Ret signaling in ureteric bud epithelial cells controls cell movements, cell clustering and bud formation.

Ret signaling promotes branching morphogenesis during kidney development, but the underlying cellular mechanisms remain unclear. While Ret-expressing progenitor cells proliferate at the ureteric bud tips, some of these cells exit the tips to generate the elongating collecting ducts, and in the process turn off Ret. Genetic ablation of Ret in tip cells promotes their exit, suggesting that Ret is required for cell rearrangements that maintain the tip compartments. Here, we examine the behaviors of ureteric bud cells that are genetically forced to maintain Ret expression. These cells move to the nascent tips, and remain there during many cycles of branching; this tip-seeking behavior may require positional signals from the mesenchyme, as it occurs in whole kidneys but not in epithelial ureteric bud organoids. In organoids, cells forced to express Ret display a striking self-organizing behavior, attracting each other to form dense clusters within the epithelium, which then evaginate to form new buds. The ability of forced Ret expression to promote these events suggests that similar Ret-dependent cell behaviors play an important role in normal branching morphogenesis.

Ret and Etv4 Promote Directed Movements of Progenitor Cells during Renal Branching Morphogenesis.

Branching morphogenesis of the epithelial ureteric bud forms the renal collecting duct system and is critical for normal nephron number, while low nephron number is implicated in hypertension and renal disease. Ureteric bud growth and branching requires GDNF signaling from the surrounding mesenchyme to cells at the ureteric bud tips, via the Ret receptor tyrosine kinase and coreceptor Gfrα1; Ret signaling up-regulates transcription factors Etv4 and Etv5, which are also critical for branching. Despite extensive knowledge of the genetic control of these events, it is not understood, at the cellular level, how renal branching morphogenesis is achieved or how Ret signaling influences epithelial cell behaviors to promote this process. Analysis of chimeric embryos previously suggested a role for Ret signaling in promoting cell rearrangements in the nephric duct, but this method was unsuited to study individual cell behaviors during ureteric bud branching. Here, we use Mosaic Analysis with Double Markers (MADM), combined with organ culture and time-lapse imaging, to trace the movements and divisions of individual ureteric bud tip cells. We first examine wild-type clones and then Ret or Etv4 mutant/wild-type clones in which the mutant and wild-type sister cells are differentially and heritably marked by green and red fluorescent proteins. We find that, in normal kidneys, most individual tip cells behave as self-renewing progenitors, some of whose progeny remain at the tips while others populate the growing UB trunks. In Ret or Etv4 MADM clones, the wild-type cells generated at a UB tip are much more likely to remain at, or move to, the new tips during branching and elongation, while their Ret-/- or Etv4-/- sister cells tend to lag behind and contribute only to the trunks. By tracking successive mitoses in a cell lineage, we find that Ret signaling has little effect on proliferation, in contrast to its effects on cell movement. Our results show that Ret/Etv4 signaling promotes directed cell movements in the ureteric bud tips, and suggest a model in which these cell movements mediate branching morphogenesis.

Nephric duct insertion requires EphA4/EphA7 signaling from the pericloacal mesenchyme.

The vesico-ureteric junction (VUJ) forms through a complex developmental program that connects the primordium of the upper urinary tract [the nephric duct (ND)] with that of the lower urinary tract (the cloaca). The signals that orchestrate the various tissue interactions in this program are poorly understood. Here, we show that two members of the EphA subfamily of receptor tyrosine kinases, EphA4 and EphA7, are specifically expressed in the mesenchyme surrounding the caudal ND and the cloaca, and that Epha4(-/-);Epha7(+/-) and Epha4(-/-);Epha7(-/-) (DKO) mice display distal ureter malformations including ureterocele, blind and ectopically ending ureters with associated hydroureter, megaureter and hydronephrosis. We trace these defects to a late or absent fusion of the ND with the cloaca. In DKO embryos, the ND extends normally and approaches the cloaca but the tip subsequently looses its integrity. Expression of Gata3 and Lhx1 and their downstream target Ret is severely reduced in the caudal ND. Conditional deletion of ephrin B2 from the ND largely phenocopies these changes, suggesting that EphA4/EphA7 from the pericloacal mesenchyme signal via ephrin B2 to mediate ND insertion. Disturbed activity of this signaling module may entail defects of the VUJ, which are frequent in the spectrum of congenital anomalies of the kidney and the urinary tract (CAKUT) in human newborns.

Mitogen-activated protein kinase (MAPK) pathway regulates branching by remodeling epithelial cell adhesion.

