Reduction of BMP4 activity by gremlin 1 enables ureteric bud outgrowth and GDNF/WNT11 feedback signalling during kidney branching morphogenesis

Antagonists act to restrict and negatively modulate the activity of secreted signals during progression of embryogenesis. In mouse embryos lacking the extra-cellular BMP antagonist gremlin 1 (Grem1), metanephric development is disrupted at the stage of initiating ureteric bud outgrowth. Treatment of mutant kidney rudiments in culture with recombinant gremlin 1 protein induces additional epithelial buds and restores outgrowth and branching. All epithelial buds express Wnt11, and Gdnf is significantly upregulated in the surrounding mesenchyme, indicating that epithelial-mesenchymal (e-m) feedback signalling is restored. In the wild type, Bmp4 is expressed by the mesenchyme enveloping the Wolffian duct and ureteric bud and Grem1 is upregulated in the mesenchyme around the nascent ureteric bud prior to initiation of its outgrowth. In agreement, BMP activity is reduced locally as revealed by lower levels of nuclear pSMAD protein in the mesenchyme. By contrast, in Grem1-deficient kidney rudiments, pSMAD proteins are detected in many cell nuclei in the metanephric mesenchyme, indicative of excessive BMP signal transduction. Indeed, genetic lowering of BMP4 levels in Grem1-deficient mouse embryos completely restores ureteric bud outgrowth and branching morphogenesis. The reduction of BMP4 levels in Grem1 mutant embryos enables normal progression of renal development and restores adult kidney morphology and functions. This study establishes that initiation of metanephric kidney development requires the reduction of BMP4 activity by the antagonist gremlin 1 in the mesenchyme, which in turn enables ureteric bud outgrowth and establishment of autoregulatory GDNF/WNT11 feedback signalling.     

Sall4 Is Transiently Expressed in the Caudal Wolffian Duct and the Ureteric Bud,but Dispensable for Kidney Development

The kidney, the metanephros, is formed by reciprocal interactions between the metanephric mesenchyme and the ureteric bud, the latter of which is derived from the Wolffian duct that elongates in the rostral-to-caudal direction. Sall1 expressed in the metanephric mesenchyme is essential for ureteric bud attraction in kidney development. Sall4, another member of the Sall gene family, is required for maintenance of embryonic stem cells and establishment of induced pluripotent stem cells, and is thus considered to be one of the stemness genes. Sall4 is also a causative gene for Okihiro syndrome and is essential for the formation of many organs in both humans and mice. However, its expression and role in kidney development remain unknown, despite the essential role of Sall1 in the metanephric mesenchyme. Here, we report that mouse Sall4 is expressed transiently in the Wolffian duct-derived lineage, and is nearly complementary to Sall1 expression. While Sall4 expression is excluded from the Wolffian duct at embryonic (E) day 9.5, Sall4 is expressed in the Wolffian duct weakly in the mesonephric region at E10.5 and more abundantly in the caudal metanephric region where ureteric budding occurs. Sall4 expression is highest at E11.5 in the Wolffian duct and ureteric bud, but disappears by E13.5. We further demonstrate that Sall4 deletion in the Wolffian duct and ureteric bud does not cause any apparent kidney phenotypes. Therefore, Sall4 is expressed transiently in the caudal Wolffian duct and the ureteric bud, but is dispensable for kidney development in mice.     

Midkine Promotes Selective Expansion of the Nephrogenic Mesenchyme during Kidney Organogenesis

