David Grote

Pax2/8-regulated Gata3 expression is necessary for morphogenesis and guidance of the nephric duct in the developing kidney

The mammalian pro- and mesonephros are transient embryonic kidneys essential for urogenital system development. The nephric (Wolffian) duct, which is a central constituent of both structures, elongates caudally along a stereotypical path to reach the hindlimb level where it induces metanephros (adult kidney) formation, while the remaining duct gives rise to the male genital tract (epidydimis, vas deferens). The transcription factors Pax2 and Pax8 are essential for the initiation of pro- and mesonephros development. In a cDNA microarray screen for genes specifically expressed in the pro/mesonephros and regulated by Pax proteins, we identified Gata3, a transcription factor gene associated with hypoparathyroidism, deafness and renal anomaly (HDR) syndrome. Gata3 is already expressed in the pronephric anlage, together with Pax2 and Pax8, suggesting that it may be a direct Pax2/8 target gene. Inactivation of Gata3 by insertion of an Ires-GFP reporter gene resulted in a massive increase in nephric duct cellularity, which was accompanied by enhanced cell proliferation and aberrant elongation of the nephric duct. Interestingly, however, the nephrogenic cord extended, with delayed kinetics, along the entire caudal path up to the level of the hindlimb bud, indicating that extension of the nephric duct and cord is controlled by different guidance cues. At the molecular level, the nephric duct of Gata3-/- embryos is characterized by the loss of Ret expression and signaling, which may contribute to the guidance defect of the nephric duct. Together, these results define Gata3 as a key regulator of nephric duct morphogenesis and guidance in the pro/mesonephric kidney.

Pax2 and Pax8 Regulate Branching Morphogenesis and Nephron Differentiation in the Developing Kidney

Pax genes are important regulators of kidney development. In the mouse, homozygous Pax2 inactivation results in renal agenesis, a phenotype that has largely precluded the analysis of Pax gene function during metanephric kidney development. To address this later function, kidney development was analyzed in embryos that were compound heterozygous for Pax2 and for Pax8, a closely related member of the Pax gene family. Both genes are coexpressed in differentiating nephrons and collecting ducts. At the morphological level, Pax2(+/-)Pax8(+/-) metanephric kidneys are severely hypodysplastic and characterized by a reduction in ureter tips and nephron number in comparison with wild-type or Pax2(+/-) kidneys. In developing nephrons, the molecular analysis of Pax2(+/-)Pax8(+/-) kidneys reveals a strong reduction in the expression levels of Lim1, a key regulator of nephron differentiation, accompanied by an increase in apoptosis. At a more mature stage, the reduction of Pax2/8 gene dosage severely affects distal tubule formation, revealing a role for Pax genes in the differentiation of specific nephron segments. At the ureter tips, the expression of Wnt11, a target of glial cell-derived neurotrophic factor-Ret signaling, is significantly reduced, whereas the expression levels of Ret and GDNF remain normal. Together, these results demonstrate a crucial role for Pax2 and Pax8 in nephron differentiation and branching morphogenesis of the metanephros.

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.

Identification of Pax2-regulated genes by expression profiling of the mid-hindbrain organizer region.

The paired domain transcription factor Pax2 is required for the formation of the isthmic organizer (IsO) at the midbrain-hindbrain boundary, where it initiates expression of the IsO signal Fgf8. To gain further insight into the role of Pax2 in mid-hindbrain patterning, we searched for novel Pax2-regulated genes by cDNA microarray analysis of FACS-sorted GFP+ mid-hindbrain cells from wild-type and Pax2–/– embryos carrying a Pax2GFP BAC transgene. Here, we report the identification of five genes that depend on Pax2 function for their expression in the mid-hindbrain boundary region. These genes code for the transcription factors En2 and Brn1 (Pou3f3), the intracellular signaling modifiers Sef and Tapp1, and the non-coding RNA Ncrms. The Brn1 gene was further identified as a direct target of Pax2, as two functional Pax2-binding sites in the promoter and in an upstream regulatory element of Brn1 were essential for lacZ transgene expression at the mid-hindbrain boundary. Moreover, ectopic expression of a dominant-negative Brn1 protein in chick embryos implicated Brn1 in Fgf8 gene regulation. Together, these data defined novel functions of Pax2 in the establishment of distinct transcriptional programs and in the control of intracellular signaling during mid-hindbrain development.