Although the growth factor (GF) signaling guiding renal branching is well characterized, the intracellular cascades mediating GF functions are poorly understood. We studied mitogen-activated protein kinase (MAPK) pathway specifically in the branching epithelia of developing kidney by genetically abrogating the pathway activity in mice lacking simultaneously dual-specificity protein kinases Mek1 and Mek2. Our data show that MAPK pathway is heterogeneously activated in the subset of G1- and S-phase epithelial cells, and its tissue-specific deletion results in severe renal hypodysplasia. Consequently to the deletion of Mek1/2, the activation of ERK1/2 in the epithelium is lost and normal branching pattern in mutant kidneys is substituted with elongation-only phenotype, in which the epithelium is largely unable to form novel branches and complex three-dimensional patterns, but able to grow without primary defects in mitosis. Cellular characterization of double mutant epithelium showed increased E-cadherin at the cell surfaces with its particular accumulation at baso-lateral locations. This indicates changes in cellular adhesion, which were revealed by electron microscopic analysis demonstrating intercellular gaps and increased extracellular space in double mutant epithelium. When challenged to form monolayer cultures, the mutant epithelial cells were impaired in spreading and displayed strong focal adhesions in addition to spiky E-cadherin. Inhibition of MAPK activity reduced paxillin phosphorylation on serine 83 while remnants of phospho-paxillin, together with another focal adhesion (FA) protein vinculin, were augmented at cell surface contacts. We show that MAPK activity is required for branching morphogenesis, and propose that it promotes cell cycle progression and higher cellular motility through remodeling of cellular adhesions.

Mosaic analysis of cell rearrangements during ureteric bud branching in dissociated/reaggregated kidney cultures and in vivo.

BACKGROUND: Cell rearrangements mediated by GDNF/Ret signaling underlie the formation of the ureteric bud (UB) tip domain during kidney development. Whether FGF signaling also influences these rearrangements is unknown. Chimeric embryos are a powerful tool for examining the genetic controls of cellular behaviors, but generating chimeras by traditional methods is expensive and laborious. Dissociated fetal kidney cells can reorganize to form complex structures including branching UB tubules, providing an easier method to generate renal chimeras. RESULTS: Cell behaviors in normal or chimeric kidney cultures were investigated using time-lapse imaging. In Spry1(-/-) ↔ wild-type chimeras, cells lacking Spry1 (a negative regulator of Ret and FGF receptor signaling) preferentially occupied the UB tips, as previously observed in traditional chimeras, thus validating this experimental system. In Fgfr2(UB-/-) ↔ wild-type chimeras, the wild-type cells preferentially occupied the tips. Independent evidence for a role of Fgfr2 in UB tip formation was obtained using Mosaic mutant Analysis with Spatial and Temporal control of Recombination (MASTR). CONCLUSIONS: Dissociation and reaggregation of fetal kidney cells of different genotypes, with suitable fluorescent markers, provides an efficient way to analyze cell behaviors in chimeric cultures. FGF/Fgfr2 signaling promotes UB cell rearrangements that form the tip domain, similarly to GDNF/Ret signaling.

ETS-related transcription factors ETV4 and ETV5 are involved in proliferation and induction of differentiation-associated genes in embryonic stem (ES) cells.

The pluripotency and self-renewal capacity of embryonic stem (ES) cells is regulated by several transcription factors. Here, we show that the ETS-related transcription factors Etv4 and Etv5 (Etv4/5) are specifically expressed in undifferentiated ES cells, and suppression of Oct3/4 results in down-regulation of Etv4/5. Simultaneous deletion of Etv4 and Etv5 (Etv4/5 double knock-out (dKO)) in ES cells resulted in a flat, epithelial cell-like appearance, whereas the morphology changed into compact colonies in a 2i medium (containing two inhibitors for GSK3 and MEK/ERK). Expression levels of self-renewal marker genes, including Oct3/4 and Nanog, were similar between wild-type and dKO ES cells, whereas proliferation of Etv4/5 dKO ES cells was decreased with overexpression of cyclin-dependent kinase inhibitors (p16/p19, p15, and p57). A differentiation assay revealed that the embryoid bodies derived from Etv4/5 dKO ES cells were smaller than the control, and expression of ectoderm marker genes, including Fgf5, Sox1, and Pax3, was not induced in dKO-derived embryoid bodies. Microarray analysis demonstrated that stem cell-related genes, including Tcf15, Gbx2, Lrh1, Zic3, and Baf60c, were significantly repressed in Etv4/5 dKO ES cells. The artificial expression of Etv4 and/or Etv5 in Etv4/5 dKO ES cells induced re-expression of Tcf15 and Gbx2. These results indicate that Etv4 and Etv5, potentially through regulation of Gbx2 and Tcf15, are involved in the ES cell proliferation and induction of differentiation-associated genes in ES cells.

