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SummaryThe ureteric bud is an epithelial tube that undergoes branching morphogenesis to form the renal collecting system. Although development of a normal kidney depends on proper ureteric bud morphogenesis, the cellular events underlying this process remain obscure. Here, we used time-lapse microscopy together with several genetic labeling methods to observe ureteric bud cell behaviors in developing mouse kidneys. We observed an unexpected cell behavior in the branching tips of the ureteric bud, which we term “mitosis-associated cell dispersal.” Premitotic ureteric tip cells delaminate from the epithelium and divide within the lumen; although one daughter cell retains a basal process, allowing it to reinsert into the epithelium at the site of origin, the other daughter cell reinserts at a position one to three cell diameters away. Given the high rate of cell division in ureteric tips, this cellular behavior causes extensive epithelial cell rearrangements that may contribute to renal branching morphogenesis.

Protein 4.1B is a membrane skeletal protein expressed in various organs, and is associated with tumor suppressor in lung cancer-1 (TSLC1) in vitro. Although involvement of 4.1B in the intercellular junctions and tumor-suppression was suggested, some controversial results posed questions to the general tumor-suppressive function of 4.1B and its relation to TSLC1 in vivo. In this study, the expression of 4.1B and its interaction with TSLC1 were examined in rodent adrenal gland, and the involvement of 4.1B in tumorigenesis and the effect of 4.1B deficiency on TSLC1 distribution were also investigated using rodent pheochromocytoma and 4.1B-knockout mice. Although plasmalemmal immunolocalization of 4.1B was shown in chromaffin cells of rodent adrenal medulla, expression of 4.1B was maintained in developed pheochromocytoma, and morphological abnormality or pheochromocytoma generation could not be found in 4.1B-deficient mice. Furthermore, molecular interaction and colocalization of 4.1B and TSLC1 were observed in mouse adrenal gland, but the immunolocalization of TSLC1 along chromaffin cell membranes was not affected in the 4.1B-deficient mice. These results suggest that the function of 4.1B as tumor suppressor might significantly differ among organs and species, and that plasmalemmal retention of TSLC1 would be maintained by molecules other than 4.1B interacting in rodent chromaffin cells.

The F-box proteins beta-TrCP1 and 2 (beta-transducin repeat containing protein) have 2 and 3 isoforms, respectively, due to alternative splicing of exons encoding the N-terminal region. We identified an extra exon in between the previously known exons 1 and 2 of beta-TrCP1 and beta-TrCP2. Interestingly, sequence analysis suggested that many more isoforms are produced than previously identified, via the alternative splicing of all possible combination of exons II to V of beta-TrCP1 and exons II to IV of beta-TrCP2. Different mouse tissues show specific expression patterns of the isoforms, and the level of expression of the isoform that has been used in most published papers was very low. Yeast two-hybrid assays show that beta-TrCP1 isoforms containing exon III, which are the most highly expressed isoforms in most tissues, do not interact with Skp1. Indirect immunofluorescence analysis of transiently expressed beta-TrCP1 isoforms suggests that the presence of exon III causes beta-TrCP1 to localize in nuclei. Consistent with the above findings, isoforms including exon III showed a reduced ability to block ectopic embryonic axes induced via injection of Wnt8 or beta-catenin in Xenopus embryos. Overall, our data suggest that isoforms of beta-TrCPs generated by alternative splicing may have different biological roles.

Ablations of the Axin family genes demonstrated that they modulate Wnt signaling in key processes of mammalian development. The ubiquitously expressed Axin1 plays an important role in formation of the embryonic neural axis, while Axin2 is essential for craniofacial skeletogenesis. Although Axin2 is also highly expressed during early neural development, including the neural tube and neural crest, it is not essential for these processes, apparently due to functional redundancy with Axin1. To further investigate the role of Wnt signaling during early neural development, and its potential regulation by Axins, we developed a mouse model for conditional gene activation in the Axin2-expressing domains. We show that gene expression can be successfully targeted to the Axin2-expressing cells in a spatially and temporally specific fashion. High levels of Axin in this domain induce a region-specific effect on the patterning of neural tube. In the mutant embryos, only the development of midbrain is severely impaired even though the transgene is expressed throughout the neural tube. Axin apparently regulates b-catenin in coordinating cell cycle progression, cell adhesion and survival of neuroepithelial precursors during development of ventricles. Our data support the conclusion that the development of embryonic neural axis is highly sensitive to the level of Wnt signaling. 2006 Elsevier Ireland Ltd. All rights reserved.