During kidney development, the growth and development of the stromal and nephrogenic mesenchyme cell populations and the ureteric bud epithelium is tightly coupled through intricate reciprocal signaling mechanisms between these three tissue compartments. Midkine, a target gene activated by retinoid signaling in the metanephros, encodes a secreted polypeptide with mitogenic and anti-apoptotic activities in a wide variety of cell types. Using immmunohistochemical methods we demonstrated that Midkine is found in the uninduced mesenchyme at the earliest stages of metanephric kidney development and only subsequently concentrated in the ureteric bud epithelium and basement membrane. The biological effects of purified recombinant Midkine were analyzed in metanephric organ culture experiments carried out in serum-free defined media. These studies revealed that Midkine selectively promoted the overgrowth of the Pax-2 and N-CAM positive nephrogenic mesenchymal cells, failed to stimulate expansion of the stromal compartment and suppressed branching morphogenesis of the ureteric bud. Midkine suppressed apoptosis and stimulated cellular proliferation of the nephrogenic mesenchymal cells, and was capable of maintaining the viability of isolated mesenchymes cultured in the absence of the ureteric bud. These results suggest that Midkine may regulate the balance of epithelial and stromal progenitor cell populations of the metanephric mesenchyme during renal organogenesis.     

Regulation of ureteric bud outgrowth by Pax2-dependent activation of the glial derived neurotrophic factor gene.

The outgrowth of the ureteric bud from the posterior nephric duct epithelium and the subsequent invasion of the bud into the metanephric mesenchyme initiate the process of metanephric, or adult kidney, development. The receptor tyrosine kinase RET and glial cell-derived neurotrophic factor (GDNF) form a signaling complex that is essential for ureteric bud growth and branching morphogenesis of the ureteric bud epithelium. We demonstrate that Pax2 expression in the metanephric mesenchyme is independent of induction by the ureteric bud. Pax2 mutants are deficient in ureteric bud outgrowth and do not express GDNF in the uninduced metanephric mesenchyme. Furthermore, Pax2 mutant mesenchyme is unresponsive to induction by wild-type heterologous inducers. In normal embryos, GDNF is sufficient to induce ectopic ureter buds in the posterior nephric duct, a process inhibited by bone morphogenetic protein 4. However, GDNF replacement in organ culture is not sufficient to stimulate ureteric bud outgrowth from Pax2 mutant nephric ducts, indicating additional defects in the nephric duct epithelium of Pax2 mutants. Pax2 can activate expression of GDNF in cell lines derived from embryonic metanephroi. Furthermore, Pax2 protein can bind to upstream regulatory elements within the GDNF promoter region and can transactivate expression of reporter genes. Thus, activation of GDNF by Pax2 coordinates the position and outgrowth of the ureteric bud such that kidney development can begin.     

Midkine Promotes Selective Expansion of the Nephrogenic Mesenchyme During Kidney Organogenesis

During kidney development, the growth and development of the stromal and nephrogenic mesenchyme cell populations and the ureteric bud epithelium is tightly coupled through intricate reciprocal signaling mechanisms between these three tissue compartments. Midkine, a target gene activated by retinoid signaling in the metanephros, encodes a secreted polypeptide with mitogenic and anti-apoptotic activities in a wide variety of cell types. Using immmunohistochemical methods we demonstrated that Midkine is found in the uninduced mesenchyme at the earliest stages of metanephric kidney development and only subsequently concentrated in the ureteric bud epithelium and basement membrane. The biological effects of purified recombinant Midkine were analyzed in metanephric organ culture experiments carried out in serum-free defined media. These studies revealed that Midkine selectively promoted the overgrowth of the Pax-2 and N-CAM positive nephrogenic mesenchymal cells, failed to stimulate expansion of the stromal compartment and suppressed branching morphogenesis of the ureteric bud. Midkine suppressed apoptosis and stimulated cellular proliferation of the nephrogenic mesenchymal cells, and was capable of maintaining the viability of isolated mesenchymes cultured in the absence of the ureteric bud. These results suggest that Midkine may regulate the balance of epithelial and stromal progenitor cell populations of the metanephric mesenchyme during renal organogenesis.Key Words: growth factor, proliferation, apoptosis, ureteric bud, branching morphogenesis, epithelial progenitor, development, signaling     

Wnt11 and Ret/Gdnf pathways cooperate in regulating ureteric branching during metanephric kidney development