Nephric duct insertion is a crucial step in urinary tract maturation that is regulated by a Gata3-Raldh2-Ret molecular network in mice

Urinary tract development depends on a complex series of events in which the ureter moves from its initial branch point on the nephric duct (ND) to its final insertion site in the cloaca (the primitive bladder and urethra). Defects in this maturation process can result in malpositioned ureters and hydronephrosis, a common cause of renal disease in children. Here, we report that insertion of the ND into the cloaca is an unrecognized but crucial step that is required for proper positioning of the ureter and that depends on Ret signaling. Analysis of Ret mutant mice at birth reveals hydronephrosis and defective ureter maturation, abnormalities that our results suggest are caused, at least in part, by delayed insertion of the ND. We find a similar set of malformations in mutants lacking either Gata3 or Raldh2. We show that these factors act in parallel to regulate ND insertion via Ret. Morphological analysis of ND extension in wild-type embryos reveals elaborate cellular protrusions at ND tips that are not detected in Ret, Gata3 or Raldh2 mutant embryos, suggesting that these protrusions may normally be important for fusion with the cloaca. Together, our studies reveal a novel Ret-dependent event, ND insertion, that, when abnormal, can cause obstruction and hydronephrosis at birth; whether ND defects underlie similar types of urinary tract abnormalities in humans is an interesting possibility.

Common variants of the glial cell-derived neurotrophic factor gene do not influence kidney size of the healthy newborn

Glial cell-derived neurotrophic factor (GDNF) plays an important role in renal development, serving as a trophic factor for outgrowth of the ureteric bud and its continued arborisation. Our previous studies have shown that common variants of the human paired-box 2 (PAX2) gene (a transcriptional activator of GDNF) and rearranged during transfection (RET) gene (encoding the cognate receptor for GDNF) are associated with a subtle reduction in the kidney size of newborns. Since heterozygosity for a mutant GDNF allele causes mild renal hypoplasia and modest hypertension in mice, we considered the possibility that common variants of the GDNF gene might also contribute to renal hypoplasia in humans. We studied the relationship between newborn renal size or umbilical cord cystatin C and 19 common GDNF gene variants [minor allele frequency (MAF) >5%], three single nucleotide polymorphisms (SNPs) related to a putative PAX binding site and one rare SNP (rs36119840 A/G) which changes an amino acid (R93W), based on data from the haplotype map of the human genome (HapMap). However, none of these 23 SNPs was associated with reduced newborn kidney size or function. Among the 163 Caucasians in our cohort, none had the R93W allele.

Live Imaging of the Developing Mouse Mesonephros

Embryonic development is a highly dynamic process involving complex tissue interactions and movements. Recent progress in cell labeling, image acquisition, and image processing technologies has brought the study of embryo morphogenesis to another level. It is now possible to visualize in real time the dynamic morphogenetic changes occurring in vivo and to reconstitute and quantify them in 4D rendering. However, extended live embryo imaging remains challenging in terms of embryo survival and minimization of phototoxicity. Here, we describe a procedure to image the developing mesonephros for up to 16 h in intact mouse embryos. This method can easily be adapted to the imaging of other structures at similar developmental stages.

Pax2 and Pax8 regulate branching morphogenesis and nephron differentiation in the developing kidney

Pax genes are important regulators of kidney development. In the mouse, homozygous Pax2 inactivation results in renal agenesis, a phenotype that has largely precluded the analysis of Pax gene function during metanephric kidney development. To address this later function, kidney development was analyzed in embryos that were compound heterozygous for Pax2 and for Pax8, a closely related member of the Pax gene family. Both genes are coexpressed in differentiating nephrons and collecting ducts. At the morphological level, Pax2(+/-)Pax8(+/-) metanephric kidneys are severely hypodysplastic and characterized by a reduction in ureter tips and nephron number in comparison with wild-type or Pax2(+/-) kidneys. In developing nephrons, the molecular analysis of Pax2(+/-)Pax8(+/-) kidneys reveals a strong reduction in the expression levels of Lim1, a key regulator of nephron differentiation, accompanied by an increase in apoptosis. At a more mature stage, the reduction of Pax2/8 gene dosage severely affects distal tubule formation, revealing a role for Pax genes in the differentiation of specific nephron segments. At the ureter tips, the expression of Wnt11, a target of glial cell-derived neurotrophic factor-Ret signaling, is significantly reduced, whereas the expression levels of Ret and GDNF remain normal. Together, these results demonstrate a crucial role for Pax2 and Pax8 in nephron differentiation and branching morphogenesis of the metanephros.