well as vas deferens hyperplasia and uterine agenesis.We further identify Gata3 as a central mediator of beta-catenin function in the nephric duct and demonstrate that the beta-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 beta-catenin, but upstream of Ret, to prevent ectopic ureter budding and premature cell differentiation in the nephric duct.

View Article: PubMed Central - PubMed

Affiliation: Goodman Cancer Centre, McGill University, Quebec, Canada.

ABSTRACT
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 beta-catenin function in the nephric duct and demonstrate that the beta-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 beta-catenin, but upstream of Ret, to prevent ectopic ureter budding and premature cell differentiation in the nephric duct.

Show MeSH

Related in: MedlinePlus

β-catenin regulates Gata3 expression in the nephric duct.(A, B) In situ hybridizations with a Gata3 cRNA probe reveal a strong downregulation of Gata3 expression in the nephric duct of E11.5 Ctnnb1ND−/− embryos. (C, D) Likewise, in situ hybridizations with a Ret probe show a strong decrease of Ret expression in Ctnnb1-deficient nephric ducts. (E, F) Adjacent sections stained for the β-catenin transcriptional target Axin2, confirm loss of β-catenin signaling in Ctnnb1ND−/− embryos. (G,H) Phosphorylated β-catenin levels are not significantly perturbed in Gata3ND−/− embryos. E-cadherin was used as a nephric duct marker. (I–L) In situ hybridizations for Axin2 (I, J) and Daple (K, L) show a normal β-catenin signaling response in Gata3ND−/− nephric ducts.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2597718&req=5

pgen-1000316-g008: β-catenin regulates Gata3 expression in the nephric duct.(A, B) In situ hybridizations with a Gata3 cRNA probe reveal a strong downregulation of Gata3 expression in the nephric duct of E11.5 Ctnnb1ND−/− embryos. (C, D) Likewise, in situ hybridizations with a Ret probe show a strong decrease of Ret expression in Ctnnb1-deficient nephric ducts. (E, F) Adjacent sections stained for the β-catenin transcriptional target Axin2, confirm loss of β-catenin signaling in Ctnnb1ND−/− embryos. (G,H) Phosphorylated β-catenin levels are not significantly perturbed in Gata3ND−/− embryos. E-cadherin was used as a nephric duct marker. (I–L) In situ hybridizations for Axin2 (I, J) and Daple (K, L) show a normal β-catenin signaling response in Gata3ND−/− nephric ducts.