Non-canonical Wnt5a/Ror2 signaling regulates kidney morphogenesis by controlling intermediate mesoderm extension.

Congenital anomalies of the kidney and urinary tract (CAKUT) affect about 1 in 500 births and are a major cause of morbidity in infants. Duplex collecting systems rank among the most common abnormalities of CAKUT, but the molecular basis for this defect is poorly understood. In mice, conditional deletion of Wnt5a in mesoderm results in bilateral duplex kidney and ureter formation. The ureteric buds (UBs) in mutants emerge as doublets from the intermediate mesoderm (IM)-derived nephric duct (ND) without anterior expansion of the glial cell line-derived neurotrophic factor (Gdnf) expression domain in the surrounding mesenchyme. Wnt5a is normally expressed in a graded manner at the posterior end of the IM, but its expression is down-regulated prior to UB outgrowth at E10.5. Furthermore, ablation of Wnt5a in the mesoderm with an inducible Cre at E7.5 results in duplex UBs, whereas ablation at E8.5 yields normal UB outgrowth, demonstrating that Wnt5a functions in IM development well before the formation of the metanephros. In mutants, the posterior ND is duplicated and surrounding Pax2-positive mesenchymal cells persist in the nephric cord, suggesting that disruption of normal ND patterning prompts the formation of duplex ureters and kidneys. Ror2 homozygous mutants, which infrequently yield duplex collecting systems, show a dramatic increase in incidence with the additional deletion of one copy of Wnt5a, implicating this receptor in non-canonical Wnt5a signaling during IM development. This work provides the first evidence of a role of Wnt5a/Ror2 signaling in IM extension and offers new insights into the etiology of CAKUT and possible involvement of Wnt5a/Ror2 mutations.

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; Fgf8(flox/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.

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.

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.

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.

Kidney development in the absence of Gdnf and Spry1 requires Fgf10.

GDNF signaling through the Ret receptor tyrosine kinase (RTK) is required for ureteric bud (UB) branching morphogenesis during kidney development in mice and humans. Furthermore, many other mutant genes that cause renal agenesis exert their effects via the GDNF/RET pathway. Therefore, RET signaling is believed to play a central role in renal organogenesis. Here, we re-examine the extent to which the functions of Gdnf and Ret are unique, by seeking conditions in which a kidney can develop in their absence. We find that in the absence of the negative regulator Spry1, Gdnf, and Ret are no longer required for extensive kidney development. Gdnf-/-;Spry1-/- or Ret-/-;Spry1-/- double mutants develop large kidneys with normal ureters, highly branched collecting ducts, extensive nephrogenesis, and normal histoarchitecture. However, despite extensive branching, the UB displays alterations in branch spacing, angle, and frequency. UB branching in the absence of Gdnf and Spry1 requires Fgf10 (which normally plays a minor role), as removal of even one copy of Fgf10 in Gdnf-/-;Spry1-/- mutants causes a complete failure of ureter and kidney development. In contrast to Gdnf or Ret mutations, renal agenesis caused by concomitant lack of the transcription factors ETV4 and ETV5 is not rescued by removing Spry1, consistent with their role downstream of both RET and FGFRs. This shows that, for many aspects of renal development, the balance between positive signaling by RTKs and negative regulation of this signaling by SPRY1 is more critical than the specific role of GDNF. Other signals, including FGF10, can perform many of the functions of GDNF, when SPRY1 is absent. But GDNF/RET signaling has an apparently unique function in determining normal branching pattern. In contrast to GDNF or FGF10, Etv4 and Etv5 represent a critical node in the RTK signaling network that cannot by bypassed by reducing the negative regulation of upstream signals.

Distal ureter morphogenesis depends on epithelial cell remodeling mediated by vitamin A and Ret.

Almost 1% of human infants are born with urogenital abnormalities, many of which are linked to irregular connections between the distal ureters and the bladder. During development, ureters migrate by an unknown mechanism from their initial integration site in the Wolffian ducts up to the base of the bladder in a process that we call ureter maturation. Rara(-/-) Rarb2(-/-) mice display impaired vitamin A signaling and develop syndromic urogenital malformations similar to those that occur in humans, including renal hypoplasia, hydronephrosis and mega-ureter, abnormalities also seen in mice with mutations in the proto-oncogene Ret. Here we show that ureter maturation depends on formation of the 'trigonal wedge', a newly identified epithelial outgrowth from the base of the Wolffian ducts, and that the distal ureter abnormalities seen in Rara(-/-) Rarb2(-/-) and Ret(-/-) mutant mice are probably caused by a failure of this process. Our studies indicate that formation of the trigonal wedge may be essential for correct insertion of the distal ureters into the bladder, and that these events are mediated by the vitamin A and Ret signaling pathways.

