Mary Taglienti

Angioblast-mesenchyme induction of early kidney development is mediated by Wt1 and Vegfa

Most studies on kidney development have considered the interaction of the metanephric mesenchyme and the ureteric bud to be the major inductive event that maintains tubular differentiation and branching morphogenesis. The mesenchyme produces Gdnf, which stimulates branching, and the ureteric bud stimulates continued growth of the mesenchyme and differentiation of nephrons from the induced mesenchyme. Null mutation of the Wt1 gene eliminates outgrowth of the ureteric bud, but Gdnf has been identified as a target of Pax2, but not of Wt1. Using a novel system for microinjecting and electroporating plasmid expression constructs into murine organ cultures, it has been demonstrated that Vegfa expression in the mesenchyme is regulated by Wt1. Previous studies had identified a population of Flk1-expressing cells in the periphery of the induced mesenchyme, and adjacent to the stalk of the ureteric bud, and that Vegfa was able to stimulate growth of kidneys in organ culture. Here it is demonstrated that signaling through Flk1 is required to maintain expression of Pax2 in the mesenchyme of the early kidney, and for Pax2 to stimulate expression of Gdnf. However, once Gdnf stimulates branching of the ureteric bud, the Flk1-dependent angioblast signal is no longer required to maintain branching morphogenesis and induction of nephrons. Thus, this work demonstrates the presence of a second set of inductive events, involving the mesenchymal and angioblast populations, whereby Wt1-stimulated expression of Vegfa elicits an as-yet-unidentified signal from the angioblasts, which is required to stimulate the expression of Pax2 and Gdnf, which in turn elicits an inductive signal from the ureteric bud.

Coordinate integrin and c-Met signaling regulate Wnt gene expression during epithelial morphogenesis

Integrin receptors for the extracellular matrix and receptor tyrosine kinase growth factor receptors represent two of the major families of receptors that transduce into cells information about the surrounding environment. Wnt proteins are a major family of signaling molecules that regulate morphogenetic events. There is presently little understanding of how the expression of Wnt genes themselves is regulated. In this study, we demonstrate that α3β1 integrin, a major laminin receptor involved in the development of the kidney, and c-Met, the receptor for hepatocyte growth factor, signal coordinately to regulate the expression of Wnt7b in the mouse. Wnt signals in turn appear to regulate epithelial cell survival in the papilla of the developing kidney, allowing for the elongation of epithelial tubules to form a mature papilla. Together, these results demonstrate how signals from integrins and growth factor receptors can be integrated to regulate the expression of an important family of signaling molecules so as to regulate morphogenetic events.

A Mammal-Specific Exon of WT1 Is Not Required for Development or Fertility

ABSTRACT The WT1 tumor suppressor gene is a zinc finger-containing transcription factor which is required for development of the kidney and gonads. A mammal-specific alternative splicing event within this gene results in the presence or absence of a 17-amino-acid sequence within the WT1 protein. To determine the function of this sequence in vivo, gene targeting was utilized to specifically eliminate the exon encoding this sequence in mice. Mice lacking WT1 exon 5 develop normally. Adult mice lacking this exon are viable and fertile, and females are capable of lactation.

c-Met and NF-κB–Dependent Overexpression of Wnt7a and -7b and Pax2 Promotes Cystogenesis in Polycystic Kidney Disease

The mechanisms of cystogenesis in autosomal dominant polycystic kidney disease (ADPKD) are not fully understood. Hyperactivation of the tyrosine kinase c-Met contributes to cyst formation, but we do not know the downstream mediators. Here, we found that hyperactivated c-Met led to increased NF-κB signaling, which in turn, drove de novo expression of Wnt7a and overexpression of Wnt7b in Pkd1(-/-) mouse kidneys. Hyperactivated Wnt signaling increased expression of the transcription factor Pax2 in the cells lining cysts. Furthermore, blocking Wnt signaling with DKK1 decreased cyst formation in an organ culture model of ADPKD. In summary, these results suggest that the c-Met/NF-κB/Wnt/Pax2 signaling transduction axis may provide pharmacological targets for the treatment of ADPKD.

Failure to ubiquitinate c-Met leads to hyperactivation of mTOR signaling in a mouse model of autosomal dominant polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is a common inherited disorder that is caused by mutations at two loci, polycystin 1 (PKD1) and polycystin 2 (PKD2). It is characterized by the formation of multiple cysts in the kidneys that can lead to chronic renal failure. Previous studies have suggested a role for hyperactivation of mammalian target of rapamycin (mTOR) in cystogenesis, but the etiology of mTOR hyperactivation has not been fully elucidated. In this report we have shown that mTOR is hyperactivated in Pkd1-null mouse cells due to failure of the HGF receptor c-Met to be properly ubiquitinated and subsequently degraded after stimulation by HGF. In Pkd1-null cells, Casitas B-lineage lymphoma (c-Cbl), an E3-ubiquitin ligase for c-Met, was sequestered in the Golgi apparatus with α₃β₁ integrin, resulting in the inability to ubiquitinate c-Met. Treatment of mouse Pkd1-null cystic kidneys in organ culture with a c-Met pharmacological inhibitor resulted in inhibition of mTOR activity and blocked cystogenesis in this mouse model of ADPKD. We therefore suggest that blockade of c-Met is a potential novel therapeutic approach to the treatment of ADPKD.

