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.     

The development of the mouse kidney--embryogenesis writ small.

Kidney development starts with an epithelial bud and a domain of committed mesenchyme inducing one another so that the former gives a bifurcating duct system and the latter nephrons. As these events take place in vitro, the mechanisms underpinning nephrogenesis can be investigated experimentally. Recent work has shown the diversity of regulatory molecules expressed during kidney development and begun to clarify the molecular basis of mesenchyme induction, but there is more to come.     

LIF, the ES-cell inhibition factor, reversibly blocks nephrogenesis in cultured mouse kidney rudiments.

Mouse kidney induction proceeds in vitro much as it does in vivo: the ureteric bud bifurcates to give collecting ducts while the mesenchyme condenses into aggregates which epithelialise and then elongate into tubules with glomerular and other nephron structures. We report here that the factor known as LIF (leukaemia inhibitory factor), which regulates the differentiation and growth of embryonic-stem (ES) and other cells in culture, has little effect in vitro on growth or on ureteric-bud morphogenesis other than to stimulate the bifurcation process. It does however exert a striking effect on the mesenchyme. At about four times the concentration required to inhibit ES-cell differentiation, LIF strongly but reversibly blocks the effects of metanephric mesenchyme induction: although mesenchyme condenses around growing duct tips, the number of mature nephrons that form over 6 days is reduced by 75% or more. The few nephrons that do develop in the presence of LIF probably come from mesenchyme already induced at the time of culture and are indistinguishable from those that form in controls as assayed by morphology, by X-gal staining of endogenous galactosidase and by antibodies to brush-border and CD15 antigens. There is a further unexpected feature of rudiments cultured in LIF which is absent in controls: they contain an unexpectedly high number of stable epithelialised aggregates that express laminin around their periphery and which do not develop further. These results argue that the process of nephrogenesis involves at least two distinct stages which can be blocked by LIF: the effect of the initial induction and the future development of epithelialised aggregates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heparin and heparan sulfate delimit nephron formation in fetal metanephric kidneys

Formation of nephrons from primitive mesenchyme in fetal kidneys is induced by ureteric buds. Nephron induction is closely coordinated with branching morphogenesis of the ureteric bud. Having previously shown that branching of the primitive ureter is associated with de novo synthesis of chondroitin sulfate proteoglycan and release of free heparan sulfate glycosaminoglycan chains, we asked whether glycosaminoglycans influence nephron development. Fetal mouse kidneys were incubated in organ cultures containing heparan sulfate, heparin, chondroitin sulfate, or hyaluronate. After 48 hr the number of nephrons at each developmental stage was enumerated by light microscopic analysis of serial tissue sections. Kidneys incubated in heparin or in heparan sulfate contained up to 10-fold fewer nephrons than did kidneys incubated in control conditions or in chondroitin sulfate or hyaluronic acid. Maturation of nephrons, however, was unaffected. Inhibition of nephron development was associated with binding of labeled heparin to primitive mesenchyme and altered tissue distribution of fibronectin. Branching morphogenesis was impaired in kidneys exposed to heparin but not to heparan sulfate or to de-N-sulfated, N-acetylated heparin. The capacity of glycosaminoglycans to inhibit nephron formation depended on sugar composition and O-sulfation but not GAG chain size or charge density. Thus, heparan sulfate may have the capacity to specifically control formation of nephrons in fetal metanephric kidneys in vitro.     

Growth and death in the developing mammalian kidney: signals, receptors and conversations.

Because the kidney (metanephros) starts to function before completing development, its patterning and morphogenesis need to be closely integrated with its growth. This is achieved by blast cells at the kidney periphery generating new nephrons that link up to the extending collecting-duct arborisation, while earlier-formed and more internal nephrons are maturing and beginning to filter serum. This pattern of development requires that cell division and apoptosis be co-ordinated in the various kidney compartments (collecting-ducts, blast cells, metanephric mesenchyme, nephrons and vascular system). The underlying regulatory networks for cell proliferation are beginning to be unravelled, mainly through expression studies, mutation analysis and experimentation in vitro. This article summarises current knowledge of kidney growth and apoptosis, and analyses some of the 80 or so ligand-receptor pairings that seem to sustain development and growth. It also points to some unanswered questions, the most intriguing being what role does apoptosis play during normal kidney development?     

