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1.
Embryonic development requires cell migration in response to positional cues. Yet, how groups of cells recognize and translate positional information into morphogenetic movement remains poorly understood. In the developing kidney, the ureteric bud epithelium grows from the nephric duct towards a group of posterior intermediate mesodermal cells, the metanephric mesenchyme, and induces the formation of the adult kidney. The secreted protein GDNF and its receptor RET are required for ureteric bud outgrowth and subsequent branching. However, it is unclear whether the GDNF–RET pathway regulates cell migration, proliferation, survival, or chemotaxis. In this report, we have used the MDCK renal epithelial cell line to show that activation of the RET pathway results in increased cell motility, dissociation of cell adhesion, and the migration towards a localized source of GDNF. Cellular responses to RET activation include the formation of lamellipodia, filopodia, and reorganization of the actin cytoskeleton. These data demonstrate that GDNF is a chemoattractant for RET-expressing epithelial cells and thus account for the developmental defects observed in RET and GDNF mutant mice. Furthermore, the RET-transfected MDCK cells described in this report are a promising model for delineating RET signaling pathways in the renal epithelial cell lineage.  相似文献   

2.
The RET receptor tyrosine kinase is activated by GDNF and controls outgrowth and invasion of the ureteric bud epithelia in the developing kidney. In renal epithelial cells and in enteric neuronal precursor cells, activation of RET results in chemotaxis as Ret expressing cells invade the surrounding GDNF expressing tissue. One potential downstream signaling pathway governing RET mediated chemotaxis may require phosphatidylinositol 3-kinase (PI3K), which generates PI(3,4,5) triphosphate. The PTEN tumor suppressor gene encodes a protein and lipid phosphatase that regulates cell growth, apoptosis and many other cellular processes. PTEN helps regulate cellular chemotaxis by antagonizing the PI3K signaling pathway through dephosphorylation of phosphotidylinositol triphosphates. In this report, we show that PTEN suppresses RET mediated cell migration and chemotaxis in cell culture assays, that RET activation results in asymmetric localization of inositol triphosphates and that loss of PTEN affects the pattern of branching morphogenesis in developing mouse kidneys. These data suggest a critical role for the PI3K/PTEN axis in shaping the pattern of epithelial branches in response to RET activation.  相似文献   

3.
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.  相似文献   

4.
Glial cell line-derived neurotrophic factor (GDNF) and hepatocyte growth factor (HGF) are multifunctional signaling molecules in embryogenesis. HGF binds to and activates Met receptor tyrosine kinase. The signaling receptor complex for GDNF typically includes both GDNF family receptor alpha1 (GFRalpha1) and Ret receptor tyrosine kinase. GDNF can also signal independently of Ret via GFRalpha1, although the mechanism has remained unclear. We now show that GDNF partially restores ureteric branching morphogenesis in ret-deficient mice with severe renal hypodysplasia. The mechanism of Ret-independent effect of GDNF was therefore studied by the MDCK cell model. In MDCK cells expressing GFRalpha1 but no Ret, GDNF stimulates branching but not chemotactic migration, whereas both branching and chemotaxis are promoted by GDNF in the cells coexpressing Ret and GFRalpha1, mimicking HGF/Met responses in wild-type MDCK cells. Indeed, GDNF induces Met phosphorylation in several ret-deficient/GFRalpha1-positive and GFRalpha1/Ret-coexpressing cell lines. However, GDNF does not immunoprecipite Met, making a direct interaction between GDNF and Met highly improbable. Met activation is mediated by Src family kinases. The GDNF-induced branching of MDCK cells requires Src activation, whereas the HGF-induced branching does not. Our data show a mechanism for the GDNF-induced branching morphogenesis in non-Ret signaling.  相似文献   

5.
Glial-Cell-Line-Derived Neurotrophic Factor (GDNF) is the major mesenchyme-derived regulator of ureteric budding and branching during nephrogenesis. The ligand activates on the ureteric bud epithelium a receptor complex composed of Ret and GFRalpha1. The upstream regulators of the GDNF receptors are poorly known. A Notch ligand, Jagged1 (Jag1), co-localises with GDNF and its receptors during early kidney morphogenesis. In this study we utilized both in vitro and in vivo models to study the possible regulatory relationship of Ret and Notch pathways. Urogenital blocks were exposed to exogenous GDNF, which promotes supernumerary ureteric budding from the Wolffian duct. GDNF-induced ectopic buds expressed Jag1, which suggests that GDNF can, directly or indirectly, up-regulate Jag1 through Ret/GFRalpha1 signalling. We then studied the role of Jag1 in nephrogenesis by transgenic mice constitutively expressing human Jag1 in Wolffian duct and its derivatives under HoxB7 promoter. Jag1 transgenic mice showed a spectrum of renal defects ranging from aplasia to hypoplasia. Ret and GFRalpha1 are normally downregulated in the Wolffian duct, but they were persistently expressed in the entire transgenic duct. Simultaneously, GDNF expression remained unexpectedly low in the metanephric mesenchyme. In vitro, exogenous GDNF restored the budding and branching defects in transgenic urogenital blocks. Renal differentiation apparently failed because of perturbed stimulation of primary ureteric budding and subsequent branching. Thus, the data provide evidence for a novel crosstalk between Notch and Ret/GFRalpha1 signalling during early nephrogenesis.  相似文献   

