Ksp-cadherin gene promoter. II. Kidney-specific activity in transgenic mice

Ciliogenesis and cystogenesis require the exocyst, a conserved eight-protein trafficking complex that traffics ciliary proteins. In culture, the small GTPase Cdc42 co-localizes with the exocyst at primary cilia and interacts with the exocyst component Sec10. The role of Cdc42 in vivo, however, is not well understood. Here, knockdown of cdc42 in zebrafish produced a phenotype similar to sec10 knockdown, including tail curvature, glomerular expansion, and mitogen-activated protein kinase (MAPK) activation, suggesting that cdc42 and sec10 cooperate in ciliogenesis. In addition, cdc42 knockdown led to hydrocephalus and loss of photoreceptor cilia. Furthermore, there was a synergistic genetic interaction between zebrafish cdc42 and sec10, suggesting that cdc42 and sec10 function in the same pathway. Mice lacking Cdc42 specifically in kidney tubular epithelial cells died of renal failure within weeks of birth. Histology revealed cystogenesis in distal tubules and collecting ducts, decreased ciliogenesis in cyst cells, increased tubular cell proliferation, increased apoptosis, increased fibrosis, and led to MAPK activation, all of which are features of polycystic kidney disease, especially nephronophthisis. Taken together, these results suggest that Cdc42 localizes the exocyst to primary cilia, whereupon the exocyst targets and docks vesicles carrying ciliary proteins. Abnormalities in this pathway result in deranged ciliogenesis and polycystic kidney disease.

Section: Kidney-specific Knockout Of Cdc42 Leads To Early Postnatal Dsupporting

confidence: 55%

Cdc42 Deficiency Causes Ciliary Abnormalities and Cystic Kidneys

Sy¹,

Mf²,

Huang³

et al. 2013

“…LoxP/LoxP mice (gift from NCI/ generated by Anton Berns), 53 with nephric lineage-specific Cre expressers Ksp-cadherin-Cre (gift from Peter Igarashi), 54,55 Hoxb7-Cre (gift from Carlton Bates), 56 and Pax3-Cre (gift from Feng Chen and Jonathan Epstein). 57,58 Transgenic Hoxb7-p53DN-IRES-GFP mice with targeted expression of p53DN in the WD/UB-derived structures (Hoxb7-p53DN) were generated at the Tulane Transgenic Mouse Facility.…”

Section: Conventional P53mentioning

confidence: 99%

p53 Regulates Metanephric Development

Saifudeen

Dipp

Stefkova

et al. 2009

p53 is best known as a tumor suppressor that regulates cell-cycle, differentiation, and apoptosis pathways, but its potential role in embryonic development and organogenesis remains controversial. Here, p53Ϫ/Ϫ embryos bred on C57Bl6 background exhibited a spectrum of congenital abnormalities of the kidney and urinary tract, including ureteric bud (UB) ectopia, double ureters/collecting systems, delayed primary branching of the UB, and hypoplastic metanephroi. We observed ectopic UB outgrowth from the Wolffian duct (WD) in one third of p53 Ϫ/Ϫ embryos. The prevalence of duplex was higher in embryos than in neonates, and ex vivo organ culture suggested that ectopic ureters can regress over time, leaving behind a dysplastic pole ("segmental dysgenesis"). Transgenic expression of dominant negative p53 or conditional inactivation of p53 in the UB but not in the metanephric mesenchyme lineage recapitulated the duplex phenotype. Mechanistically, p53 inactivation in the WD associated with enhanced sensitivity to glial cell line-derived neurotrophic factor (GDNF)-induced ectopic budding and potentiated phosphatidylinositol-3 kinase activation by GDNF in UB cells. Unlike several other models of UB ectopia, hypersensitivity of p53 Ϫ/Ϫ WD to GDNF is not accompanied by reduced Sprouty-1 or anterior expansion of the GDNF domain. In summary, our data lend support for a restrictive role for p53 activity in UB outgrowth from the WD. Organogenesis is dependent on a multitude of growth factors, signaling molecules, and transcription factors that temporally and spatially define a developmental program. Metanephric development is dependent on formation of the Wolffian duct (WD) and the adjacent metanephric mesenchyme (MM) from the intermediate mesoderm. In mice, the ureteric bud (UB) develops at embryonic day 10.5 (E10.5) from the caudal end of the WD. Inductive interactions between the UB and the MM ensure cell survival and subsequent onset of metanephrogenesis. Emergence of the UB from a specific site in the WD adjacent to the MM is controlled by the glial cell line-derived neurotrophic factor (GDNF)-cRet signaling pathway. 1-6 GDNF is a growth factor that is secreted by the MM and binds to its receptor c-Ret and co-receptor GFR␣1 that are expressed along the WD and UB tips. Stimulation of the GDNF-cRet pathway results in cell proliferation and chemotaxis. 7,8 After the initial broad distribution of the GDNF field along the posterior half of the WD, the GDNF expression domain is progressively restricted to the caudal end of the WD in the immediate vicinity of the site of UB outgrowth 9 -11 ; however, the potential for bud emergence remains along the length of the WD, which continues to express the c-Ret/GFR␣1 receptor/co-receptor. Evidence from mutant mouse models and metanephric organ culture implicates impaired restriction of the GDNF field and the GDNF/c-Ret signaling pathway in ureter duplica-