Mesenchymal to epithelial conversion in rat metanephros is induced by LIF

Inductive signals cause conversion of mesenchyme into epithelia during the formation of many organs. Yet a century of study has not revealed the inducing molecules. Using a standard model of induction, we found that ureteric bud cells secrete factors that convert kidney mesenchyme to epithelia that, remarkably, then form nephrons. Purification and sequencing of one such factor identified it as leukemia inhibitory factor (LIF). LIF acted on epithelial precursors that we identified by the expression of Pax2 and Wnt4. Other IL-6 type cytokines acted like LIF, and deletion of their shared receptor reduced nephron development. In situ, the ureteric bud expressed LIF, and metanephric mesenchyme expressed its receptors. The data suggest that IL-6 cytokines are candidate regulators of mesenchymal to epithelial conversion during kidney development.     

Antibodies to cell surface ganglioside GD3 perturb inductive epithelial-mesenchymal interactions

Most epithelial sheets emerge during embryogenesis by a branching and growth of the epithelium. The surrounding mesenchyme is crucial for this process. We report that branching morphogenesis and the formation of a new epithelium from the mesenchyme in the embryonic kidney can be blocked by a monoclonal antibody reacting with a surface glycolipid, disialoganglioside GD3. In contrast, a more than 10-fold excess of antibodies to adhesive glycoproteins (N-CAM, L-CAM, fibronectin) fails to inhibit morphogenesis. Although the anti-GD3 antibody affected epithelial development, the disialoganglioside GD3 was expressed not in the epithelium, but in the mesenchyme surrounding the developing epithelia. The data raise the intriguing possibility that the anti-GD3 antibody inhibits epithelial development by interfering with epithelial-mesenchymal interactions.     

Cell lineages in the embryonic kidney: their inductive interactions and signalling molecules.

The first signalling genes acting in the inductive interactions in the kidney have now been identified. Differentiation of the permanent kidney or the metanephros is critically dependent on inductive signalling between the nephrogenic mesenchyme and ureteric bud epithelium. Further inductive interactions occur between developing nephrons, interstitial stroma, endothelial cells and neurones. Glial-cell-line-derived neurotrophic factor is a signal for the ureteric bud initiation and branching, and Wnt4 is an autocrine epithelializing signal at the pretubular stage of nephron formation. The signals for renal angiogenesis and innervation are less well defined, but seem to include vascular endothelial growth factor and neurotrophins, at least. The ureteric-bud-derived signal for induction of the nephrogenic mesenchyme (to bring the cells to the condensate stage) is not yet known, but fibroblast growth factor 2 is a good candidate. None of the signalling genes identified from the embryonic kidney is specific to the organ, which raises some general questions. How do the organs develop from similar rudiments to various patterns with different cell types and functions? Does the information for organ-specific differentiation pathways retain in the epithelial or mesenchymal compartment? The present, rather fragmentary molecular data would favour the view that similar molecules acting in different combinations and developmental sequences, rather than few organ-specific master genes, could be responsible for the divergence of patterning.     

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.     

Mechanisms of ectodermal organogenesis

All ectodermal organs, e.g. hair, teeth, and many exocrine glands, originate from two adjacent tissue layers: the epithelium and the mesenchyme. Similar sequential and reciprocal interactions between the epithelium and mesenchyme regulate the early steps of development in all ectodermal organs. Generally, the mesenchyme provides the first instructive signal, which is followed by the formation of the epithelial placode, an early signaling center. The placode buds into or out of the mesenchyme, and subsequent proliferation, cell movements, and differentiation of the epithelium and mesenchyme contribute to morphogenesis. The molecular signals regulating organogenesis, such as molecules in the FGF, TGFbeta, Wnt, and hedgehog families, regulate the development of all ectodermal appendages repeatedly during advancing morphogenesis and differentiation. In addition, signaling by ectodysplasin, a recently identified member of the TNF family, and its receptor Edar is required for ectodermal organ development across vertebrate species. Here the current knowledge on the molecular regulation of the initiation, placode formation, and morphogenesis of ectodermal organs is discussed with emphasis on feathers, hair, and teeth.     

