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BACKGROUND: Topiroxostat, an inhibitor of xanthine oxidoreductase (XOR) was shown to reduce urinary albumin excretion of hyperuricemic patients with chronic kidney disease. However, its pharmacological mechanism is not well understood. In this study, we examined the effects of topiroxostat on glomerular podocytes. Podocyte is characterized by foot process and a unique cell-cell junction slit diaphragm functioning as a final barrier to prevent proteinuria. METHODS: The effects of topiroxostat on the expressions of podocyte functional molecules were analysed in db/db mice, a diabetic nephropathy model, anti-nephrin antibody-induced rat podocyte injury model and cultured podocytes treated with adriamycin. RESULTS: Topiroxostat treatment ameliorated albuminuria in db/db mice. The expression of desmin, a podocyte injury marker was increased, and nephrin and podocin, key molecules of slit diaphragm, and podoplanin, an essential molecule in maintaining foot process were downregulated in db/db mice. Topiroxostat treatment prevented the alterations in the expressions of these molecules in db/db mice. XOR activity in kidney was increased in rats with anti-nephrin antibody-induced podocyte injury. Topiroxostat treatment reduced XOR activity and restored the decreased expression of nephrin, podocin and podoplanin in the podocyte injury. Furthermore, topiroxostat enhanced the expression of podoplanin in injured human cultured podocytes. CONCLUSIONS: Podocyte injury was evident in db/db mice. Topiroxostat ameliorated albuminuria in diabetic nephropathy model by preventing podocyte injury. Increase of XOR activity in kidney contributes to development of podocyte injury caused by stimulation to slit diaphragm. Topiroxostat has an effect to stabilize slit diaphragm and foot processes by inhibiting the reduction of nephrin, podocin and podoplanin.

DOI： 10.1016/j.nefro.2020.10.011

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Dysfunction of slit diaphragm, a cell-cell junction of glomerular podocytes, is involved in the development of proteinuria in several glomerular diseases. Slit diaphragm should be a target of a novel therapy for proteinuria. Nephrin, NEPH1, P-cadherin, FAT, and ephrin-B1 were reported to be extracellular components forming a molecular sieve of the slit diaphragm. Several cytoplasmic proteins such as ZO-1, podocin, CD2AP, MAGI proteins and Par-complex molecules were identified as scaffold proteins linking the slit diaphragm to the cytoskeleton. In this article, new insights into these molecules and the pathogenic roles of the dysfunction of these molecules were introduced. The slit diaphragm functions not only as a barrier but also as a signaling platform transfer the signal to the inside of the cell. For maintaining the slit diaphragm function properly, the phosphorylation level of nephrin is strictly regulated. The recent studies on the signaling pathway from nephrin, NEPH1, and ephrin-B1 were reviewed. Although the mechanism regulating the function of the slit diaphragm had remained unclear, recent studies revealed TRPC6 and angiotensin II-regulating mechanisms play a critical role in regulating the barrier function of the slit diaphragm. In this review, recent investigations on the regulation of the slit diaphragm function were reviewed, and a strategy for the establishment of a novel therapy for proteinuria was proposed.

DOI： 10.1007/s10157-020-01854-3

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Ephrin-B1 plays a critical role at slit diaphragm. Partitioning-defective (Par)-6 is down-regulated in podocyte of ephrin-B1 knockout mouse, suggesting that Par-6 is associated with ephrin-B1. Par polarity complex, consisting of Par-6, Par-3, and atypical protein kinase C, is essential for tight junction formation. In this study, the expression of Par-6 was analyzed in the normal and nephrotic syndrome model rats, and the molecular association of Par-6, Par-3, ephrin-B1, and nephrin was assessed with the human embryonic kidney 293 cell expression system. Par-6 was concentrated at slit diaphragm. Par 6 interacted with ephrin-B1 but not with nephrin, and Par-3 interacted with nephrin but not with ephrin-B1. The complexes of Par-6-ephrin-B1 and Par-3-nephrin were linked via extracellular sites of ephrin-B1 and nephrin. The Par-6-ephrin-B1 complex was delinked from the Par-3-nephrin complex, and Par-6 and ephrin-B1 were clearly down-regulated already at early phase of nephrotic model. The alteration of Par-6/ephrin-B1 advanced that of Par-3/nephrin. Stimulation to nephrin phosphorylated not only nephrin but also ephrin-B1, and consequently inhibited the interaction between ephrin-B1 and Par-6. Par-6 appeared at presumptive podocyte of early developmental stage and moved to basal area at capillary loop stage to participate in slit diaphragm formation at the final stage. Par-6-ephrin-B1 interaction is crucial for formation and maintenance of slit diaphragm of podocyte.