Reciprocal cell-cell interactions between the ureteric epithelium and the metanephric mesenchyme are needed to drive growth and differentiation of the embryonic kidney to completion. Branching morphogenesis of the Wolffian duct derived ureteric bud is integral in the generation of ureteric tips and the elaboration of the collecting duct system. Wnt11, a member of the Wnt superfamily of secreted glycoproteins, which have important regulatory functions during vertebrate embryonic development, is specifically expressed in the tips of the branching ureteric epithelium. In this work, we explore the role of Wnt11 in ureteric branching and use a targeted mutation of the Wnt11 locus as an entrance point into investigating the genetic control of collecting duct morphogenesis. Mutation of the Wnt11 gene results in ureteric branching morphogenesis defects and consequent kidney hypoplasia in newborn mice. Wnt11 functions, in part, by maintaining normal expression levels of the gene encoding glial cell-derived neurotrophic factor (Gdnf). Gdnf encodes a mesenchymally produced ligand for the Ret tyrosine kinase receptor that is crucial for normal ureteric branching. Conversely, Wnt11 expression is reduced in the absence of Ret/Gdnf signaling. Consistent with the idea that reciprocal interaction between Wnt11 and Ret/Gdnf regulates the branching process, Wnt11 and Ret mutations synergistically interact in ureteric branching morphogenesis. Based on these observations, we conclude that Wnt11 and Ret/Gdnf cooperate in a positive autoregulatory feedback loop to coordinate ureteric branching by maintaining an appropriate balance of Wnt11-expressing ureteric epithelium and Gdnf-expressing mesenchyme to ensure continued metanephric development.     

Lrp4 Regulates Initiation of Ureteric Budding and Is Crucial for Kidney Formation – A Mouse Model for Cenani-Lenz Syndrome

Background

Development of the kidney is initiated when the ureteric bud (UB) branches from the Wolffian duct and invades the overlying metanephric mesenchyme (MM) triggering the mesenchymal/epithelial interactions that are the basis of organ formation. Multiple signaling pathways must be integrated to ensure proper timing and location of the ureteric bud formation.

Methods and Principal Findings

We have used gene targeting to create an Lrp4 null mouse line. The mutation results in early embryonic lethality with a subpenetrant phenotype of kidney agenesis. Ureteric budding is delayed with a failure to stimulate the metanephric mesenchyme in a timely manner, resulting in failure of cellular differentiation and resulting absence of kidney formation in the mouse as well as comparable malformations in humans with Cenani-Lenz syndrome.

Conclusion

Lrp4 is a multi-functional receptor implicated in the regulation of several molecular pathways, including Wnt and Bmp signaling. Lrp4^−/− mice show a delay in ureteric bud formation that results in unilateral or bilateral kidney agenesis. These data indicate that Lrp4 is a critical regulator of UB branching and lack of Lrp4 results in congenital kidney malformations in humans and mice.     

Dominant effects of RET receptor misexpression and ligand-independent RET signaling on ureteric bud development

During kidney development, factors from the metanephric mesenchyme induce the growth and repeated branching of the ureteric bud, which gives rise to the collecting duct system and also induces nephrogenesis. One signaling pathway known to be required for this process includes the receptor tyrosine kinase RET and co-receptor GFR(&agr;)-1, which are expressed in the ureteric bud, and the secreted ligand GDNF produced in the mesenchyme. To examine the role of RET signaling in ureteric bud morphogenesis, we produced transgenic mice in which the pattern of RET expression was altered, or in which a ligand-independent form of RET kinase was expressed. The Hoxb7 promoter was used to express RET throughout the ureteric bud branches, in contrast to its normal expression only at the bud tips. This caused a variable inhibition of ureteric bud growth and branching reminiscent of, but less severe than, the RET knockout phenotype. Manipulation of the level of GDNF, in vitro or in vivo, suggested that this defect was due to insufficient rather than excessive RET signaling. We propose that RET receptors expressed ectopically on ureteric bud trunk cells sequester GDNF, reducing its availability to the normal target cells at the bud tips. When crossed to RET knockout mice, the Hoxb7/RET transgene, which encoded the RET9 isoform, supported normal kidney development in some RET-/- animals, indicating that the other major isoform, RET51, is not required in this organ. Expression of a Hoxb7/RET-PTC2 transgene, encoding a ligand-independent form of RET kinase, caused the development of abnormal nodules, outside the kidney or at its periphery, containing branched epithelial tubules apparently formed by deregulated growth of the ureteric bud. This suggests that RET signaling is not only necessary but is sufficient to induce ureteric bud growth, and that the orderly, centripetal growth of the bud tips is controlled by the spatially and temporally regulated expression of GDNF and RET.     

