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.     

Comprehensive microarray analysis of Hoxa11/Hoxd11 mutant kidney development

The Hox11 paralogous genes play critical roles in kidney development. They are expressed in the early metanephric mesenchyme and are required for the induction of ureteric bud formation and its subsequent branching morphogenesis. They are also required for the normal nephrogenesis response of the metanephric mesenchyme to inductive signals from the ureteric bud. In this report, we use microarrays to perform a comprehensive gene expression analysis of the Hoxa11/Hoxd11 mutant kidney phenotype. We examined E11.5, E12.5, E13.5 and E16.5 developmental time points. A novel high throughput strategy for validation of microarray data is described, using additional biological replicates and an independent microarray platform. The results identified 13 genes with greater than 3-fold change in expression in early mutant kidneys, including Hoxa11s, GATA6, TGFbeta2, chemokine ligand 12, angiotensin receptor like 1, cytochrome P450, cadherin5, and Lymphocyte antigen 6 complex, Iroquois 3, EST A930038C07Rik, Meox2, Prkcn, and Slc40a1. Of interest, many of these genes, and others showing lower fold expression changes, have been connected to processes that make sense in terms of the mutant phenotype, including TGFbeta signaling, iron transport, protein kinase C function, growth arrest and GDNF regulation. These results identify the multiple molecular pathways downstream of Hox11 function in the developing kidney.     

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.     

The ECM protein nephronectin promotes kidney development via integrin alpha8beta1-mediated stimulation of Gdnf expression

Development of the metanephric kidney crucially depends on proper interactions between cells and the surrounding extracellular matrix. For example, we showed previously that in the absence of alpha8beta1 integrin, invasion by the ureteric bud into the metanephric mesenchyme is inhibited, resulting in renal agenesis. Here we present genetic evidence that the extracellular matrix protein nephronectin is an essential ligand that engages alpha8beta1 integrin during early kidney development. We show that embryos lacking a functional nephronectin gene frequently display kidney agenesis or hypoplasia, which can be traced to a delay in the invasion of the metanephric mesenchyme by the ureteric bud at an early stage of kidney development. Significantly, we detected no defects in extracellular matrix organization in the nascent kidneys of the nephronectin mutants. Instead, we found that Gdnf expression was dramatically reduced in both nephronectin- and alpha8 integrin-null mutants specifically in the metanephric mesenchyme at the time of ureteric bud invasion. We show that this reduction is sufficient to explain the agenesis and hypoplasia observed in both mutants. Interestingly, the reduction in Gdnf expression is transient, and its resumption presumably enables the nephronectin-deficient ureteric buds to invade the metanephric mesenchyme and begin branching. Our results thus place nephronectin and alpha8beta1 integrin in a pathway that regulates Gdnf expression and is essential for kidney development.     

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.     

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.     

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.     

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.     

TGFbeta superfamily signals are required for morphogenesis of the kidney mesenchyme progenitor population

The TGFbeta superfamily plays diverse and essential roles in kidney development. Gdf11 and Bmp4 are essential for outgrowth and positioning of the ureteric bud, the inducer of metanephric mesenchyme. During nephrogenesis, Bmp7 is required for renewal of the mesenchyme progenitor population. Additionally, in vitro studies demonstrate inhibitory effects of BMPs and TGFbetas on collecting duct branching and growth. Here, we explore the predicted models of TGFbeta superfamily function by cell-specific inactivation of Smad4, a key mediator of TGFbeta signaling. Using a HoxB7cre transgene expressed in ureteric bud and collecting duct, we find that development of the collecting duct is Smad4 independent. By contrast, removal of Smad4 in nephrogenic mesenchyme using the Bmp7(cre/+) allele leads to disorganization of the nephrogenic mesenchyme and impairment of mesenchyme induction. Smad4-deficient metanephric mesenchyme does not display defects in inducibility in LiCl or spinal cord induction assays. However, in situ hybridization and lineage analysis of Smad4 null mesenchyme cells at E11.5 show that the nephrogenic mesenchyme does not aggregate tightly around the ureteric bud tips, but remains loosely associated, embedded within a population of cells expressing markers of both nephrogenic mesenchyme and peripheral stroma. We conclude that the failure of recruitment of nephrogenic mesenchyme leaves a primitive population of mesenchyme at the periphery of the kidney. This population is gradually depleted, and by E16.5 the periphery is composed of cells of stromal phenotype. This study uncovers a novel role for TGFbeta superfamily signaling in the recruitment and/or organization of the nephrogenic mesenchyme at early time-points of kidney development. Additionally, we present conclusive genetic lineage mapping of the collecting duct and nephrogenic mesenchyme.     

Identification of kidney mesenchymal genes by a combination of microarray analysis and Sall1-GFP knockin mice

SALL1, a causative gene for Townes-Brocks syndrome, encodes a zinc finger protein, and its mouse homolog (Sall1) is essential for metanephros development, as noted during gene targeting. In the embryonic kidney, Sall1 is expressed abundantly in mesenchyme-derived structures from condensed mesenchyme, S-, comma-shaped bodies, to renal tubules and podocytes. We generated mice in which a green fluorescent protein (GFP) gene was inserted into the Sall1 locus and we isolated the GFP-positive population from embryonic kidneys of these mice by fluorescein-activated cell sorting. The GFP-positive population indeed expressed mesenchymal genes, while the negative population expressed genes in the ureteric bud. To systematically search for genes expressed in the mesenchyme-derived cells, we compared gene expression profiles in the GFP-positive and -negative populations using microarray analysis, followed by in situ hybridization. We detected many genes known to be important for metanephros development including Sall1, GDNF, Raldh2, Pax8 and FoxD1, and genes expressed abundantly in the metanephric mesenchyme such as Unc4.1, Six2, Osr-2 and PDGFc. We also found groups of genes including SSB-4, Smarcd3, micro-Crystallin, TRB-2, which are not known to be expressed in the metanephric mesenchyme. Therefore a combination of microarray technology and Sall1-GFP mice is useful for systematic identification of genes expressed in the developing kidney.     

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.     

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.     

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.     

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.     

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.     

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     

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.