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.     

Induced repatterning of type XVIII collagen expression in ureter bud from kidney to lung type: association with sonic hedgehog and ectopic surfactant protein C

Epithelial-mesenchymal tissue interactions regulate the formation of signaling centers that play a role in the coordination of organogenesis, but it is not clear how their activity leads to differences in organogenesis. We report that type XVIII collagen, which contains both a frizzled and an endostatin domain, is expressed throughout the respective epithelial bud at the initiation of lung and kidney organogenesis. It becomes localized to the epithelial tips in the lung during the early stages of epithelial branching, while its expression in the kidney is confined to the epithelial stalk region and is lost from the nearly formed ureter tips, thus displaying the reverse pattern to that in the lung. In recombinants, between ureter bud and lung mesenchyme, type XVIII collagen expression pattern in the ureter bud shifts from the kidney to the lung type, accompanied by a shift in sonic hedgehog expression in the epithelium. The lung mesenchyme is also sufficient to induce ectopic lung surfactant protein C expression in the ureter bud. Moreover, the shift in type XVIII collagen expression is associated with changes in ureter development, thus resembling aspects of early lung type epigenesis in the recombinants. Respecification of collagen is necessary for the repatterning process, as type XVIII collagen antibody blocking had no effect on ureter development in the intact kidney, whereas it reduced the number of epithelial tips in the lung and completely blocked ureter development with lung mesenchyme. Type XVIII collagen antibody blocking also led to a notable reduction in the expression of Wnt2, which is expressed in the lung mesenchyme but not in that of the kidney, suggesting a regulatory interaction between this collagen and Wnt2. Respecification also occurred in a chimeric organ containing the ureter bud and both kidney and lung mesenchymes, indicating that the epithelial tips can integrate the morphogenetic signals independently. A glial cell line-derived neurotrophic factor signal induces loss of type XVIII collagen from the ureter tips and renders the ureter bud competent for repatterning by lung mesenchyme-derived signals. Our data suggest that differential organ morphogenesis is regulated by an intra-organ patterning process that involves coordination between inductive signals and matrix molecules, such as type XVIII collagen.     

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     

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.     

Rat metanephric organ culture in terato-embryology

The development of the permanent mammalian kidney, or metanephros, depends on mesenchymal-epithelial interactions, leading to branching morphogenesis of the ureteric bud that forms the collecting ducts and to conversion of the metanephric mesenchyme into epithelium that forms the nephrons. Rat metanephric organ culture in which these interactions are maintained is a valuable in vitro model system for investigating normal and abnormal renal organogenesis. Methods were designed to evaluate either the capacity of the ureteric bud to branch or that of the mesenchyme to form nephrons. Both are based on specific staining of the ureteric bud and the glomeruli with lectins. Using this approach, we have shown that retinoids are potent stimulating factors of nephrogenesis, acting through an increase in the branching capacity of the ureteric bud. On the other hand, several drugs such as gentamicin and cyclosporin A were found to reduce the number of nephrons formed in vitro. While gentamicin affects the early branching pattern of the ureteric bud, cyclosporin may affect the capacity of the mesencyme to convert into epithelium. This methodology therefore appears a potentially useful tool for toxicological studies new drugs.     

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.     

The tip-top branching ureter

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.     

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.     

Erk MAP kinase regulates branching morphogenesis in the developing mouse kidney.

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.     

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.     

Distinct and sequential tissue-specific activities of the LIM-class homeobox gene Lim1 for tubular morphogenesis during kidney development

Kidney organogenesis requires the morphogenesis of epithelial tubules. Inductive interactions between the branching ureteric buds and the metanephric mesenchyme lead to mesenchyme-to-epithelium transitions and tubular morphogenesis to form nephrons, the functional units of the kidney. The LIM-class homeobox gene Lim1 is expressed in the intermediate mesoderm, nephric duct, mesonephric tubules, ureteric bud, pretubular aggregates and their derivatives. Lim1-null mice lack kidneys because of a failure of nephric duct formation, precluding studies of the role of Lim1 at later stages of kidney development. Here, we show that Lim1 functions in distinct tissue compartments of the developing metanephros for both proper development of the ureteric buds and the patterning of renal vesicles for nephron formation. These observations suggest that Lim1 has essential roles in multiple steps of epithelial tubular morphogenesis during kidney organogenesis. We also demonstrate that the nephric duct is essential for the elongation and maintenance of the adjacent Mullerian duct, the anlage of the female reproductive tract.     

