Postnatal prolongation of mammalian nephrogenesis by excess fetal GDNF.

Nephron endowment, defined during the fetal period, dictates renal and related cardiovascular health throughout life. We show here that, despite its negative effects on kidney growth, genetic increase of GDNF prolongs the nephrogenic program beyond its normal cessation. Multi-stage mechanistic analysis revealed that excess GDNF maintains nephron progenitors and nephrogenesis through increased expression of its secreted targets and augmented WNT signaling, leading to a two-part effect on nephron progenitor maintenance. Abnormally high GDNF in embryonic kidneys upregulates its known targets but also Wnt9b and Axin2, with concomitant deceleration of nephron progenitor proliferation. Decline of GDNF levels in postnatal kidneys normalizes the ureteric bud and creates a permissive environment for continuation of the nephrogenic program, as demonstrated by morphologically and molecularly normal postnatal nephron progenitor self-renewal and differentiation. These results establish that excess GDNF has a bi-phasic effect on nephron progenitors in mice, which can faithfully respond to GDNF dosage manipulation during the fetal and postnatal period. Our results suggest that sensing the signaling activity level is an important mechanism through which GDNF and other molecules contribute to nephron progenitor lifespan specification.

Corepressor SMRT is required to maintain Hox transcriptional memory during somitogenesis.

Nuclear hormone receptors (NRs), such as retinoic acid receptors (RARs), play critical roles in vertebrate development and homeostasis by regulating target gene transcription. Their activity is controlled by ligand-dependent release of corepressors and subsequent recruitment of coactivators, but how these individual receptor modes contribute to development are unknown. Here, we show that mice carrying targeted knockin mutations in the corepressor Silencing Mediator of Retinoid and Thyroid hormone receptor (SMRT) that specifically disable SMRT function in NR signaling (SMRTmRID), display defects in cranial neural crest cell-derived structures and posterior homeotic transformations of axial vertebrae. SMRTmRID embryos show enhanced transcription of RAR targets including Hox loci, resulting in respecification of vertebral identities. Up-regulated histone acetylation and decreased H3K27 methylation are evident in the Hox loci whose somitic expression boundaries are rostrally shifted. Furthermore, enhanced recruitment of super elongation complex is evident in rapidly induced non-Pol II-paused targets in SMRTmRID embryonic stem cells. These results demonstrate that SMRT-dependent repression of RAR is critical to establish and maintain the somitic Hox code and segmental identity during fetal development via epigenetic marking of target loci.

Signals and Receptors.

Communication between cells in a multicellular organism occurs by the production of ligands (proteins, peptides, fatty acids, steroids, gases, and other low-molecular-weight compounds) that are either secreted by cells or presented on their surface, and act on receptors on, or in, other target cells. Such signals control cell growth, migration, survival, and differentiation. Signaling receptors can be single-span plasma membrane receptors associated with tyrosine or serine/threonine kinase activities, proteins with seven transmembrane domains, or intracellular receptors. Ligand-activated receptors convey signals into the cell by activating signaling pathways that ultimately affect cytosolic machineries or nuclear transcriptional programs or by directly translocating to the nucleus to regulate transcription.

Dissection of embryonic mouse kidney, culture in vitro, and imaging of the developing organ.

INTRODUCTION: In this article, we outline procedures for the dissection of intact kidneys and the isolation of the ureteric bud (UB) and the metanephric mesenchyme (MM) from mouse embryos. The apparatus required for the culture of these tissues in vitro is described in detail as well as the equipment necessary for performing time-lapse imaging studies of the developing kidney.

Imaging kidney development.

INTRODUCTION: Development of the kidney involves interactions between several cell lineages and complex morphogenetic processes, such as branching of the ureteric bud (UB) to form the collecting duct system and condensation and differentiation of the mesenchymal progenitors to form the nephron epithelia. One of the advantages of the mouse kidney as an experimental system is that it can develop in culture, from the stage of initial branching of the UB (E11.5) for up to a week (although it achieves the size and degree of development of only an E13.5-E14.5 kidney in vivo). The availability of fluorescent proteins (FPs) has provided powerful tools for visualizing the morphogenesis of specific renal structures in organ cultures. Two categories of genetically modified mice that express FPs are useful for visualizing different cell lineages and developmental processes in these organ cultures: (1) transgenic mice that express a fluorescent reporter in the pattern of a specific gene; and (2) Cre reporter mice, which turn on an FP in cells with Cre recombinase activity (and their daughter cells), used in conjunction with cell type-specific Cre transgenic mice. Here, we describe some of the currently available Cre and FP transgenic lines that are useful for the study of kidney development.

