Autosomal dominant polycystic kidney disease (ADPKD) is characterized by cyst formation in the kidney, liver, and pancreas and is associated often with cardiovascular abnormalities such as hypertension, mitral valve prolapse, and intracranial aneurysms. It is caused by mutations in PKD1 or PKD2, encoding polycystin-1 and -2, which together form a cell surface nonselective cation ion channel. Pkd2؊͞؊ mice have cysts in the kidney and pancreas and defects in cardiac septation, whereas Pkd1 del34 ؊͞؊ and Pkd1 L ؊͞؊ mice have cysts but no cardiac abnormalities, although vascular fragility was reported in the latter. Here we describe mice carrying a targeted mutation in Pkd1 (Pkd1 del17-21geo ), which defines its expression pattern by using a lacZ reporter gene and may identify novel functions for polycystin-1. Although Pkd1 del17-21geo ؉͞؊ adult mice develop renal and hepatic cysts, Pkd1 del17-21geo ؊͞؊ embryos die at embryonic days 13.5-14.5 from a primary cardiovascular defect that includes double outflow right ventricle, disorganized myocardium, and abnormal atrio-ventricular septation. Skeletal development is also severely compromised. These abnormalities correlate with the major sites of Pkd1 expression. During nephrogenesis, Pkd1 is expressed in maturing tubular epithelial cells from embryonic day 15.5. This expression coincides with the onset of cyst formation in Pkd1 del34 ؊͞؊, Pkd1 L ؊͞؊, and Pkd2؊͞؊ mice, supporting the hypothesis that polycystin-1 and polycystin-2 interact in vivo and that their failure to do so leads to abnormalities in tubule morphology and function.
BackgroundSignals from the extracellular environment control many aspects of cell behaviour including proliferation, survival, differentiation, adhesion and migration. It is increasingly evident that these signals can be modulated by a group of matricellular proteins called the CCN family. CCN proteins have multiple domains through which they regulate the activities of a variety of signalling molecules including TGFβ, BMPs and integrins, thereby influencing a wide range of processes in development and disease. Whilst the developmental roles of CCN1 and CCN2 have been elucidated, very little is known about the function of CCN3 (NOV). To investigate this, we have generated mice carrying a targeted mutation in the Nov gene (Novdel3) which reveal for the first time its diverse functions in embryos and adults.ResultsBy replacing Nov exon 3 with a TKneomycin cassette, we have generated Novdel3-/- mice which produce no full length NOV protein and express at a barely detectable level a mutant NOV protein that lacks the VWC domain. In Novdel3-/- embryos, and to a lesser extent in Novdel3+/- embryos, development of the appendicular and axial skeleton was affected with enlarged vertebrae, elongated long bones and digits, delayed ossification, increased bone mineralization and severe joint malformations. Primary embryo fibroblasts from Novdel3-/- mutant embryos showed enhanced chondrogenesis and osteogenesis. Cardiac development was also influenced leading to enlargement and abnormal modelling of the endocardial cushions, associated with septal defects and delayed fusion. In adults, cardiomyopathy was apparent, with hypertrophy and calcification of the septum and left ventricle dilation. Muscle atrophy was seen by 5 months of age, associated with transdifferentiation to fat. Premature tissue degeneration was also seen in the lens, with cataracts present from 6 months.ConclusionWe have generated the first mice with a mutation in the Nov gene (Novdel3). Our data demonstrate that NOV is a regulator of skeletal and cardiac development, and implicates NOV in various disease processes including cardiomyopathy, muscle atrophy and cataract formation. Novdel3 mutants represent a valuable resource for studying NOV's role in the modulation and co-ordination of multiple signalling pathways that underpin organogenesis and tissue homeostasis.
We have constructed a plasmid pXTl which can be used to derive replication-defective retroviruses capable of efficient constitutive expression of foreign genes in embryonic as well as adult cells. This plasmid is based on pXm5 (used to derive the retrovirus N2 (ref. 1)), and has the LTRs and part of the gag region of Moloney Murine Leukaemia Virus. A selectable neomycin (neo) gene, conferring G418 resistance, is under the control of the LTR, which although normally repressed in embryonic cells (2), can become transcriptionally active under selective conditions (3). An internal herpes simplex virus thymidine kinase (TK) promoter is used for expression of the exogenous gene; this promoter is active in embryonic stem cells (4) and in a wide variety of tissues in transgenic mice (5). Infectious, helper-free replication-defective retroviruses can be obtained either by direct transfection onto the P2 packaging cell line (6), or via transfection of the amphotrophic cell line PA317 (7), and infection of T2 cells. Titres of viral supematants on NIH3T3 fibroblasts range from 1-5x105 G418R cfu/ml ('2 transfection) to 1-5x106 G418R cfu/ml ('2 infection). Titres on embryonic stem (EC or ES) cells are 103-105 fold lower (depending on cell line) due to repression of the viral LTR. Viruses based on pXTI, carrying a transforming gene under the control of the TK promoter, have been used to infect fibroblasts, EC and ES cells, bone marrow and primary embryonic cerebellum cultures; in all cases both neo and exogenous genes are efficiently expressed.
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