DOI： 10.1016/j.ajpath.2019.10.015

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BACKGROUND: Given that amyloid-β (Aβ) peptide is produced and released at synapses, synaptic Aβ is one of the promising therapeutic targets to prevent synaptic dysfunction in Alzheimer's disease (AD). Although Aβ production begins with the cleavage of the amyloid-β protein precursor (AβPP) by β-site AβPP cleaving enzyme 1 (BACE1), the mechanism on how BACE1 is involved in AβPP processing at synapses remains unclear. OBJECTIVE: This study aimed to identify novel BACE1 interacting proteins regulating Aβ production at the synapse. METHODS: BACE1 interacting proteins were pulled down using a mass spectrometry-based proteomics of wild-type (WT) rat brain synaptoneurosome lysates utilizing anti-BACE1 antibody. Then, a novel BACE1 interactor was identified and characterized using experimental systems that utilized transfected cells and knockout (KO) mice. RESULTS: Synaptic vesicle protein 2B (SV2B) was identified as a novel presynaptic interaction partner of BACE1. In HEK293 cells, co-overexpression of SV2B with BACE1 significantly reduced the sAβPPβ and Aβ levels released in the media; thus, SV2B overexpression negatively affected the AβPP cleavage by BACE1. Compared with those of WT mice, the hippocampal lysates of SV2B knockout mice had significantly elevated Aβ levels, whereas the β-secretase activity and the AβPP and BACE1 protein levels remained unchanged. Finally, a fractionation assay revealed that BACE1 was mislocalized in SV2B KO mice; hence, SV2B may be involved in BACE1 trafficking downregulating the amyloidogenic pathway of AβPP. CONCLUSION: SV2B has a novel role of negatively regulating the amyloidogenic processing of AβPP at the presynapses.

DOI： 10.3233/JAD-200071

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society of Nephrology  

Background B-type ephrins are membrane-bound proteins that maintain tissue function in several organs. We previously reported that ephrin-B1 is localized at the slit diaphragm of glomerular podocytes. However, the function of ephrin-B1 at this location is unclear. Methods We analyzed the phenotype of podocyte-specific ephrin-B1 knockout mice and assessed the molecular association of ephrin-B1 and nephrin, a key molecule of the slit diaphragm, in HEK293 cells and rats with anti-nephrin antibody-induced nephropathy. Results Compared with controls, ephrin-B1 conditional knockoutmice displayed altered podocytemorphology, disarrangement of the slit diaphragm molecules, and proteinuria. Ephrin-B1 expressed in HEK293 cells immunoprecipitated with nephrin, which required the basal regions of the extracellular domains of both proteins. Treatment of cells with an anti-nephrin antibody promoted the phosphorylation of nephrin and ephrin-B1. However, phosphorylation of ephrin-B1 did not occur in cells expressing amutant nephrin lacking the ephrin-B1 binding site or in cells treated with an Src kinase inhibitor. The phosphorylation of ephrin-B1 enhanced the phosphorylation of nephrin and promoted the phosphorylation of c-Jun N-terminal kinase (JNK), which was required for ephrin-B1- promoted cell motility in wound-healing assays. Notably, phosphorylated JNK was detected in the glomeruli of control mice but not ephrin-B1 conditional knockout mice. In rats, the phosphorylation of ephrin-B1, JNK, and nephrin occurred in the early phase (24 hours) of anti-nephrin antibody-induced nephropathy. Conclusions Through interactions with nephrin, ephrin-B1 maintains the structure and barrier function of the slit diaphragm. Moreover, phosphorylation of ephrin-B1 and, consequently, JNK are involved in the development of podocyte injury.