SOX9 controls epithelial branching by activating RET effector genes during kidney development.

Congenital abnormalities of the kidney and urinary tract are some of the most common defects detected in the unborn child. Kidney growth is controlled by the GDNF/RET signalling pathway, but the molecular events required for the activation of RET downstream targets are still poorly understood. Here we show that SOX9, a gene involved in campomelic dysplasia (CD) in humans, together with its close homologue SOX8, plays an essential role in RET signalling. Expression of SOX9 can be found from the earliest stages of renal development within the ureteric tip, the ureter mesenchyme and in a segment-specific manner during nephrogenesis. Using a tissue-specific knockout approach, we show that, in the ureteric tip, SOX8 and SOX9 are required for ureter branching, and double-knockout mutants exhibit severe kidney defects ranging from hypoplastic kidneys to renal agenesis. Further genetic analysis shows that SOX8/9 are required downstream of GDNF signalling for the activation of RET effector genes such as Sprouty1 and Etv5. At later stages of development, SOX9 is required to maintain ureteric tip identity and SOX9 ablation induces ectopic nephron formation. Taken together, our study shows that SOX9 acts at multiple steps during kidney organogenesis and identifies SOX8 and SOX9 as key factors within the RET signalling pathway. Our results also explain the aetiology of kidney hypoplasia found in a proportion of CD patients.

Actin depolymerizing factors cofilin1 and destrin are required for ureteric bud branching morphogenesis.

The actin depolymerizing factors (ADFs) play important roles in several cellular processes that require cytoskeletal rearrangements, such as cell migration, but little is known about the in vivo functions of ADFs in developmental events like branching morphogenesis. While the molecular control of ureteric bud (UB) branching during kidney development has been extensively studied, the detailed cellular events underlying this process remain poorly understood. To gain insight into the role of actin cytoskeletal dynamics during renal branching morphogenesis, we studied the functional requirements for the closely related ADFs cofilin1 (Cfl1) and destrin (Dstn) during mouse development. Either deletion of Cfl1 in UB epithelium or an inactivating mutation in Dstn has no effect on renal morphogenesis, but simultaneous lack of both genes arrests branching morphogenesis at an early stage, revealing considerable functional overlap between cofilin1 and destrin. Lack of Cfl1 and Dstn in the UB causes accumulation of filamentous actin, disruption of normal epithelial organization, and defects in cell migration. Animals with less severe combinations of mutant Cfl1 and Dstn alleles, which retain one wild-type Cfl1 or Dstn allele, display abnormalities including ureter duplication, renal hypoplasia, and abnormal kidney shape. The results indicate that ADF activity, provided by either cofilin1 or destrin, is essential in UB epithelial cells for normal growth and branching.

Twist1 activity thresholds define multiple functions in limb development.

The basic helix-loop-helix transcription factor Twist1 is essential for normal limb development. Twist1(-/-) embryos die at midgestation. However, studies on early limb buds found that Twist1(-/-) mutant limb mesenchyme has an impaired response to FGF signaling from the apical ectodermal ridge, which disrupts the feedback loop between the mesenchyme and AER, and reduces and shifts anteriorly Shh expression in the zone of polarizing activity. We have combined Twist1 null, hypomorph and conditional alleles to generate a Twist1 allelic series that survives to birth. As Twist1 activity is reduced, limb skeletal defects progress from preaxial polydactyly to girdle reduction combined with hypoplasia, aplasia or mirror symmetry of all limb segments. With reduced Twist1 activity there is striking and progressive upregulation of ectopic Shh expression in the anterior of the limb, combined with an anterior shift in the posterior Shh domain, which is expressed at normal intensity, and loss of the posterior AER. Consequently limb outgrowth is initially impaired, before an ectopic anterior Shh domain expands the AER, promoting additional growth and repatterning. Reducing the dosage of FGF targets of the Etv gene family, which are known repressors of Shh expression in anterior limb mesenchyme, strongly enhances the anterior skeletal phenotype. Conversely this and other phenotypes are suppressed by reducing the dosage of the Twist1 antagonist Hand2. Our data support a model whereby multiple Twist1 activity thresholds contribute to early limb bud patterning, and suggest how particular combinations of skeletal defects result from differing amounts of Twist1 activity.

The transcription factors Etv4 and Etv5 mediate formation of the ureteric bud tip domain during kidney development.