6.
Crosstalk between VEGF-A/VEGFR2 and GDNF/RET signaling pathways   总被引:1,自引:0,他引:1  
Vascular endothelial growth factor (VEGF-A) plays multiple roles in kidney development: stimulates cell proliferation, survival, tubulogenesis, and branching morphogenesis. However, the mechanism that mediates VEGF-A induced ureteric bud branching is unclear. Glial-derived neurotrophic factor (GDNF) signaling through tyrosine kinase c-RET is the major regulator of ureteric bud branching. Here we examined whether VEGF-A regulates RET signaling. We determined that ureteric bud-derived cells express the main VEGF-A signaling receptor, VEGFR2 and RET, by RT-PCR, immunoblotting, and immunocytochemistry. We show that the VEGF-A isoform VEGF(165) induces RET-tyr(1062) phosphorylation in addition to VEGFR2 autophosphorylation, that VEGF(165) and GDNF have additive effects on RET-tyr(1062) phosphorylation, and that VEGFR2 and RET co-immunoprecipitate. Functionally, VEGF(165) induces ureteric bud cell proliferation and branching morphogenesis. Similarly, in embryonic kidney explants VEGF(165) induces RET-tyr(1062) phosphorylation and upregulates GDNF. These findings provide evidence for a novel cooperative interaction between VEGFR2 and RET that mediates VEGF-A functions in ureteric bud cells.  相似文献   

7.
The role of GDNF in patterning the excretory system   总被引:5,自引:0,他引:5  
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.  相似文献   

8.
9.
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.  相似文献   

10.
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.  相似文献   

11.
Branching of ureteric bud-derived epithelial tubes is a key morphogenetic process that shapes development of the kidney. Glial cell line-derived neurotrophic factor (GDNF) initiates ureteric bud formation and promotes subsequent branching morphogenesis. Exactly how GDNF coordinates branching morphogenesis is unclear. Here we show that the absence of the receptor tyrosine kinase antagonist Sprouty1 (Spry1) results in irregular branching morphogenesis characterized by both increased number and size of ureteric bud tips. Deletion of Spry1 specifically in the epithelium is associated with increased epithelial Wnt11 expression as well as increased mesenchymal Gdnf expression. We propose that Spry1 regulates a Gdnf/Ret/Wnt11-positive feedback loop that coordinates mesenchymal-epithelial dialogue during branching morphogenesis. Genetic experiments indicate that the positive (GDNF) and inhibitory (Sprouty1) signals have to be finely balanced throughout renal development to prevent hypoplasia or cystic hyperplasia. Epithelial cysts develop in Spry1-deficient kidneys that share several molecular characteristics with those observed in human disease, suggesting that Spry1 null mice may be useful animal models for cystic hyperplasia.  相似文献   

12.
GDNF/Ret signaling and the development of the kidney   总被引:6,自引:0,他引:6  
Signaling by GDNF through the Ret receptor is required for normal growth of the ureteric bud during kidney development. However, the precise role of GDNF/Ret signaling in renal branching morphogenesis and the specific responses of ureteric bud cells to GDNF remain unclear. Recent studies have provided new insight into these issues. The localized expression of GDNF by the metanephric mesenchyme, together with several types of negative regulation, is important to elicit and correctly position the initial budding event from the Wolffian duct. GDNF also promotes the continued branching of the ureteric bud. However, it does not provide the positional information required to specify the pattern of ureteric bud growth and branching, as its site of synthesis can be drastically altered with minimal effects on kidney development. Cells that lack Ret are unable to contribute to the tip of the ureteric bud, apparently because GDNF-driven proliferation is required for the formation and growth of this specialized epithelial domain.  相似文献   

