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Deficiency of phospholipase C-1 impairs renal development and hematopoiesis

¹ Department of Product Research and
² Department of Molecular Oncology, Nippon Roche Research Center, Kajiwara 200, Kamakura, Kanagawa 247-8530, Japan
³ Laboratory of Molecular and Cellular Pathology, Hokkaido University Graduate School of Medicine, CREST, JST, N15W7, Kita-Ku, Sapporo 060-8638, Japan
⁴ Department of Molecular Cell Biology, Osaka University Research Institute for Microbial Diseases, Yamadaoka 3-1, Suita, Osaka 565-0871, Japan
⁵ Department of Cell Biology, Institute for Virus Research, Kyoto University, Seigoinkawara-machi 53, Sakyo-Ku, Kyoto 606-8507, Japan
⁶ Department of Dermatology, Gunma University School of Medicine, Showa-machi 3-39-22, Maebashi, Gunma 371-8511, Japan

View larger version (42K): [in a new window]   Fig. 1. Disruption of the PLC-1 gene in mouse ES cells. (A) Genomic structure coding the functional domains of the mouse PLC-1 protein, the structure of the targeting vector and predicted structure of the targeted plc-1 locus. The location of the hybridization probe, 0.4 kb XhoI-EcoRI fragment, and the expected sizes of the EcoRI and the KpnI fragments that hybridize with the probe are indicated. E, EcoRI; Xh, XhoI; Xb, XbaI; H, HindIII; K, KpnI. (B) Southern blot analysis of the ES cell clones. The plc-1–/– ES cells were selected by culturing the plc-1+/– ES cells in the increased dose of G418 as described in Materials and Methods. Genomic DNA isolated from wild-type (+/+), plc-1+/– (+/–) and plc-1–/– (–/–) ES cells were digested with EcoRI (left panel) or Kpn I (right panel) and hybridized with the probe shown in (A). The expected sizes of the wild-type and the mutated fragments are 3.3 kb and 1.3 kb, respectively, when digested with EcoRI, 8.0 kb and 5.2 kb, respectively, with KpnI. (C) Expression of the PLC-1 (left) and PLC-2 (right) proteins in the wild-type (+/+), plc-1+/– (+/–) and plc-1–/– (–/–) ES clones.

View larger version (75K): [in a new window]   Fig. 2. Abnormal appearance of the kidneys of plc-1–/– chimeric mouse. (A) Abdominal cavities of the control (right) and plc-1–/– (left) chimeras. (B) Histopathology of the kidney from plc-1–/– chimeric mice. The specimens of the kidney from plc-1–/– chimeric mice were stained with Haematoxylin and Eosin. (C) In situ hybridization of the kidney with a Y-chromosome-specific probe. Dark spots show digoxigenin-positive plc-1–/– cells. *Normal looking tubules were negative for digoxigenin signals. (D,E) Representative kidney glomeruli in the plc-1–/– and control chimeras. The arrow indicates a sclerotic glomeruli.

View larger version (66K): [in a new window]   Fig. 3. GPI isozyme analysis. (A) Representative pattern of GPI assay in the control and plc-1–/– chimeric mice. Lysates from several tissues isolated from the chimeric mice were separated by cellulose acetate electrophoresis and stained for GPI activity as described in Materials and Methods. RB, red blood cells; BM, bone marrow, SM, skeletal muscle; Te, testis; Ep, epididymis; Bl, bladder; Ap, appendix; Lin, large intestine; Sin, small intestine; Sp, spleen; Pa, pancreas; St, stomach; Ki, kidney; Ad, adrenal; Li, liver; Di, diaphragm; Lu, lung; He, heart; Th, thymus; Es, esophagus; SG, submaxillay gland; TG, thyroid gland; To, tongue; Ey, Eye; FB, forebrain; Ce, cerebellum; SC, spinal cord and Sk, skin. (B) Quantification of the tissue distribution of the wild-type (open bars; n=8) and plc-1–/– (filled bars; n=12) ES cells. Relative levels of the GPI isozymes were quantified by densitometry software. Each column shows the percentage contribution of wild-type or plc-1–/– ES cells to the representative tissues isolated from the control and plc-1–/– chimeric mice and is expressed as the mean±s.e.m.

View larger version (25K): [in a new window]   Fig. 4. Flow cytometric analysis of lymphocytes. Thymocytes, lymph node (LN) cells, splenocytes and bone marrow (BM) cells isolated from control (upper panel; n=7) and plc-1–/– chimeric mice (lower panel; n=12) were stained for Ly9.1. The dotted lines indicate the staining pattern of the cells isolated from C57/BL6 mice. Each value was expressed as the mean percentages±s.e.m. of the Ly9.1+ cells.

View larger version (80K): [in a new window]   Fig. 5. Hematopoiesis in the PLC-1-deficient mice at E9.5.

View larger version (17K): [in a new window]   Fig. 6. In vitro differentiation of the wild-type (+/+), plc-1+/– (+/–) and plc-1–/– (–/–) ES cells. (A) ES cells differentiated into primitive erythrocytes (EryP) and definitive erythrocytes (EryD) with or without erythropoietin (EPO). EryP production at the 7th day (left) and EryD production at the 13th day (right) were investigated. (B) ES cells were differentiated to monocytes/macrophage with or without macrophage colony-stimulating factor (M-CSF) by co-culture with the stromal cell line, OP9. Total numbers of adherent cell on the tenthth day were counted. Data represent mean±s.e.m. from six independent experiments.

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