Branching of ureteric bud-derived epithelial tubes is a key morphogenetic process that shapes development of the kidney. Glial cell line-derived neurotrophic factor (GDNF) initiates ureteric bud formation and promotes subsequent branching morphogenesis. Exactly how GDNF coordinates branching morphogenesis is unclear. Here we show that the absence of the receptor tyrosine kinase antagonist Sprouty1 (Spry1) results in irregular branching morphogenesis characterized by both increased number and size of ureteric bud tips. Deletion of Spry1 specifically in the epithelium is associated with increased epithelial Wnt11 expression as well as increased mesenchymal Gdnf expression. We propose that Spry1 regulates a Gdnf/Ret/Wnt11-positive feedback loop that coordinates mesenchymal–epithelial dialogue during branching morphogenesis. Genetic experiments indicate that the positive (GDNF) and inhibitory (Sprouty1) signals have to be finely balanced throughout renal development to prevent hypoplasia or cystic hyperplasia. Epithelial cysts develop in Spry1-deficient kidneys that share several molecular characteristics with those observed in human disease, suggesting that Spry1 null mice may be useful animal models for cystic hyperplasia.

Studies have suggested that continuous Wnt/beta-catenin signaling in nascent cartilaginous skeletal elements blocks chondrocyte hypertrophy and endochondral ossification, whereas signaling starting at later stages stimulates hypertrophy and ossification, indicating that Wnt/beta-catenin roles are developmentally regulated. To test this conclusion further, we created transgenic mice expressing a fusion mutant protein of beta-catenin and LEF (CA-LEF) in nascent chondrocytes. Transgenic mice had severe skeletal defects, particularly in limbs. Growth plates were totally disorganized, lacked maturing chondrocytes expressing Indian hedgehog and collagen X, and failed to undergo endochondral ossification. Interestingly, the transgenic cartilaginous elements were ill defined, intermingled with surrounding connective and vascular tissues, and even displayed abnormal joints. However, when activated beta-catenin mutant (delta-beta-catenin) was expressed in chondrocytes already engaged in maturation such as those present in chick limbs, chondrocyte maturation and bone formation were greatly enhanced. Differential responses to Wnt/beta-catenin signaling were confirmed in cultured chondrocytes. Activation in immature cells blocked maturation and actually de-stabilized their phenotype, as revealed by reduced expression of chondrocyte markers, abnormal cytoarchitecture, and loss of proteoglycan matrix. Activation in mature cells instead stimulated hypertrophy, matrix mineralization, and expression of terminal markers such as metalloprotease (MMP)-13 and vascular endothelial growth factor. Because proteoglycans are crucial for cartilage function, we tested possible mechanisms for matrix loss. Delta-beta-catenin expression markedly increased expression of MMP-2, MMP-3, MMP-7, MMP-9, MT3-MMP, and ADAMTS5. In conclusion, Wnt/beta-catenin signaling regulates chondrocyte phenotype, maturation, and function in a developmentally regulated manner, and regulated action by this pathway is critical for growth plate organization, cartilage boundary definition, and endochondral ossification.