Low nephron endowment increases susceptibility to renal stress and chronic kidney disease.

Preterm birth results in low nephron endowment and increased risk of acute kidney injury (AKI) and chronic kidney disease (CKD). To understand the pathogenesis of AKI and CKD in preterm humans, we generated potentially novel mouse models with a 30%-70% reduction in nephron number by inhibiting or deleting Ret tyrosine kinase in the developing ureteric bud. These mice developed glomerular and tubular hypertrophy, followed by the transition to CKD, recapitulating the renal pathological changes seen in humans born preterm. We injected neonatal mice with gentamicin, a ubiquitous nephrotoxic exposure in preterm infants, and detected more severe proximal tubular injury in mice with low nephron number compared with controls with normal nephron number. Mice with low nephron number had reduced proliferative repair with more rapid development of CKD. Furthermore, mice had more profound inflammation with highly elevated levels of MCP-1 and CXCL10, produced in part by damaged proximal tubules. Our study directly links low nephron endowment with postnatal renal hypertrophy, which in this model is maladaptive and results in CKD. Underdeveloped kidneys are more susceptible to gentamicin-induced AKI, suggesting that AKI in the setting of low nephron number is more severe and further increases the risk of CKD in this vulnerable population.

The tyrosine phosphatase Shp2 acts downstream of GDNF/Ret in branching morphogenesis of the developing mouse kidney.

The tyrosine phosphatase Shp2 acts downstream of various growth factors, hormones or cytokine receptors. Mutations of the Shp2 gene are associated with several human diseases. Here we have ablated Shp2 in the developing kidneys of mice, using the ureteric bud epithelium-specific Hoxb7/Cre. Mutant mice produced a phenotype that is similar to mutations of the genes of the GDNF/Ret receptor system, that is: strongly reduced ureteric bud branching and downregulation of the Ret target genes Etv4 and Etv5. Shp2 mutant embryonic kidneys also displayed reduced cell proliferation at the branch tips and branching defects, which could not be overcome by GDNF in organ culture. We also examined compound mutants of Shp2 and Sprouty1, which is an inhibitor of receptor tyrosine kinase signaling in the kidney. Sprouty1 single mutants produce supernumerary ureteric buds, which branch excessively. Sprouty1 mutants rescued branching deficits in Ret(-/-) and GDNF(-/-) kidneys. Sprouty1; Shp2 double mutants showed no rescue of kidney branching. Our data thus indicate an intricate interplay of Shp2 and Sprouty1 in signaling downstream of receptor tyrosine kinases during kidney development. Apparently, Shp2 mediates not only GDNF/Ret but also signaling by other receptor tyrosine kinases in branching morphogenesis of the embryonic kidney.

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.

SUMO-specific protease 2 is essential for modulating p53-Mdm2 in development of trophoblast stem cell niches and lineages.

SUMO-specific protease 2 (SENP2) modifies proteins by removing SUMO from its substrates. Although SUMO-specific proteases are known to reverse sumoylation in many defined systems, their importance in mammalian development and pathogenesis remains largely elusive. Here we report that SENP2 is highly expressed in trophoblast cells that are required for placentation. Targeted disruption of SENP2 in mice reveals its essential role in development of all three trophoblast layers. The mutation causes a deficiency in cell cycle progression. SENP2 has a specific role in the G-S transition, which is required for mitotic and endoreduplication cell cycles in trophoblast proliferation and differentiation, respectively. SENP2 ablation disturbs the p53-Mdm2 pathway, affecting the expansion of trophoblast progenitors and their maturation. Reintroducing SENP2 into the mutants can reduce the sumoylation of Mdm2, diminish the p53 level and promote trophoblast development. Furthermore, downregulation of p53 alleviates the SENP2-null phenotypes and stimulation of p53 causes abnormalities in trophoblast proliferation and differentiation, resembling those of the SENP2 mutants. Our data reveal a key genetic pathway, SENP2-Mdm2-p53, underlying trophoblast lineage development, suggesting its pivotal role in cell cycle progression of mitosis and endoreduplication.

Gata3 acts downstream of beta-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 beta-catenin function in the nephric duct and demonstrate that the beta-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 beta-catenin, but upstream of Ret, to prevent ectopic ureter budding and premature cell differentiation in the nephric duct.

Dispensable role of protein 4.1B/DAL-1 in rodent adrenal medulla regarding generation of pheochromocytoma and plasmalemmal localization of TSLC1.

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.