Xuan Chi

Ret-Dependent Cell Rearrangements in the Wolffian Duct Epithelium Initiate Ureteric Bud Morphogenesis

While the genetic control of renal branching morphogenesis has been extensively described, the cellular basis of this process remains obscure. GDNF/RET signaling is required for ureter and kidney development, and cells lacking Ret are excluded from the tips of the branching ureteric bud in chimeric kidneys. Here, we find that this exclusion results from earlier Ret-dependent cell rearrangements in the caudal Wolffian duct, which generate a specialized epithelial domain that later emerges as the tip of the primary ureteric bud. By juxtaposing cells with elevated or reduced RET activity, we find that Wolffian duct cells compete, based on RET signaling levels, to contribute to this domain. At the same time, the caudal Wolffian duct transiently converts from a simple to a pseudostratified epithelium, a process that does not require Ret. Thus, both Ret-dependent cell movements and Ret-independent changes in the Wolffian duct epithelium contribute to ureteric bud formation.

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.

Gata3 Acts Downstream of β-Catenin Signaling to Prevent Ectopic Metanephric Kidney Induction

Metanephric kidney induction critically depends on mesenchymal–epithelial interactions in the caudal region of the nephric (or Wolffian) duct. Central to this process, GDNF secreted from the metanephric mesenchyme induces ureter budding by activating the Ret receptor expressed in the nephric duct epithelium. A failure to regulate this pathway is believed to be responsible for a large proportion of the developmental anomalies affecting the urogenital system. Here, we show that the nephric duct-specific inactivation of the transcription factor gene Gata3 leads to massive ectopic ureter budding. This results in a spectrum of urogenital malformations including kidney adysplasia, duplex systems, and hydroureter, as well as vas deferens hyperplasia and uterine agenesis. The variability of developmental defects is reminiscent of the congenital anomalies of the kidney and urinary tract (CAKUT) observed in human. We show that Gata3 inactivation causes premature nephric duct cell differentiation and loss of Ret receptor gene expression. These changes ultimately affect nephric duct epithelium homeostasis, leading to ectopic budding of interspersed cells still expressing the Ret receptor. Importantly, the formation of these ectopic buds requires both GDNF/Ret and Fgf signaling activities. We further identify Gata3 as a central mediator of β-catenin function in the nephric duct and demonstrate that the β-catenin/Gata3 pathway prevents premature cell differentiation independently of its role in regulating Ret expression. Together, these results establish a genetic cascade in which Gata3 acts downstream of β-catenin, but upstream of Ret, to prevent ectopic ureter budding and premature cell differentiation in the nephric duct.

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.

A transgenic mouse that reveals cell shape and arrangement during ureteric bud branching

Understanding the cellular events that underlie epithelial morphogenesis is a key problem in developmental biology. Here, we describe a new transgenic mouse line that makes it possible to visualize individual cells specifically in the Wolffian duct and ureteric bud, the epithelial structures that give rise to the collecting system of the kidney. myr‐Venus, a membrane‐associated form of the fluorescent protein Venus, was expressed in the ureteric bud lineage under the control of the Hoxb7 promoter. In Hoxb7/myr‐Venus mice, the outlines of all Wolffian duct and ureteric bud epithelial cells are strongly labeled at all stages of urogenital development, allowing the shapes and arrangements of individual cells to be readily observed by confocal microscopy of freshly excised or cultured kidneys. This strain should be extremely useful for studies of cell behavior during ureteric bud branching morphogenesis in wild type and mutant mouse lines. genesis 47:61–66, 2009.

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.

FGF8 is essential for formation of the ductal system in the male reproductive tract

During development of the urogenital tract, fibroblast growth factor 8 (Fgf8) is expressed in mesonephric tubules, but its role in this tissue remains undefined. An evaluation of previously generated T-Cre-mediated Fgf8-deficient mice (T-Cre; Fgf8flox/Δ2,3 mice), which lack Fgf8 expression in the mesoderm, revealed that the cranial region of the Wolffian duct degenerated prematurely and the cranial mesonephric tubules were missing. As a result, the epididymis, vas deferens and efferent ductules were largely absent in mutant mice. Rarb2-Cre was used to eliminate FGF8 from the mesonephric tubules but to allow expression in the adjacent somites. These mutants retained the cranial end of the Wolffian duct and formed the epididymis and vas deferens, but failed to elaborate the efferent ductules, indicating that Fgf8 expression by the mesonephric tubules is required specifically for the formation of the ductules. Ret knockout mice do not form the ureteric bud, a caudal outgrowth of the Wolffian duct and progenitor for the collecting duct network in the kidney, but they do develop the cranial end normally. This indicates that Fgf8, but not Ret, expression is essential to the outgrowth of the cranial mesonephric tubules from the Wolffian duct and to the development of major portions of the sex accessory tissues in the male reproductive tract. Mechanistically, FGF8 functions upstream of Lhx1 expression in forming the nephron, and analysis of Fgf8 mutants similarly shows deficient Lhx1 expression in the mesonephric tubules. These results demonstrate a multifocal requirement for FGF8 in establishing the male reproductive tract ducts and implicate Lhx1 signaling in tubule elongation.

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.