DOI： 10.1681/ASN.2017090993

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SAGE PUBLICATIONS LTD  

Background and objective: Renin-angiotensin system (RAS) inhibitors reduce glomerular injury and proteinuria, indicating that angiotensin II (Ang II) is involved in glomerular diseases. Although the local RAS is reported to play an essential role in maintaining local tissue functions, the role of the local RAS in regulating glomerular function is not well evaluated. In this study, we analyzed the glomerular expression of RAS components in nephrotic models and the effect of Ang II receptor blockers (ARB) on the expression of angiotensinogen (AGT).
Methods: The levels of glomerular expression of RAS components were analyzed in two nephrotic models: anti-nephrin antibody-induced nephropathy and PAN nephropathy, a mimic of human minimal change nephrotic syndrome. The effect of the ARB irbesartan on the expression of AGT in the nephrotic model was analyzed.
Results: Glomerular expression of AGT and the receptors for Ang II was clearly increased in the nephrotic models, while the expression levels of renin, ACE and ACE2 were decreased. ARB treatment suppressed the increase of glomerular expression of AGT in the nephrotic model.
Conclusion: It is conceivable that the promoted local RAS action participated in the glomerular dysfunction, and that ARB treatment ameliorated slit diaphragm injury by inhibiting the positive feedback loop of the activated local Ang II action.

DOI： 10.1177/1470320316681223

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley-Blackwell Publishing Ltd  

Although calcineurin (CN) is distributed in many cell types and functions in regulating cell functions, the precise roles of CN remained in each type of the cells are not well understood yet. A CN inhibitor (CNI) has been used for steroid-resistant nephrotic syndrome. A CNI is assumed to ameliorate proteinuria by preventing the overproduction of T-cell cytokines. However, recent reports suggest that CNI has a direct effect on podocyte. It is accepted that a slit diaphragm (SD), a unique cell-cell junction of podocytes, is a critical barrier preventing a leak of plasma protein into urine. Therefore, we hypothesized that CNI has an effect on the SD. In this study, we analyzed the expression of CN in physiological and in the nephrotic model caused by the antibody against nephrin, a critical component of the SD. We observed that CN is expressed at the SD in normal rat and human kidney sections and has an interaction with nephrin. The staining of CN at the SD was reduced in the nephrotic model, while CN activity in glomeruli was increased. We also observed that the treatment with tacrolimus, a CNI, in this nephrotic model suppressed the redistribution of CN, nephrin, and other SD components and ameliorated proteinuria. These observations suggested that the redistribution and the activation of CN may participate in the development of the SD injury.

DOI： 10.14814/phy2.12679

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Podoplanin was identified as a protein associated with the transformation of arborized foot processes of glomerular epithelial cells (podocytes) to flat feet. However, the function of podoplanin in the podocyte is not yet fully clarified. In this study, we analyzed the molecular nature of podoplanin, and its expression in rat nephrotic models and patients with minimal change nephrotic syndrome (MCNS). We demonstrated here that podoplanin has two forms: one contains abundant sialic acid and the other a lesser amount of sialic acid. Podoplanin bound ezrin to interact with the cytoskeleton. The silencing of podoplanin in cultured podocytes caused a change in the cell shape and the distribution of ezrin and actin. The expression of podoplanin was clearly reduced before the onset of proteinuria in puromycin aminonucleoside (PAN) nephropathy, a mimic of MCNS, and the decrease in the expression of podoplanin became more evident at the proteinuric stage. Podoplanin was detected in normal urine samples, and the amount of urinary podoplanin markedly increased on day 1 of PAN nephropathy. Urinary ezrin was also detected. The amount of the phosphorylated ezrin was reduced, while the amount of the podoplanin-interacting ezrin increased. The podoplanin expression was reduced in a patient with active-phase MCNS. It is conceivable that the alteration of the podoplanin-ezrin-cytoskeleton linkage is an important event of the podocyte injury in MCNS.