A secreted BMP antagonist, Cer1, fine tunes the spatial organization of the ureteric bud tree during mouse kidney development

The epithelial ureteric bud is critical for mammalian kidney development as it generates the ureter and the collecting duct system that induces nephrogenesis in dicrete locations in the kidney mesenchyme during its emergence. We show that a secreted Bmp antagonist Cerberus homologue (Cer1) fine tunes the organization of the ureteric tree during organogenesis in the mouse embryo. Both enhanced ureteric expression of Cer1 and Cer1 knock out enlarge kidney size, and these changes are associated with an altered three-dimensional structure of the ureteric tree as revealed by optical projection tomography. Enhanced Cer1 expression changes the ureteric bud branching programme so that more trifid and lateral branches rather than bifid ones develop, as seen in time-lapse organ culture. These changes may be the reasons for the modified spatial arrangement of the ureteric tree in the kidneys of Cer1+ embryos. Cer1 gain of function is associated with moderately elevated expression of Gdnf and Wnt11, which is also induced in the case of Cer1 deficiency, where Bmp4 expression is reduced, indicating the dependence of Bmp expression on Cer1. Cer1 binds at least Bmp2/4 and antagonizes Bmp signalling in cell culture. In line with this, supplementation of Bmp4 restored the ureteric bud tip number, which was reduced by Cer1+ to bring it closer to the normal, consistent with models suggesting that Bmp signalling inhibits ureteric bud development. Genetic reduction of Wnt11 inhibited the Cer1-stimulated kidney development, but Cer1 did not influence Wnt11 signalling in cell culture, although it did inhibit the Wnt3a-induced canonical Top Flash reporter to some extent. We conclude that Cer1 fine tunes the spatial organization of the ureteric tree by coordinating the activities of the growth-promoting ureteric bud signals Gndf and Wnt11 via Bmp-mediated antagonism and to some degree via the canonical Wnt signalling involved in branching.     

Six1 and Six4 are essential for Gdnf expression in the metanephric mesenchyme and ureteric bud formation, while Six1 deficiency alone causes mesonephric-tubule defects

Interaction between the ureteric-bud epithelium and the metanephric mesenchyme is important for kidney development. Six1 and Six4 are the mammalian homologs of Drosophila sine oculis, and they are coexpressed in the nephrogenic mesenchyme. Six1-deficient mice show varying kidney defects, while Six4-deficient mice have no apparent abnormalities. Here, we report Six1/Six4-deficient mice that we generated in order to elucidate the functions of Six4 in Six1-deficient kidney development. The Six1/Six4-deficient mice exhibited more severe kidney phenotypes than the Six1-deficient mice; kidney and ureter agenesis was observed in all the neonates examined. The Six1/Six4-deficient metanephric mesenchyme cells were directed toward kidney lineage but failed to express Pax2, Pax8, or Gdnf, whereas the expression of these genes was partially reduced or unchanged in the case of Six1 deficiency. Thus, Six4 cooperates with Six1 in the metanephric mesenchyme to regulate the level of Gdnf expression; this could explain the absence of the ureteric bud in the Six1/Six4-deficient mice. In contrast, Six1 deficiency alone caused defects in mesonephric-tubule formation, and these defects were not exacerbated in the Six1/Six4-deficient mesonephros. These results highlight the fact that Six1 and Six4 have collaborative functions in the metanephros but not in the mesonephros.     