Summary Intercellular signaling molecules and their receptors, whose expression must be tightly regulated in time and space, coordinate organogenesis. Regulators of intracellular signaling pathways provide an additional level of control. Here we report that loss of the receptor tyrosine kinase (RTK) antagonist, Sprouty1 (Spry1) , causes defects in kidney development in mice. Spry1 −/− embryos have supernumerary ureteric buds, resulting in the development of multiple ureters and multiplex kidneys. These defects are due to increased sensitivity of the Wolffian duct to GDNF/RET signaling, and reducing Gdnf gene dosage correspondingly rescues the Spry1 null phenotype. We conclude that the function of Spry1 is to modulate GDNF/RET signaling in the Wolffian duct, ensuring that kidney induction is restricted to a single site. These results demonstrate the importance of negative feedback regulation of RTK signaling during kidney induction and suggest that failures in feedback control may underlie some human congenital kidney malformations.

While GDNF signaling through the Ret receptor is critical for kidney development, its specific role in branching morphogenesis of the epithelial ureteric bud (UB) is unclear. Ret expression defines a population of UB “tip cells” distinct from cells of the tubular “trunks,” but how these cells contribute to UB growth is unknown. We have used time-lapse mosaic analysis to investigate normal cell fates within the growing UB and the developmental potential of cells lacking Ret. We found that normal tip cells are bipotential, contributing to both tips and trunks. Cells lacking Ret are specifically excluded from the tips, although they contribute to the trunks, revealing that the tips form and expand by GDNF-driven cell proliferation. Surprisingly, the mutant cells assumed an asymmetric distribution in the UB trunks, suggesting a model of branching in which the epithelium of the tip and the adjacent trunk is remodeled to form new branches.

Adenomatous polyposis coli (APC) protein and Axin form a complex that mediates the down-regulation of beta-catenin, a key effector of Wnt signaling. Truncation mutations in APC are responsible for familial and sporadic colorectal tumors due to failure in the down-regulation of beta-catenin. While the regulation of beta-catenin by APC has been extensively studied, the regulation of APC itself has received little attention. Here we show that the level of APC is down-regulated by the ubiquitin-proteasome pathway and that Wnt signaling inhibits the process. The domain responsible for the down-regulation and direct ubiquitination was identified. We also show an unexpected role for Axin in facilitating the ubiquitination-proteasome-mediated down-regulation of APC through the oligomerization of Axin. Our results suggest a new mechanism for the regulation of APC by Axin and Wnt signaling.

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 supplementary materials) 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.

Upon binding of a Wnt to its receptor, GSK3beta is inhibited through an unknown mechanism involving Dishevelled (Dsh), resulting in the dephosphorylation and stabilization of beta-catenin, which translocates to the nucleus and interacts with Lef/Tcf transcription factors to activate target gene expression. Axin is a scaffold protein which binds beta-catenin and GSK3beta (as well as several other proteins) and thus promotes the phosphorylation of beta-catenin. Here we report that Axin is phosphorylated on Ser and Thr residues in several regions in vivo, while only one region (amino acids 600-672) is efficiently phosphorylated by GSK3beta in vitro. Site-directed mutagenesis, together with in vitro and in vivo phosphorylation assays, demonstrates that Axin residues T609 and S614 are physiological GSK3beta targets. Substitutions for one or more of these residues, which lie within a beta-catenin binding site, reduce the ability of Axin to modulate Wnt-induced signaling in a Lef/Tcf reporter assay. These amino acid substitutions also reduce the binding between Axin and beta-catenin. We propose a model in which inhibition of GSK3beta activity upon Wnt signaling leads to the dephosphorylation of GSK3beta sites in Axin, resulting in the release of beta-catenin from the phosphorylation complex.