FGF-10 induces SP-C and Bmp4 and regulates proximal-distal patterning in embryonic tracheal epithelium

The induction, growth, and differentiation of epithelial lung buds are regulated by the interaction of signals between the lung epithelium and its surrounding mesenchyme. Fibroblast growth factor-10 (FGF-10), which is expressed in the mesenchyme near the distal tips, and bone morphogenetic protein 4 (BMP4), which is expressed in the most distal regions of the epithelium, are important molecules in lung morphogenesis. In the present study, we used two in vitro systems to examine the induction, growth, and differentiation of lung epithelium. Transfilter cultures were used to determine the effect of diffusible factors from the distal lung mesenchyme (LgM) on epithelial branching, and FGF-10 bead cultures were used to ascertain the effect of a high local concentration of a single diffusible molecule on the epithelium. Embryonic tracheal epithelium (TrE) was induced to grow in both culture systems and to express the distal epithelial marker surfactant protein C at the tips nearest the diffusible protein source. TrE cultured on the opposite side of a filter to LgM branched in a pattern resembling intact lungs, whereas TrE cultured in apposition to an FGF-10 bead resembled a single elongating epithelial bud. Examination of the role of BMP4 on lung bud morphogenesis revealed that BMP4 signaling suppressed expression of the proximal epithelial genes Ccsp and Foxj1 in both types of culture and upregulated the expression of Sprouty 2 in TrE cultured with an FGF-10 bead. Antagonizing BMP signaling with Noggin, however, increased expression of both Ccsp and Foxj1.     

Cell-adhesion molecule uvomorulin during kidney development

We studied the expression of a cell adhesion molecule during morphogenesis of the embryonic kidney. The 120-kDa glycoprotein, called uvomorulin, is known to be present on a number of epithelia. During the development of the kidney, a mesenchyme is converted into an epithelium when it is properly induced. The uninduced mesenchyme did not express uvomorulin, as judged by immunofluorescence and immunoblotting using previously characterized antibodies. Uvomorulin does not appear in the mesenchyme as a direct consequence of induction. Rather it becomes detectable approximately 12 hr after completion of induction, at 30-36 hr in vitro when the cells adhere to each other. Distinct differences in uvomorulin expression were seen in the different parts of the nephron. In the mesenchymally derived epithelia (glomeruli, tubules), uvomorulin could be detected only in the tubules, whereas the epithelium of the glomeruli remained negative at all stages of development. Our embryonic studies show that these differences arise very early, as soon as the different parts of the nephron can be distinguished morphologically. It is likely that uvomorulin plays a role in the initial adhesion of the differentiating tubule cells. However, we failed to disrupt histogenesis by applying antibodies to the organ cultures of developing tubules although the antibodies penetrated the tissues well and bound to the differentiating cells.     

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.     

Ureteric bud controls multiple steps in the conversion of mesenchyme to epithelia

Conversion of renal mesenchyme into epithelia depends on the ureteric bud, but its specific actions are not established. From conditioned media of ureteric bud cells, we have identified molecules that mimic the growth and epithelialization of mesenchyme in vivo. LIF targets late epithelial progenitors surrounding the ureteric bud, and in combination with survival factors, converts them into nephrons. In contrast, 24p3/Ngal targets early progenitors at the kidney's periphery through an iron-mediated, but a transferrin-independent mechanism. Hence, the ureteric bud controls many steps of cell conversion. A genome wide search for ureteric bud-specific molecules will identify additional pathways that induce morphogenesis.     

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.     

Kidney development in cadherin-6 mutants: delayed mesenchyme-to-epithelial conversion and loss of nephrons

During nephrogenesis, dynamic changes in the expression of cell adhesion molecules are evident as epithelial structures differentiate from the induced mesenchyme. The cadherins are thought to play an important role in the metanephric mesenchyme, when cells aggregate to form the renal vesicle, a polarized epithelial structure which eventually fuses with the ureteric bud to generate a continuous nascent nephron. We have generated and analyzed mice with a targeted mutation in the gene encoding cadherin-6 (Cad-6), a type II cadherin expressed during early stages of nephrogenesis. These mice are viable and fertile, and they complete both early and late aspects of nephrogenesis. However, upon closer examination in vitro and in vivo, a fraction of the induced metanephric mesenchyme in Cad-6 mutant kidneys fails to form a fully polarized epithelium on schedule. Moreover, a significant number of the renal vesicles in Cad-6 mutant kidneys apparently fail to fuse to the ureteric bud. These alterations in epithelialization and fusion apparently lead to a loss of nephrons in the adult. These studies support the idea that cadherins play an essential role in the formation of epithelial structures and underscore the importance of timing in orchestrating the morphogenesis of complex epithelial tissues.