13.
Organ rudiments with their epithelial bud and adjacent mesenchyme look much the same at their initial stage of differentiation. The subsequent branching of the epithelial anlagen determines the final pattern of the organs, but the mesenchyme provides essential signals for epithelial differentiation. Glial cell line derived neurotrophic factor (GDNF) has recently been shown to regulate ureteric branching morphogenesis and is thereby the first defined signalling molecule in the embryonic metanephric kidney. GDNF is expressed by the mesenchyme, binds to the tip of the ureteric bud and functions in both bud induction and bud orientation. The active receptor complex for GDNF includes the receptor tyrosine kinase Ret and a novel class of glycosylphosphatidylinositol-linked receptors, called GDNF family receptor αs.  相似文献   

14.
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.  相似文献   

15.
During chick liver development, the liver bud arises from the foregut, invaginates into the septum transversum, and elongates along and envelops the ductus venosus. However, the mechanism of liver bud migration is only poorly understood. Here, we demonstrate that a GDNF family ligand involved in neuronal outgrowth and migration, neurturin (NRTN), and its receptor, GFRalpha2, are essential for liver bud migration. In the chick embryo, we found that GFRalpha2 was expressed in the liver bud and that NRTN was expressed in the endothelial cells of the ductus venosus. Inhibition of GFRalpha2 signaling suppressed liver bud elongation along the ductus venous without affecting cell proliferation and apoptosis. Moreover, ectopic expression of NRTN perturbed the directional migration along the ductus venosus, leading to splitting or ectopic branching of the liver. We showed that liver buds selectively migrated toward an NRTN-soaked bead in vitro. These data represent a new model for liver bud migration: NRTN secreted from endothelial cells functions as a chemoattractant to direct the migration of the GFRalpha2-expressing liver bud in early liver development.  相似文献   

16.
Branching morphogenesis of epithelium is a common and important feature of organogenesis; it is, for example, responsible for development of renal collecting ducts, lung airways, milk ducts of mammary glands and seminal ducts of the prostate. In each case, epithelial development is controlled by a variety of mesenchyme-derived molecules, both soluble (e.g. growth factors) and insoluble (e.g. extracellular matrix). Little is known about how these varied influences are integrated to produce a coherent morphogenetic response, but integration is likely to be achieved at least partly by cytoplasmic signal transduction networks. Work in other systems (Drosophila tracheae, MDCK models) suggests that the mitogen-activated protein (MAP) kinase pathway might be important to epithelial branching. We have investigated the role of the MAP kinase pathway in one of the best characterised mammalian examples of branching morphogenesis, the ureteric bud of the metanephric kidney. We find that Erk MAP kinase is normally active in ureteric bud, and that inhibiting Erk activation with the MAP kinase kinase inhibitor, PD98059, reversibly inhibits branching in a dose-dependent manner, while allowing tubule elongation to continue. When Erk activation is inhibited, ureteric bud tips show less cell proliferation than controls and they also produce fewer laminin-rich processes penetrating the mesenchyme and fail to show the strong concentration of apical actin filaments typical of controls; apoptosis and expression of Ret and Ros, are, however, normal. The activity of the Erk MAP kinase pathway is dependent on at least two known regulators of ureteric bud branching; the GDNF-Ret signalling system and sulphated glycosaminoglycans. MAP kinase is therefore essential for normal branching morphogenesis of the ureteric bud, and lies downstream of significant extracellular regulators of ureteric bud development.  相似文献   

17.
Class 3 semaphorins are guidance proteins involved in axon pathfinding, vascular patterning and lung branching morphogenesis in the developing mouse embryo. Semaphorin3a (Sema3a) is expressed in renal epithelia throughout kidney development, including podocytes and ureteric bud cells. However, the role of Sema3a in ureteric bud branching is unknown. Here we demonstrate that Sema3a plays a role in patterning the ureteric bud tree in both metanephric organ cultures and Sema3a mutant mice. In vitro ureteric bud injection with Sema3a antisense morpholino resulted in increased branching, whereas recombinant SEMA3A inhibited ureteric bud branching and decreased the number of developing glomeruli. Additional studies revealed that SEMA3A effects on ureteric bud branching involve downregulation of glial cell-line derived neurotrophic factor (GDNF) signaling, competition with vascular endothelial growth factor A (VEGF-A) and decreased activity of Akt survival pathways. Deletion of Sema3a in mice is associated with increased ureteric bud branching, confirming its inhibitory role in vivo. Collectively, these data suggest that Sema3a is an endogenous antagonist of ureteric bud branching and hence, plays a role in patterning the renal collecting system as a negative regulator.  相似文献   