DOI： 10.1007/s00441-015-2178-8

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC NEUROSCIENCE  

Wingless-related MMTV integration site 1 (WNT1)/beta-catenin signaling plays a crucial role in the generation of mesodiencephalic dopaminergic (mdDA) neurons, including the substantia nigra pars compacta (SNc) subpopulation that preferentially degenerates in Parkinson's disease (PD). However, the precise functions of WNT1/beta-catenin signaling in this context remain unknown. Stem cell-based regenerative (transplantation) therapies for PD have not been implemented widely in the clinical context, among other reasons because of the heterogeneity and incomplete differentiation of the transplanted cells. This might result in tumor formation and poor integration of the transplanted cells into the dopaminergic circuitry of the brain. Dickkopf 3 (DKK3) is a secreted glycoprotein implicated in the modulation of WNT/beta-catenin signaling. Using mutant mice, primary ventral midbrain cells, and pluripotent stem cells, we show that DKK3 is necessary and sufficient for the correct differentiation of a rostrolateral mdDA neuron subset. Dkk3 transcription in the murine ventral midbrain coincides with the onset of mdDA neurogenesis and is required for the activation and/or maintenance of LMX1A (LIM homeobox transcription factor 1 alpha) and PITX3 (paired-like homeodomain transcription factor 3) expression in the wcorresponding mdDA precursor subset, without affecting the proliferation or specification of their progenitors. Notably, the treatment of differentiating pluripotent stem cells with recombinant DKK3 and WNT1 proteins also increases the proportion of mdDA neurons with molecular SNc DA cell characteristics in these cultures. The specific effects of DKK3 on the differentiation of rostrolateral mdDA neurons in the murine ventral midbrain, together with its known prosurvival and anti-tumorigenic properties, make it a good candidate for the improvement of regenerative and neuroprotective strategies in the treatment of PD.

DOI： 10.1523/JNEUROSCI.1722-15.2015

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The glomerular visceral epithelial cell (podocyte) is characterized as a specialized structure of the interdigitating foot processes, covering the outer side of the glomerular basement membrane (GBM). The neighboring foot processes are connected by a slit diaphragm, which is a key structure regulating the barrier function of the glomerular capillary wall to prevent proteinuria. We have previously reported that synaptic vesicle protein 2 B (SV2B) is expressed in the podocyte and that the expression is clearly decreased in nephrotic models. However, the precise function of SV2B in the podocyte is unclear. To investigate the role of SV2B in maintaining the podocyte function and to better understand the function of the neuron-like vesicle expressing SV2B in the podocyte, we analyzed them with SV2B knockout (KO) mice. An increase in the amount of proteinuria, effacement of the foot process of the podocyte, and alterations of the GBM were detected in SV2B KO mice. It was also found that the expression of CD2AP, nephrin, and NEPH1, the functional molecules of the slit diaphragm, and laminin, a critical component of the GBM, is clearly altered in SV2B KO mice. Synaptotagmin and neurexin, which have a role in the synaptic vesicle docking in neurons, are downregulated in the kidney cortex of SV2B KO mice. We have previously reported that neurexin interacts with CD2AP, and the present study shows that SV2B interacts with CD2AP. These findings suggest that the SV2B-neurexin complex is involved in the formation and maintenance of the slit diaphragm. In addition, SV2B is densely expressed close to the cell surface in the presumptive podocyte in the early stage of glomerulogenesis. These results suggest that SV2B has an essential role in the formation and maintenance of the glomerular capillary wall.

DOI： 10.1038/labinvest.2015.39.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WICHTIG EDITORE  

The precise pathogenic mechanism and role of angiotensin II (Ang II) action in the development of proteinuria in minimal change nephrotic syndrome (MCNS) is uncertain.
The glomerular expressions of the slit diaphragm (SD) molecules nephrin, podocin and NEPH1 in rat puromycin aminonucleoside (PAN) nephropathy, a mimic of MCNS, were analyzed. The effects of Ang II receptor blockade (ARB) (irbesartan 15 mg/kg body weight/day) on proteinuria and on the expression of the SD molecules were analyzed.
mRNA expressions of nephrin, podocin and NEPH1 were decreased to an undetectable level at 1 h. The staining of these SD molecules shifted to a discontinuous pattern, and their intensity was reduced. NEPH1 staining was reduced to an undetectable level on day 10. ARB treatment ameliorated the peak value of proteinuria (237.6 +/- A 97.0 vs. 359.0 +/- A 63.3 mg/day, p < 0.05), and prevented the decrease in the mRNA expression of the SD molecules (nephrin 66.96 %, podocin 60.40 %, NEPH1 77.87 % of normal level). The immunofluorescence staining of NEPH1 was restored by ARB. ARB treatment enhanced the expression of NEPH1 of normal rats.
Dysfunction of the SD molecules including NEPH1 is a crucial initiation event of PAN nephropathy. ARB treatment ameliorates proteinuria in PAN nephropathy by inhibiting the reduction of NEPH1 and nephrin. Ang II action regulates the expression of NEPH1 and nephrin in not only the pathological but also physiological state.