Localization and quantitation of 125I-epidermal growth factor binding in mouse embryonic tooth and other embryonic tissues at different developmental stages

We have shown earlier that epidermal growth factor (EGF) inhibits morphogenesis and cell differentiation in mouse embryonic teeth in organ culture. This inhibition depends on the stage of tooth development so that only teeth at early developmental stages respond to EGF (A-M. Partanen, P. Ekblom, and I. Thesleff (1985) Dev. Biol. 111, 84-94). We have now studied the quantity and pattern of EGF binding in teeth at various stages of development by incubating the dissected tooth germs with 125I-labeled EGF. Although the quantity of 125I-EGF binding per microgram DNA stays at the same level, localization of 125I-EGF binding by autoradiography reveals that the distribution of binding sites changes dramatically. In bud stage the epithelial tooth bud that is intruding into the underlying mesenchyme has binding sites for EGF, but the condensation of dental mesenchymal cells around the bud does not bind EGF. At the cap stage of development the dental mesenchyme binds EGF, but the dental epithelium shows no binding. This indicates that the dental mesenchyme is the primary target tissue for the inhibitory effect of EGF on tooth morphogenesis during early cap stage. During advanced morphogenesis the binding sites of EGF disappear also from the dental papilla mesenchyme, but the dental follicle which consists of condensed mesenchymal cells surrounding the tooth germ, binds EGF abundantly. We have also studied EGF binding during the development of other embryonic organs, kidney, salivary gland, lung, and skin, which are all formed by mesenchymal and epithelial components. The patterns of EGF binding in various tissues suggest that EGF may have a role in the organogenesis of epitheliomesenchymal organs as a stimulator of epithelial proliferation during initial epithelial bud formation and branching morphogenesis. The results of this study indicate that EGF stimulates or maintains proliferation of undifferentiated cells during embryonic development and that the expression of EGF receptors in different organs is not related to the age of the embryo, but is specific to the developmental stage of each organ.     

Cessation of renal morphogenesis in mice

The kidney develops by cycles of ureteric bud branching and nephron formation. The cycles begin and are sustained by reciprocal inductive interactions and feedback between ureteric bud tips and the surrounding mesenchyme. Understanding how the cycles end is important because it controls nephron number. During the period when nephrogenesis ends in mice, we examined the morphology, gene expression, and function of the domains that control branching and nephrogenesis. We found that the nephrogenic mesenchyme, which is required for continued branching, was gone by the third postnatal day. This was associated with an accelerated rate of new nephron formation in the absence of apoptosis. At the same time, the tips of the ureteric bud branches lost the typical appearance of an ampulla and lost Wnt11 expression, consistent with the absence of the capping mesenchyme. Surprisingly, expression of Wnt9b, a gene necessary for mesenchyme induction, continued. We then tested the postnatal day three bud branch tip and showed that it maintained its ability both to promote survival of metanephric mesenchyme and to induce nephrogenesis in culture. These results suggest that the sequence of events leading to disruption of the cycle of branching morphogenesis and nephrogenesis began with the loss of mesenchyme that resulted from its conversion into nephrons.     

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.     

Involvement of laminin binding integrins and laminin-5 in branching morphogenesis of the ureteric bud during kidney development.