18.
In mammals, secretion of GDNF by the metanephrogenic mesenchyme is essential for branching morphogenesis of the ureteric bud and, thus, metanephric development. However, the expression pattern of GDNF and its receptor complex-the GPI-linked ligand-binding protein, GFRalpha-1, and the Ret tyrosine kinase signaling protein-indicates that it could operate at early steps in kidney development as well. Furthermore, the developing nephric systems of fish and amphibian embryos express components of the GDNF signaling system even though they do not make a metanephros. We provide evidence that GDNF signaling through GFRalpha-1 is sufficient to direct pathfinding of migrating pronephric duct cells in axolotl embryos by: (1) demonstrating that application of soluble GFRalpha-1 to an embryo lacking all GPI-linked proteins rescues PND migration in a dose-dependent fashion, (2) showing that application of excess soluble GFRalpha-1 to a normal embryo inhibits migration and that inhibition is dependent upon GDNF-binding activity, and (3) showing that the PND will migrate toward a GDNF-soaked bead in vivo, but will fail to migrate when GDNF is applied uniformly to the flank. These data suggest that PND pathfinding is accomplished by migration up a gradient of GDNF.  相似文献   

19.
Glial cell line-derived neurotrophic factor (GDNF) binds a coreceptor GDNF family receptor α1 (GFRα1) and forms a signaling complex with the receptor tyrosine kinase RET. GDNF-GFRα1-RET signaling activates cellular pathways that are required for normal induction of the ureteric bud (UB) from the Wolffian duct (WD). Failure of UB formation results in bilateral renal agenesis and perinatal lethality. Gfrα1 is expressed in both the epithelial and mesenchymal compartments of the developing kidney while Ret expression is specific to the epithelium. The biological importance of Gfrα1’s wider tissue expression and its role in later kidney development are unclear. We discovered that conditional loss of Gfrα1 in the WD epithelium prior to UB branching is sufficient to cause renal agenesis. This finding indicates that Gfrα1 expressed in the nonepithelial structures cannot compensate for this loss. To determine Gfrα1’s role in branching morphogenesis after UB induction we used an inducible Gfrα1-specific Cre-deletor strain and deleted Gfrα1 from the majority of UB tip cells post UB induction in vivo and in explant kidney cultures. We report that Gfrα1 excision from the epithelia compartment after UB induction caused a modest reduction in branching morphogenesis. The loss of Gfrα1 from UB-tip cells resulted in reduced cell proliferation and decreased activated ERK (pERK). Further, cells without Gfrα1 expression are able to populate the branching UB tips. These findings delineate previously unclear biological roles of Gfrα1 in the urinary tract and demonstrate its cell-type and stage-specific requirements in kidney development.  相似文献   

20.
Glial cell line-derived neurotrophic factor (GDNF) family, consisting of GDNF, neurturin, artemin and persephin are distant members of the transforming growth factor-beta (TGF-beta) superfamily. Unlike other members of the TGF-beta superfamily, which signal through the receptor serine-threonine kinases, GDNF family ligands activate intracellular signalling cascades via the receptor tyrosine kinase Ret. GDNF family ligands first bind to the glycosylphosphatidylinositol (GPI)-anchored GDNF family receptor alpha (GFRalpha) and then the GDNF family ligand-GFRalpha complex binds to and stimulates autophosphorylation of Ret. Alternatively, a preassociated complex between GFRalpha and Ret could form the binding site for the GDNF family ligand. GFRalpha1, GFRalpha2, GFRalpha3 and GFRalpha4 are the physiological coreceptors for GDNF, neurturin, artemin and persephin, respectively. Although all GDNF family ligands signal via activated Ret, GDNF can signal also via GFRalpha1 in the absence of Ret. GPI-anchored GFRalpha receptors are localized in plasma membrane to lipid rafts. GDNF binding to GFRalpha1 also recruits Ret to the lipid rafts and triggers association with Src, which is required for effective downstream signalling, leading to differentiation and neuronal survival. GDNF family ligands are potent survival factors for midbrain dopamine neurons, motoneurons, noradrenergic neurons, as well as for sympathetic, parasympathetic and sensory neurons. However, for most neuronal populations, except for motoneurons, TGF-beta is required as a cofactor for GDNF family ligand signalling. Because GDNF and neurturin can rescue dopamine neurons in the animal models of Parkinson disease, as well as motoneurons in vivo, hopes have been raised that GDNF family ligands may be new drugs for the treatment of neurodegenerative diseases. GDNF also has distinct functions outside the nervous system, promoting ureteric branching in kidney development and regulating spermatogenesis.  相似文献   

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