Axin is a negative regulator of embryonic axis formation in vertebrates, which acts through a Wnt signal transduction pathway involving the serine/threonine kinase GSK-3 and beta-catenin. Axin has been shown to have distinct binding sites for GSK-3 and beta-catenin and to promote the phosphorylation of beta-catenin and its consequent degradation. This provides an explanation for the ability of Axin to inhibit signaling through beta-catenin. In addition, a more N-terminal region of Axin binds to adenomatous polyposis coli (APC), a tumor suppressor protein that also regulates levels of beta-catenin. Here, we report the results of a yeast two-hybrid screen for proteins that interact with the C-terminal third of Axin, a region in which no binding sites for other proteins have previously been identified. We found that Axin can bind to the catalytic subunit of the serine/threonine protein phosphatase 2A through a domain between amino acids 632 and 836. This interaction was confirmed by in vitro binding studies as well as by co-immunoprecipitation of epitope-tagged proteins expressed in cultured cells. Our results suggest that protein phosphatase 2A might interact with the Axin.APC.GSK-3.beta-catenin complex, where it could modulate the effect of GSK-3 on beta-catenin or other proteins in the complex. We also identified a region of Axin that may allow it to form dimers or multimers. Through two-hybrid and co-immunoprecipitation studies, we demonstrated that the C-terminal 100 amino acids of Axin could bind to the same region as other Axin molecules.

Extracellular matrix molecules, including laminin, affect the development of enteric neurons and accumulate in the aganglionic colon of ls/ls mice. Quantitative Northern analysis revealed that mRNAs encoding the beta 1 and gamma 1 subunits of laminin and collagens alpha 1(IV) and alpha 2(IV) are increased in the colons of ls/ls mice. Transcripts of laminin alpha 1 were evaluated quantitatively with reverse transcription and the competitive polymerase chain reaction (RT-cPCR). The abundance of laminin alpha 1 transcripts was developmentally regulated, but greater in the ls/ls than the wild-type colon at each age examined. In situ hybridization revealed that transcripts in the colon encoding laminin alpha 1 and beta 1 and collagen alpha 2(IV) were initially expressed in the endoderm, but by E15, expression shifted to cells of the colonic mesenchyme (ls/ls > wild type) where crest-derived cells migrate. The expression of laminin alpha 1 was examined in the totally aganglionic intestine of E15 and newborn c-ret -/- mice, to determine whether an increase occurs when neurogenesis fails independently of the ls/ls defect. RT-cPCR revealed no difference from control in mRNA encoding laminin alpha 1 in the c-ret -/- colon in either E15 or newborn animals. The accumulation of immunohistochemically demonstrable laminin that is prominent in the newborn ls/ls colon could not be detected in that of c-ret -/- animals. These observations suggest that transcripts encoding laminin-1 and collagen (IV) are increased in the colon and surrounding pelvic mesenchyme of ls/ls mice because of an intrinsic lesion, rather than a secondary consequence of aganglionosis. The data are compatible with the hypothesis that the increased expression of laminin-1 contributes to the failure of crest-derived cells to complete their colonization of the ls/ls colon.

The beta S12 transgenic line carries an insertion of exogenous DNA and a deletion of approximately 2 cM on chromosome 1. Transgenic heterozygotes (beta S12/+) were viable and fertile, but homozygous mice (beta S12/beta S12) could not be produced. To determine the stage at which developmental arrest occurred in homozygotes, embryos derived from intercrosses among heterozygotes or from control crosses were examined at various stages. No homozygotes could be detected at postimplantation stages, suggesting that the mutation affected embryogenesis before implantation. Recovery of embryos at 1.5 or 3.5 days postcoitum, followed by culture in vitro, revealed an abnormal class of embryos which constituted one-fourth of the progeny from intercrosses, and thus appeared to represent the beta S12/beta S12 homozygotes. These embryos cleaved normally to the 8-cell stage and formed compacted morulae, but then decompacted without forming a blastocyst, and ceased dividing at approximately the 16-cell stage. Nearly all blastomeres were viable at the time of decompaction, as demonstrated by trypan blue exclusion, indicating that the loss of compaction is not simply a consequence of cell death. When labeled with a short-term lineage marker and aggregated with normal embryos at the early 8-cell stage, homozygous mutant embryos failed to contribute to the resulting blastocysts, indicating that the defect in the maintenance of compaction is cell autonomous. Based on the mutant phenotype, we conclude that this genetic lesion affects a gene (or genes) required for the maintenance of compaction, and propose that it be named morula decompaction (mdn). The phenotype of mdn/mdn embryos appears to be distinct from those caused by other previously described preimplantation lethal mutations or chromosomal deletions.