Branching morphogenesis of the ureteric bud (UB) [induced by the metanephric mesenchyme (MM)] is necessary for normal kidney development. The role of integrins in this complex developmental process is not well understood. However, the recent advent of in vitro model systems to study branching of UB cells and isolated UB tissue makes possible a more detailed analysis of the integrins involved. We detected integrin subunits alpha3, alpha6, beta1, and beta4 in both the UB and cells derived from the early UB. Blocking the function of each of these integrin subunits individually markedly inhibited branching morphogenesis in cell culture models. However, inhibiting individual integrin function with blocking antibodies in whole kidney and isolated UB culture only partially inhibited UB branching morphogenesis, suggesting that, in these more complex in vitro systems, multiple integrins are involved in the branching program. In whole organ and isolated bud culture, marked retardation of UB branching was observed only when both alpha3 and alpha6 integrin subunits were inhibited. The alpha6 integrin subunit can be expressed as both alpha6beta1 and alpha6beta4, and both of these beta subunits are important for UB branching morphogenesis in both cell and organ culture. Furthermore, laminin-5, a common ligand for integrins alpha3beta1 and alpha6beta4, was detected in the developing UB and shown to be required for normal UB branching morphogenesis in whole embryonic kidney organ culture as well as isolated UB culture. Together, these data from UB cell culture, organ culture, and isolated UB culture models indicate that both integrin alpha3 and alpha6 subunits play a direct role in UB branching morphogenesis, as opposed to being modulators of the inductive effects of mesenchyme on UB development. Furthermore the data are consistent with a role for laminin-5, acting through its alpha3beta1 and/or alpha6beta4 integrin receptors, in UB branching during nephrogenesis. These data may help to partially explain the renal phenotype seen in integrin alpha3 and alpha3/alpha6 subunit-deficient animals.     

Hoxa11 and Hoxd11 regulate branching morphogenesis of the ureteric bud in the developing kidney

Hoxa11 and Hoxd11 are functionally redundant during kidney development. Mice with homozygous null mutation of either gene have normal kidneys, but double mutants have rudimentary, or in extreme cases, absent kidneys. We have examined the mechanism for renal growth failure in this mouse model and find defects in ureteric bud branching morphogenesis. The ureteric buds are either unbranched or have an atypical pattern characterized by lack of terminal branches in the midventral renal cortex. The mutant embryos show that Hoxa11 and Hoxd11 control development of a dorsoventral renal axis. By immunohistochemical analysis, Hoxa11 expression is restricted to the early metanephric mesenchyme, which induces ureteric bud formation and branching. It is not found in the ureteric bud. This suggests that the branching defect had been caused by failure of mesenchyme to epithelium signaling. In situ hybridizations with Wnt7b, a marker of the metanephric kidney, show that the branching defect was not simply the result of homeotic transformation of metanephros to mesonephros. Absent Bf2 and Gdnf expression in the midventral mesenchyme, findings that could by themselves account for branching defects, shows that Hoxa11 and Hoxd11 are necessary for normal gene expression in the ventral mesenchyme. Attenuation of normal gene expression along with the absence of a detectable proliferative or apoptotic change in the mutants show that one function of Hoxa11 and Hoxd11 in the developing renal mesenchyme is to regulate differentiation necessary for mesenchymal-epithelial reciprocal inductive interactions.     

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.     

Real-time analysis of ureteric bud branching morphogenesis in vitro

While it is clear that the normal branching morphogenesis of the ureteric bud (UB) is critical for development of the metanephric kidney, the specific patterns of branching and growth have heretofore only been inferred from static images. Here, we present a systematic time-lapse analysis of UB branching morphogenesis during the early development of the mouse kidney in organ culture. Metanephric primordia from Hoxb7/GFP transgenic embryos were cultured for 3-4 days, and GFP images of the UB taken every 30 min were assembled into movies. Analysis of these movies (available as )revealed that the UB is a highly plastic structure, which can branch in a variety of complex patterns, including terminal bifid, terminal trifid, and lateral branching. To examine kinetic parameters of branching and elongation, skeletal representations of the UB were used to measure the number of segments and branch points and the length of each segment as a function of time and of branch generation. These measurements provide a baseline for future studies on mutant kidneys with defects in renal development. To illustrate how these quantitative methods can be applied to the analysis of abnormal kidney development, we examined the effects of the MEK1 inhibitor PD98059 on renal organ cultures and confirmed a previous report that the drug has a specific inhibitory effect on UB branching as opposed to elongation.     

GDNF/Ret signaling and the development of the kidney

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.