Dynamic regulation of Smad expression during mesenchyme to epithelium transition in the metanephric kidney

Genetic studies have revealed a role for the transforming growth factor beta (TGFbeta) superfamily growth factors in development of the mouse kidney. In this study, we have characterized developmental expression of Smad proteins, the downstream effectors of TGFbeta superfamily ligands. Immunohistochemistry detecting Smads 1-5 and 8 was performed on 11.25-15.5 dpc kidney sections. We find that Smad proteins are readily detected in metanephric mesenchyme but are coordinately down-regulated in condensing mesenchyme and derivative structures such as renal vesicles. In order to define the stage at which down-regulation occurs, we used the heterologous inducer lithium chloride to promote condensation in isolated cultured mesenchymal explants and subsequently assayed expression of Smad 4. Down-regulation is complete in lithium chloride induced mesenchymal condensates, indicating that Smad regulation takes place at an early stage, prior to mesenchyme to epithelium transition.     

Regulation of metanephric kidney development by growth/differentiation factor 11

Growth/differentiation factor 11 (Gdf11) is a transforming growth factor beta family member previously shown to control anterior/posterior patterning of the axial skeleton. We now report that Gdf11 also regulates kidney organogenesis. Mice carrying a targeted deletion of Gdf11 possess a spectrum of renal abnormalities with the majority of mutant animals lacking both kidneys. Histological analysis revealed a failure in ureteric bud formation at the initial stage of metanephric development in most Gdf11 mutant embryos examined. The metanephric mesenchyme of mutant embryos lacking a ureteric bud was found to be defective in the expression of glial cell line-derived neurotrophic factor (Gdnf), a gene known to direct ureteric bud outgrowth. The addition of Gdnf protein to urogenital tracts taken from Gdf11 null embryos induced ectopic ureteric bud formation along the Wolffian duct. Our studies suggest that Gdf11 may be important in directing the initial outgrowth of the ureteric bud from the Wolffian duct by controlling the expression of Gdnf in the metanephric mesenchyme.     

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

Differentiation 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.     

The role of GDNF in patterning the excretory system

Mesenchymal-epithelial interactions are an important source of information for pattern formation during organogenesis. In the developing excretory system, one of the secreted mesenchymal factors thought to play a critical role in patterning the growth and branching of the epithelial ureteric bud is GDNF. We have tested the requirement for GDNF as a paracrine chemoattractive factor by altering its site of expression during excretory system development. Normally, GDNF is secreted by the metanephric mesenchyme and acts via receptors on the Wolffian duct and ureteric bud epithelium. Misexpression of GDNF in the Wolffian duct and ureteric buds resulted in formation of multiple, ectopic buds, which branched independently of the metanephric mesenchyme. This confirmed the ability of GDNF to induce ureter outgrowth and epithelial branching in vivo. However, in mutant mice lacking endogenous GDNF, kidney development was rescued to a substantial degree by GDNF supplied only by the Wolffian duct and ureteric bud. These results indicate that mesenchymal GDNF is not required as a chemoattractive factor to pattern the growth of the ureteric bud within the developing kidney, and that any positional information provided by the mesenchymal expression of GDNF may provide for renal branching morphogenesis is redundant with other signals.     

Recent genetic studies of mouse kidney development

Recent functional studies in mouse further illustrate the importance of the epithelial-mesenchymal interaction between the ureteric bud epithelium and the metanephric mesenchyme in kidney formation. Genetic ablation of Gdf11, Six1, Slit2/Robo2 reveal a role of these genes in regulating the outgrowth of a single ureteric bud from the Wolffian duct. Studies of Wnt11 and Fras1/Grip1, all expressed in the ureteric bud, show a role for these genes in regulating events in the adjacent metanephric mesenchyme. Furthermore, various approaches were used to address the function of Pod1, Pbx1, the Notch pathway and Brn1 in nephron formation.     