Signaling by the Ret receptor tyrosine kinase promotes cell movements in the Wolffian duct that give rise to the first ureteric bud tip, initiating kidney development. Although the ETS transcription factors Etv4 and Etv5 are known to be required for mouse kidney development and to act downstream of Ret, their specific functions are unclear. Here, we examine their role by analyzing the ability of Etv4 Etv5 compound mutant cells to contribute to chimeric kidneys. Etv4(-/-);Etv5(+/-) cells show a limited distribution in the caudal Wolffian duct and ureteric bud, similar to Ret(-/-) cells, revealing a cell-autonomous role for Etv4 and Etv5 in the cell rearrangements promoted by Ret. By contrast, Etv4(-/-);Etv5(-/-) cells display more severe developmental limitations, suggesting a broad role for Etv4 and Etv5 downstream of multiple signals, which are together important for Wolffian duct and ureteric bud morphogenesis.

Non-cell-autonomous retinoid signaling is crucial for renal development.

In humans and mice, mutations in the Ret gene result in Hirschsprung's disease and renal defects. In the embryonic kidney, binding of Ret to its ligand, Gdnf, induces a program of epithelial cell remodeling that controls primary branch formation and branching morphogenesis within the kidney. Our previous studies showed that transcription factors belonging to the retinoic acid (RA) receptor family are crucial for controlling Ret expression in the ureteric bud; however, the mechanism by which retinoid-signaling acts has remained unclear. In the current study, we show that expression of a dominant-negative RA receptor in mouse ureteric bud cells abolishes Ret expression and Ret-dependent functions including ureteric bud formation and branching morphogenesis, indicating that RA-receptor signaling in ureteric bud cells is crucial for renal development. Conversely, we find that RA-receptor signaling in ureteric bud cells depends mainly on RA generated in nearby stromal cells by retinaldehyde dehydrogenase 2, an enzyme required for most fetal RA synthesis. Together, these studies suggest that renal development depends on paracrine RA signaling between stromal mesenchyme and ureteric bud cells that regulates Ret expression both during ureteric bud formation and within the developing collecting duct system.

Etv4 and Etv5 are required downstream of GDNF and Ret for kidney branching morphogenesis.

Glial cell line-derived neurotrophic factor signaling through the Ret receptor tyrosine kinase is crucial for ureteric bud branching morphogenesis during kidney development, yet few of the downstream genes are known. Here we show that the ETS transcription factors Etv4 and Etv5 are positively regulated by Ret signaling in the ureteric bud tips. Mice lacking both Etv4 alleles and one Etv5 allele show either renal agenesis or severe hypodysplasia, whereas kidney development fails completely in double homozygotes. We identified several genes whose expression in the ureteric bud depends on Etv4 and Etv5, including Cxcr4, Myb, Met and Mmp14. Thus, Etv4 and Etv5 are key components of a gene network downstream of Ret that promotes and controls renal branching morphogenesis.

Novel regulators of kidney development from the tips of the ureteric bud.

Mammalian nephrogenesis depends on the interaction between the ureteric bud and the metanephric mesenchyme. As the ureteric bud undergoes branching and segmentation, the stalks differentiate into the collecting system of the mature kidney, while the tip cells interact with the adjacent cells of the metanephric mesenchyme, inducing their conversion into nephrons. This induction is mediated by secreted factors. For identifying novel mediators, the tips of the ureteric tree were isolated and microarray analyses were performed using manually refined, multistep gene ontology annotations. For identifying conserved factors, two databases were developed, one from mouse E12.5 and one from rat E13.5 ureteric buds. The overlap of mouse and rat data sets yielded 20 different transcripts that were enriched in the ureteric bud compared with metanephric mesenchyme and predicted to code for secreted proteins. Real-time reverse transcriptase-PCR and in situ hybridization confirmed these identifications. One of the genes that was highly specific to the ureteric bud tip was cytokine-like factor 1 (CLF-1). Recombinant CLF-1 in complex with its physiologic ligand, cardiotrophin-like cytokine (CLC), triggered phosphorylation of signal transducer and activator of transcription 3 in mesenchyme, a pathway characteristic of mesenchymal-to-epithelial conversion. Indeed, when applied to isolated rat metanephric mesenchyme, CLF-1/CLC (3 nM) induced mature nephron structures expressing glomerular and tubular markers. These results underline the power of this first comprehensive gene expression analysis of the ureteric bud tip to identify bioactive molecules.