“…EDTA], and sterilized by filtration through 0.2-m filters. Transgene DNA was microinjected into the pronuclei of fertilized oocytes from ICR donors as described previously (9). Pronuclear injection was performed by the University of Texas Southwestern Transgenic Mouse Core Facility.…”

Section: Plasmid Constructionmentioning

confidence: 99%

“…Immunostaining and indirect immunofluorescence were performed using a modification of a previously described method (9). Tissues and embryos were perfusion-fixed and/or immersed for 1 h on ice in phosphate-buffered saline (PBS) containing 4% paraformaldehyde.…”

Section: Immunostainingmentioning

confidence: 99%

“…Reporter gene assays in both transiently transfected cells and transgenic mice have shown that the activity of the Ksp-cadherin promoter is tissue-specific. Transgenic mice carrying 3425 bp of the Kspcadherin 5' flanking region linked to a lacZ reporter gene express ␤-galactosidase activity exclusively in the kidney, indicating that the promoter is tissue-specific in vivo (9). However, expression of the lacZ reporter gene is restricted to renal collecting ducts, whereas Ksp-cadherin is endogenously expressed in all nephron segments.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Epithelial-Specific Cre/lox Recombination in the Developing Kidney and Genitourinary Tract

Shao

Somlo

Igarashi

2002

Self Cite

285

320

Abstract. Ksp-cadherin is a unique, tissue-specific member of the cadherin family of cell adhesion molecules that is expressed in tubular epithelial cells in the kidney and developing genitourinary (GU) tract. It has recently been shown that a 1341-bp fragment of the 5' flanking region containing the Ksp-cadherin gene promoter can recapitulate the complete expression pattern of the gene in the developing kidney and GU tract. Similar to the endogenous Ksp-cadherin gene, transgenes containing 1341 bp of the 5' flanking region are expressed in developing nephrons, ureteric bud, mesonephric tubules, Wolffian duct, and Müllerian duct. In adult mice, the expression is restricted to renal tubules. In the current study, Ksp1.3/Cre transgenic mice carrying 1329 bp of the Kspcadherin 5' flanking region linked to the Cre recombinase gene were produced. Adult transgenic mice expressed Cre recombinase in renal tubules, especially collecting ducts and thick ascending limbs of Henle's loops. Transgenic embryos expressed Cre recombinase in the branching ureteric bud, developing renal tubules, and sex ducts. Ksp1.3/Cre transgenic mice were crossed with mice carrying a lacZ reporter gene that is activated by Cre/lox-mediated genetic recombination. Bitransgenic progeny expressed lacZ exclusively in renal tubules, mesonephric tubules, ureteric bud, developing ureter, and Wolffian duct. These results demonstrate that Ksp1.3/Cre transgenic mice express Cre recombinase exclusively in the kidney and developing GU tract and can mediate epithelialspecific Cre/lox recombination in these tissues. Ksp1.3/Cre transgenic mice should be useful for cell lineage studies and kidney-specific gene targeting.Cre recombinase (cyclization recombination) is a site-specific DNA recombinase from bacteriophage P1 that mediates genetic recombination at specific 34-bp recognition sites (called loxP) without any requirement for accessory proteins or highenergy cofactors (1). Cre-mediated intramolecular recombination between two directly repeated loxP sites flanking a DNA segment results in deletion of the intervening segment, leaving a single loxP site. If two loxP sites are introduced into the genomic DNA flanking an essential exon