Stromal progenitors are important for patterning epithelial and mesenchymal cell types in the embryonic kidney

Growth and expansion of the embryonic kidney is driven in large part by continuous branching morphogenesis and nephron induction that occurs in a restricted domain beneath the renal capsule called the nephrogenic zone. Here, new ureteric bud branches and nephron aggregates form surrounded by a layer of cortical stromal cell progenitors. The boundaries and inductive activities of the nephrogenic zone are maintained as the kidney grows. As new ureteric bud branches and nephrogenic aggregates form, older generations of ureteric bud branches, renal vesicles and stromal progenitors are displaced from the nephrogenic zone and undergo further differentiation surrounded by medullary stroma, a different population of stromal cells. Recent studies suggest that cortical and medullary stromal progenitors may be an important source of signals that maintain outer and inner zones of differentiation in the embryonic kidney, and regulate distinct events important for differentiation of nephrons and the collecting duct system.     

SLIT2-mediated ROBO2 signaling restricts kidney induction to a single site

Kidney development occurs in a stereotypic position along the body axis. It begins when a single ureteric bud emerges from the nephric duct in response to GDNF secreted by the adjacent nephrogenic mesenchyme. Posterior restriction of Gdnf expression is considered critical for correct positioning of ureteric bud development. Here we show that mouse mutants lacking either SLIT2 or its receptor ROBO2, molecules known primarily for their function in axon guidance and cell migration, develop supernumerary ureteric buds that remain inappropriately connected to the nephric duct, and that the SLIT2/ROBO2 signal is transduced in the nephrogenic mesenchyme. Furthermore, we show that Gdnf expression is inappropriately maintained in anterior nephrogenic mesenchyme in these mutants. Thus our data identify an intercellular signaling system that restricts, directly or indirectly, the extent of the Gdnf expression domain, thereby precisely positioning the site of kidney induction.     

Identification of pleiotrophin as a mesenchymal factor involved in ureteric bud branching morphogenesis

Branching morphogenesis is central to epithelial organogenesis. In the developing kidney, the epithelial ureteric bud invades the metanephric mesenchyme, which directs the ureteric bud to undergo repeated branching. A soluble factor(s) in the conditioned medium of a metanephric mesenchyme cell line is essential for multiple branching morphogenesis of the isolated ureteric bud. The identity of this factor had proved elusive, but it appeared distinct from factors such as HGF and EGF receptor ligands that have been previously implicated in branching morphogenesis of mature epithelial cell lines. Using sequential column chromatography, we have now purified to apparent homogeneity an 18 kDa protein, pleiotrophin, from the conditioned medium of a metanephric mesenchyme cell line that induces isolated ureteric bud branching morphogenesis in the presence of glial cell-derived neurotrophic factor. Pleiotrophin alone was also found to induce the formation of branching tubules in an immortalized ureteric bud cell line cultured three-dimensionally in an extracellular matrix gel. Consistent with an important role in ureteric bud morphogenesis during kidney development, pleiotrophin was found to localize to the basement membrane of the developing ureteric bud in the embryonic kidney. We suggest that pleiotrophin could act as a key mesenchymally derived factor regulating branching morphogenesis of the ureteric bud and perhaps other embryonic epithelial structures.     

Role of BMP family members during kidney development.

Members of the Bone morphogenetic protein (BMP) family have been shown to be important signaling molecules throughout mouse development. Accordingly, many BMPs are also expressed during organogenesis of the metanephric kidney. However, only BMP7 has been shown to be absolutely required for proper formation of the kidney, thus the majority of information known involves this family member. BMP7 is expressed in both the ureteric epithelium and the mesenchyme throughout embryonic development and has been shown to function as a survival factor for the nephrogenic mesenchyme. However, there has been some controversy over the role of BMP7 as an inducing molecule for the metanephric mesenchyme. Recent studies have shown that BMP7 functions as an anti-differentiation factor for this mesenchyme cell population. The function of BMPs in the ureter and in the more differentiated epithelial structures of the nephron is less well understood.     

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.