Expression regulation and function of heparan sulfate 6-O-endosulfatases in the spermatogonial stem cell niche.

Glial cell line-derived neurotrophic factor (GDNF) is a heparan sulfate (HS)-binding factor. GDNF is produced by somatic Sertoli cells, where it signals to maintain spermatogonial stem cells (SSCs) and reproduction. Here, we investigate the roles of extracellular HS 6-O-endosulfatases (Sulfs), Sulf1 and Sulf2, in the matrix transmission of GDNF from Sertoli cells to SSCs. Although Sulfs are not required for testis formation, Sulf deficiency leads to the accelerated depletion of SSCs, a testis phenotype similar to that of GDNF+/- mice. Mechanistically, we show that Sulfs are expressed in GDNF-producing Sertoli cells. In addition, reduced Sulf activity profoundly worsens haplo-deficient GDNF phenotypes in our genetic studies. These findings establish a critical role of Sulfs in promoting GDNF signaling and support a model in which Sulfs regulate the bioavailability of GDNF by enzymatically remodeling HS 6-O-desulfation to release GDNF from matrix sequestration. Further, Sertoli cell-specific transcriptional factor Wilms tumor 1 (WT1) directly activates the transcription of both Sulf1 and Sulf2 genes. Together, our studies not only identify Sulfs as essential regulators of GDNF signaling in the SSC niche, but also as direct downstream targets of WT1, thus establishing a physiological role of WT1 in Sertoli cells.

Eed, a member of the Polycomb group, is required for nephron differentiation and the maintenance of nephron progenitor cells

ABSTRACT Epigenetic regulation of gene expression has a crucial role allowing for the self-renewal and differentiation of stem and progenitor populations during organogenesis. The mammalian kidney maintains a population of self-renewing stem cells that differentiate to give rise to thousands of nephrons, which are the functional units that carry out filtration to maintain physiological homeostasis. The polycomb repressive complex 2 (PRC2) epigenetically represses gene expression during development by placing the H3K27me3 mark on histone H3 at promoter and enhancer sites, resulting in gene silencing. To understand the role of PRC2 in nephron differentiation, we conditionally inactivated the Eed gene, which encodes a nonredundant component of the PRC2 complex, in nephron progenitor cells. Resultant kidneys were smaller and showed premature loss of progenitor cells. The progenitors in Eed mutant mice that were induced to differentiate did not develop into properly formed nephrons. Lhx1, normally expressed in the renal vesicle, was overexpressed in kidneys of Eed mutant mice. Thus, PRC2 has a crucial role in suppressing the expression of genes that maintain the progenitor state, allowing nephron differentiation to proceed. Summary: Conditional inactivation of the polycomb gene Eed in nephron progenitor cells results in their premature loss and prevents normal nephron differentiation beyond the S-shaped tubule stage.

Ectopic Phosphorylated Creb Marks Dedifferentiated Proximal Tubules in Cystic Kidney Disease

Ectopic cAMP signaling is pathologic in polycystic kidney disease; however, its spatiotemporal actions are unclear. We characterized the expression of phosphorylated Creb (p-Creb), a target and mediator of cAMP signaling, in developing and cystic kidney models. We also examined tubule-specific effects of cAMP analogs in cystogenesis in embryonic kidney explants. In wild-type mice, p-Creb marked nephron progenitors (NP), early epithelial NP derivatives, ureteric bud, and cortical stroma; p-Creb was present in differentiated thick ascending limb of Henle, collecting duct, and stroma; however, it disappeared in mature NP-derived proximal tubules. In Six2cre;Frs2αFl/Fl mice, a renal cystic model, ectopic p-Creb stained proximal tubule-derived cystic segments that lost the differentiation marker lotus tetragonolobus lectin. Furthermore, lotus tetragonolobus lectin-negative/p-Creb-positive cyst segments (re)-expressed Ncam1, Pax2, and Sox9 markers of immature nephron structures and dedifferentiated proximal tubules after acute kidney injury. These dedifferentiation markers were co-expressed with p-Creb in renal cysts in Itf88 knockout mice subjected to ischemia and Six2cre;Pkd1Fl/Fl mice, other renal cystogenesis models. 8-Br-cAMP addition to wild-type embryonic kidney explants induced proximal tubular cystogenesis and p-Creb expression; these effects were blocked by co-addition of protein kinase A inhibitor. Thus p-Creb/cAMP signaling is appropriate in NP and early nephron derivatives, but disappears in mature proximal tubules. Moreover, ectopic p-Creb expression/cAMP signaling marks dedifferentiated proximal tubular cystic segments. Furthermore, proximal tubules are predisposed to become cystic after cAMP stimulation.