Mentions: The developmental defects of Gata3ND−/− embryos described above show striking similarities with the phenotypes resulting from the conditional inactivation of β-catenin (ctnnb1) in the nephric duct [45]. The fact that both animal models have the same spectrum of genital tract and kidney defects, prompted us to verify whether Gata3 and β-catenin act in the same genetic pathway. For this, we first performed in situ hybridization against Gata3 in E11.5 HoxB7-Cre; Ctnnb1flox/− embryos (Ctnnb1ND−/−) and found a strong downregulation of Gata3 expression in the nephric duct in comparison to control embryos (Figure 8A,B). Accordingly, in situ hybridizations with a Ret cRNA probe revealed a loss of Ret expression in Ctnnb1ND−/− embryos (Figure 8C,D), which mimics the loss of Ret expression in Gata3ND−/− embryos (Figure 5). Staining of adjacent tissue sections with in situ probes for the canonical Wnt target genes Axin2, Sp5 and Daple [46],[47] confirmed the loss of β-catenin transcriptional response in Ctnnb1ND−/− embryos (Figure 8E,F and data not shown). In order to clarify the genetic hierarchy between Gata3 and β-catenin, we next stained Gata3ND−/− embryos with antibodies against β-catenin and phospho-β-catenin. These experiments failed to show any modification of β-catenin expression levels or activity following Gata3 inactivation (Figure 8G,H and data not shown). In support of this, the expression of the canonical Wnt-signaling target genes Axin2, Daple and Sp5 was unchanged in Gata3ND−/− embryos (Figure 8I–L and data not shown). From these data, we conclude that Gata3 acts genetically downstream of β-catenin but upstream of Ret in the nephric duct. To further characterize the molecular basis of the β-catenin-Gata3-Ret pathway, we first performed a bioinformatics analysis of the 50 kb genomic region upstream of the mouse and human Gata3 genes. Highly conserved sequences shared by the two species were further analyzed for putative binding sites for TCF/Lef. In total, 10 putative TCF/Lef binding sites, located in 5 of the 8 conserved regions, were identified (Figure 9A). In this analysis, we additionally included a Gata3 urogenital enhancer located at −110 kb [48], but failed to detect any conserved TCF/Lef binding sites in this element. A similar bioinformatics analysis of the Ret regulatory region revealed putative Gata3 binding sites in 4 of the 11 conserved regions 50 kb upstream of the Ret-ATG. (Figure 9B). The potential of β-catenin to regulate endogenous Gata3 expression was further evaluated in mouse IMCD3 collecting duct-derived cells. Using the GSK3β inhibitor BIO to stabilize the β-catenin protein [49], we observed a significant increase of Gata3 expression levels in those cells (Figure 9C). In order to assess the activity of Gata3 on the Ret regulatory region, we took advantage of the fact that one of the Gata3 binding sites mapped to a region previously reported to drive reporter gene expression in the zebrafish pronephros (Figure 9B, large asterisk) [50]. To test whether Gata3 acts on this site, we isolated the 1.2 kb conserved fragment and introduced a specific point mutation in the Gata3 binding site (Figure 9B). The wild-type and mutated fragments cloned upstream of a β-Gal reporter construct were transfected in IMCD3 cells stably expressing Gata3. The inactivation of the Gata3 binding site led to a significant downregulation of β-Gal expression relative to the wild-type control (Figure 9D), thereby suggesting that Gata3 may regulate Ret expression directly from this binding site. Together these data are consistent with a β-catenin-Gata3-Ret genetic cascade in the nephric duct.


Gata3 acts downstream of beta-catenin signaling to prevent ectopic metanephric kidney induction.

Grote D, Boualia SK, Souabni A, Merkel C, Chi X, Costantini F, Carroll T, Bouchard M - PLoS Genet. (2008)

β-catenin regulates Gata3 expression in the nephric duct.(A, B) In situ hybridizations with a Gata3 cRNA probe reveal a strong downregulation of Gata3 expression in the nephric duct of E11.5 Ctnnb1ND−/− embryos. (C, D) Likewise, in situ hybridizations with a Ret probe show a strong decrease of Ret expression in Ctnnb1-deficient nephric ducts. (E, F) Adjacent sections stained for the β-catenin transcriptional target Axin2, confirm loss of β-catenin signaling in Ctnnb1ND−/− embryos. (G,H) Phosphorylated β-catenin levels are not significantly perturbed in Gata3ND−/− embryos. E-cadherin was used as a nephric duct marker. (I–L) In situ hybridizations for Axin2 (I, J) and Daple (K, L) show a normal β-catenin signaling response in Gata3ND−/− nephric ducts.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2597718&req=5