c-myc as an inducer of polycystic kidney disease in transgenic mice. In this study, a genetic model of polycystic kidney disease (PKD) has been produced in transgenic mice bearing the murine c-mycgene driven by the SV40 enhancer and the adult β-globin promoter. These animals reproducibly develop PKD and die of renal failure. The phenotype appears to result from the overexpression of c-myc in the renal tubular epithelium and consequent abnormal cell proliferation. These transgenic mice represent a genetic model of PKD which bears similarities to human autosomal dominant PKD (ADPKD) with respect to renal morphology, renal functional alterations and dominant transmission. Study of these transgenic mice may offer valuable insights into the pathogenesis of PKD.

The possibility that the degeneration of hippocampal neurons can be caused by mis-regulation of Wnt/β-catenin signaling was tested. Downregulation of Wnt signaling by the inducible expression of Axin, ICAT, and dnTcf4E causes degeneration of hippocampal neurons, while upregulation of Wnt signaling by the inducible expression of Dvl and β-catenin has a negligible effect. Treatment with ICG-001, a small molecule known to inhibit Wnt signaling, causes degeneration of hippocampal neurons, while the treatment with a JNK specific inhibitor does not show any effect. The results from LDH and TUNEL assays suggest that degeneration occurs via apoptotic processes. Inhibition of Wnt signaling reduced IGF-1 expression and the addition of IGF-1 blocked degeneration, which suggests that downregulation of IGF-1/Akt signaling is partially responsible for the degeneration. Inducible expression of Axin in the hippocampal neurons isolated from Axin2P-rtTA/pBI-EGFP-Axin double transgenic mice also causes degeneration. Consistent with the findings, these mice had more neuronal cell death in hippocampus and had differences in contextual conditioning upon the inducible expression of Axin. In summary, our data strongly support the idea that downregulation of Wnt/β-catenin signaling causes degeneration of hippocampal neurons in vivo and may be a cause of neurodegenerative diseases related to an anxiety related response.

Branching morphogenesis is a central developmental mechanism utilized in the morphogenesis of several organs including lung, liver, salivary gland and kidney. The molecular control of the basic process of renal differentiation, the ureteric bud (UB) branching, has been in the focus of extensive studies while the cellular events, such as migration and cell shape changes, underlying this process are poorly understood. The actin depolymerizing factors (ADFs) play important roles in actin cytoskeletal dynamics, but little is known about the in vivo functions of ADFs in developmental events like branching morphogenesis. Therefore we studied the functional requirements for the closely related ADFs cofilin1 ( Cfl1 ) and destrin ( Dstn ) during mouse development. Either deletion of Cfl11 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.

Signaling by GDNF through the Ret receptor tyrosine kinase is required for the normal formation, growth and branching of the ureteric bud (UB) during kidney development. However, the precise role of GDNF/Ret signaling in this process, and the specific responses of UB cells to GDNF, remain to be fully elucidated. Recent studies provide new insight into the effects of Ret signaling on cell behavior, the functional overlap between GDNF and other growth factors, and the genes functioning downstream of Ret. Lineage studies show that the UB tip cells, which express Ret, are the progenitors for UB growth, while GDNF-expressing mesenchymal cells are the progenitors of nephron epithelia. Time-lapse studies of chimeric embryos reveal that the earliest role of Ret signaling is in the Wolffian duct, where it promotes cell movements that give rise to the first ureteric bud tip. The requirement for GDNF/Ret signaling can be largely relieved by removing the negative regulator Sprouty1, implicating other growth factors, in particular FGF10, in the support of UB growth and branching. However, the kidneys that develop in the absence of GDNF/Ret and Sprouty1 display branching abnormalities, suggesting a unique role for GDNF in determining UB branching pattern. A number of genes whose expression is induced in the UB by GDNF has been identified, including two ETS transcription factors Etv4 and Etv5. These genes are required downstream of Ret for the Wolffian duct cell movements that form the UB tip domain, as well as for later UB growth and branching.