DOI： 10.1007/s40620-014-0147-z

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BACKGROUND: The precise pathogenic mechanism and role of angiotensin II (Ang II) action in the development of proteinuria in minimal change nephrotic syndrome (MCNS) is uncertain. METHODS: The glomerular expressions of the slit diaphragm (SD) molecules nephrin, podocin and NEPH1 in rat puromycin aminonucleoside (PAN) nephropathy, a mimic of MCNS, were analyzed. The effects of Ang II receptor blockade (ARB) (irbesartan 15 mg/kg body weight/day) on proteinuria and on the expression of the SD molecules were analyzed. RESULTS: mRNA expressions of nephrin, podocin and NEPH1 were decreased to an undetectable level at 1 h. The staining of these SD molecules shifted to a discontinuous pattern, and their intensity was reduced. NEPH1 staining was reduced to an undetectable level on day 10. ARB treatment ameliorated the peak value of proteinuria (237.6 ± 97.0 vs. 359.0 ± 63.3 mg/day, p < 0.05), and prevented the decrease in the mRNA expression of the SD molecules (nephrin 66.96 %, podocin 60.40 %, NEPH1 77.87 % of normal level). The immunofluorescence staining of NEPH1 was restored by ARB. ARB treatment enhanced the expression of NEPH1 of normal rats. CONCLUSIONS: Dysfunction of the SD molecules including NEPH1 is a crucial initiation event of PAN nephropathy. ARB treatment ameliorates proteinuria in PAN nephropathy by inhibiting the reduction of NEPH1 and nephrin. Ang II action regulates the expression of NEPH1 and nephrin in not only the pathological but also physiological state.

DOI： 10.1007/s40620-014-0147-z

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The slit diaphragm bridging the neighboring foot processes functions as a final barrier of glomerular capillary wall for preventing the leak of plasma proteins into primary urine. It is now accepted that the dysfunction of the sit diaphragm contributes to the development of proteinuria in several glomerular diseases. Nephrin, a gene product of NPHS1, a gene for a congenital nephrotic syndrome of Finnish type, constitutes an extracellular domain of the slit diaphragm. Podocin was identified as a gene product of NPHS2, a gene for a familial steroid-resistant nephrotic syndrome of French. Podocin binds the cytoplasmic domain of nephrin. After then, CD2 associated protein, NEPH1 and transient receptor potential-6 were also found as crucial molecules of the slit diaphragm. In order to explore other novel molecules contributing to the development of proteinuria, we performed a subtraction hybridization assay with a normal rat glomerular RNA and a glomerular RNA of rats with a puromycin aminonucleoside nephropathy, a mimic of a human minimal change type nephrotic syndrome. Then we have found that synaptic vesicle protein 2B, ephrin-B1 and neurexin were already downregulated at the early stage of puromycin aminonucleoside nephropathy, and that these molecules were localized close to nephrin. It is conceivable that these molecules are the slit diaphragm associated molecules, which participate in the regulation of the barrier function. These molecules could be targets to establish a novel therapy for nephrotic syndrome.

DOI： 10.5527/wjn.v3.i3.77.

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Glycosphingolipids (GSLs) are important for various biological functions in the nervous system, the immune system, embryogenesis and in other tissues and processes. Lactosylceramide (LacCer), which is synthesized from glucosylceramide (GlcCer) by LacCer synthase, is a core structure of GSLs, including gangliosides. LacCer synthase was reported to be synthesized by the beta4-galactosyltransferase-6 (beta4GalT-6) gene in the rat brain. However, the existence of another LacCer synthase gene was shown in cultured cells lacking beta4GalT-6. Here, we report that LacCer synthase is mainly synthesized by the beta4GalT-5 gene during early mouse embryogenesis, and its disruption is embryonic lethal. beta4GalT-5-deficient embryos showed developmental retardation from E7.5 and died by E10.5 as reported previously. LacCer synthase activity was significantly reduced in beta4GalT-5-deficient embryos and extra-embryonic endoderm (XEN) cells derived from blastocysts, and it was recovered when beta4GalT-5 cDNA was introduced into beta4GalT-5-deficient XEN cells. The amounts of LacCer and GM3 ganglioside were drastically reduced, while GlcCer accumulated in the beta4GalT-5-deficient XEN cells. Hematoma and ectopically accumulated trophoblast giant cells were observed in the anti-mesometrial pole of the extra-embryonic tissues, although all three embryonic layers formed. beta4GalT-5-deficient embryos developed until E12.5 as chimeras with wild-type tetraploid cells, which formed the extra-embryonic membranes, indicating that extra-embryonic defects caused the early embryonic lethality. Our results suggest that beta4GalT-5 is essential for extra-embryonic development during early mouse embryogenesis.