pgen-1000316-g008: β-catenin regulates Gata3 expression in the nephric duct.(A, B) In situ hybridizations with a Gata3 cRNA probe reveal a strong downregulation of Gata3 expression in the nephric duct of E11.5 Ctnnb1ND−/− embryos. (C, D) Likewise, in situ hybridizations with a Ret probe show a strong decrease of Ret expression in Ctnnb1-deficient nephric ducts. (E, F) Adjacent sections stained for the β-catenin transcriptional target Axin2, confirm loss of β-catenin signaling in Ctnnb1ND−/− embryos. (G,H) Phosphorylated β-catenin levels are not significantly perturbed in Gata3ND−/− embryos. E-cadherin was used as a nephric duct marker. (I–L) In situ hybridizations for Axin2 (I, J) and Daple (K, L) show a normal β-catenin signaling response in Gata3ND−/− nephric ducts.
Mentions: The developmental defects of Gata3ND−/− embryos described above show striking similarities with the phenotypes resulting from the conditional inactivation of β-catenin (ctnnb1) in the nephric duct [45]. The fact that both animal models have the same spectrum of genital tract and kidney defects, prompted us to verify whether Gata3 and β-catenin act in the same genetic pathway. For this, we first performed in situ hybridization against Gata3 in E11.5 HoxB7-Cre; Ctnnb1flox/− embryos (Ctnnb1ND−/−) and found a strong downregulation of Gata3 expression in the nephric duct in comparison to control embryos (Figure 8A,B). Accordingly, in situ hybridizations with a Ret cRNA probe revealed a loss of Ret expression in Ctnnb1ND−/− embryos (Figure 8C,D), which mimics the loss of Ret expression in Gata3ND−/− embryos (Figure 5). Staining of adjacent tissue sections with in situ probes for the canonical Wnt target genes Axin2, Sp5 and Daple [46],[47] confirmed the loss of β-catenin transcriptional response in Ctnnb1ND−/− embryos (Figure 8E,F and data not shown). In order to clarify the genetic hierarchy between Gata3 and β-catenin, we next stained Gata3ND−/− embryos with antibodies against β-catenin and phospho-β-catenin. These experiments failed to show any modification of β-catenin expression levels or activity following Gata3 inactivation (Figure 8G,H and data not shown). In support of this, the expression of the canonical Wnt-signaling target genes Axin2, Daple and Sp5 was unchanged in Gata3ND−/− embryos (Figure 8I–L and data not shown). From these data, we conclude that Gata3 acts genetically downstream of β-catenin but upstream of Ret in the nephric duct. To further characterize the molecular basis of the β-catenin-Gata3-Ret pathway, we first performed a bioinformatics analysis of the 50 kb genomic region upstream of the mouse and human Gata3 genes. Highly conserved sequences shared by the two species were further analyzed for putative binding sites for TCF/Lef. In total, 10 putative TCF/Lef binding sites, located in 5 of the 8 conserved regions, were identified (Figure 9A). In this analysis, we additionally included a Gata3 urogenital enhancer located at −110 kb [48], but failed to detect any conserved TCF/Lef binding sites in this element. A similar bioinformatics analysis of the Ret regulatory region revealed putative Gata3 binding sites in 4 of the 11 conserved regions 50 kb upstream of the Ret-ATG. (Figure 9B). The potential of β-catenin to regulate endogenous Gata3 expression was further evaluated in mouse IMCD3 collecting duct-derived cells. Using the GSK3β inhibitor BIO to stabilize the β-catenin protein [49], we observed a significant increase of Gata3 expression levels in those cells (Figure 9C). In order to assess the activity of Gata3 on the Ret regulatory region, we took advantage of the fact that one of the Gata3 binding sites mapped to a region previously reported to drive reporter gene expression in the zebrafish pronephros (Figure 9B, large asterisk) [50]. To test whether Gata3 acts on this site, we isolated the 1.2 kb conserved fragment and introduced a specific point mutation in the Gata3 binding site (Figure 9B). The wild-type and mutated fragments cloned upstream of a β-Gal reporter construct were transfected in IMCD3 cells stably expressing Gata3. The inactivation of the Gata3 binding site led to a significant downregulation of β-Gal expression relative to the wild-type control (Figure 9D), thereby suggesting that Gata3 may regulate Ret expression directly from this binding site. Together these data are consistent with a β-catenin-Gata3-Ret genetic cascade in the nephric duct.

Bottom Line: This results in a spectrum of urogenital malformations including kidney adysplasia, duplex systems, and hydroureter, as well as vas deferens hyperplasia and uterine agenesis.We further identify Gata3 as a central mediator of beta-catenin function in the nephric duct and demonstrate that the beta-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 beta-catenin, but upstream of Ret, to prevent ectopic ureter budding and premature cell differentiation in the nephric duct.

View Article: PubMed Central - PubMed

Affiliation: Goodman Cancer Centre, McGill University, Quebec, Canada.

ABSTRACT
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 beta-catenin function in the nephric duct and demonstrate that the beta-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 beta-catenin, but upstream of Ret, to prevent ectopic ureter budding and premature cell differentiation in the nephric duct.

Show MeSH
Related in: MedlinePlus