DOI： 10.1093/glycob/cwq098

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Glycosphingolipids (GSLs) are important for various biological functions in the nervous system, the immune system, embryogenesis and in other tissues and processes. Lactosylceramide (LacCer), which is synthesized from glucosylceramide (GlcCer) by LacCer synthase, is a core structure of GSLs, including gangliosides. LacCer synthase was reported to be synthesized by the β4-galactosyltransferase-6 (β4GalT-6) gene in the rat brain. However, the existence of another LacCer synthase gene was shown in cultured cells lacking β4GalT-6. Here, we report that LacCer synthase is mainly synthesized by the β4GalT-5 gene during early mouse embryogenesis, and its disruption is embryonic lethal. β4GalT-5-deficient embryos showed developmental retardation from E7.5 and died by E10.5 as reported previously. LacCer synthase activity was significantly reduced in β4GalT-5-deficient embryos and extra-embryonic endoderm (XEN) cells derived from blastocysts, and it was recovered when β4GalT-5 cDNA was introduced into β4GalT-5-deficient XEN cells. The amounts of LacCer and GM3 ganglioside were drastically reduced, while GlcCer accumulated in the β4GalT-5- deficient XEN cells. Hematoma and ectopically accumulated trophoblast giant cells were observed in the anti-mesometrial pole of the extra-embryonic tissues, although all three embryonic layers formed. β4GalT-5-deficient embryos developed until E12.5 as chimeras with wild-type tetraploid cells, which formed the extra-embryonic membranes, indicating that extra-embryonic defects caused the early embryonic lethality. Our results suggest that β4GalT-5 is essential for extra-embryonic development during early mouse embryogenesis. © 2010 The Author.

DOI： 10.1093/glycob/cwq098

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Authorship：Lead author   Language：English   Publishing type：Thesis (other)   Publisher：WILEY-BLACKWELL  

Wilms&apos; tumor 1-associating protein (WTAP) was previously identified as a protein associated with Wilms&apos; tumor-1 (WT-1) protein that is essential for the development of the genitourinary system. Although WTAP has been suggested to function in alternative splicing, stabilization of mRNA, and cell growth, its in vivo function is still unclear. We generated Wtap mutant mice using a novel gene-trap approach and showed that Wtap mutant embryos exhibited defective egg-cylinder formation at the gastrulation stage and died by embryonic day 10.5. Although they could form extraembryonic tissues and anterior visceral endoderm, Wtap mutant embryos and embryonic stem cells failed to differentiate into endoderm. and mesoderm. The chimera analysis showed that Wtap in extraembryonic tissues was required for the formation of mesoderm and endoderm in embryonic tissues. Taken together, our findings indicate that Wtap is indispensable for differentiation of mesoderm and endoderm in the mouse embryo.

DOI： 10.1002/dvdy.21444

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Gene trapping is a powerful method for identifying novel genes and for analyzing their functions. It is, however, difficult to select trapped genes on the basis of their function. To identify genes regulated by transcription factors that are important in the mesodermal formation, we selected trapped ES clones by infection of adenoviral vectors expressing Pax1, Brachyury, and Foxa2. Among 366 trapped genes, seven seemed to be controlled by these transcription factors in the first screening. The trapped genes were identified by 5' RACE, and a Northern blotting revealed that expressions of three trapped genes were regulated by these transcription factors. Expression patterns of Cx43 and HPI gamma implicated their functional relationships to Foxa2 in the formation of the notochord and the neural tube. Furthermore, Wtap mutant mice derived from the trapped clone showed defects in the mesendoderm formation. Our results indicate that trapped ES clones could be selected effectively using transcription factors. (c) 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2007.06.161

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