Fig 1.
Expression of Ifng increases in the kidneys of the Ifng GOF mouse.
A. Schematic depicting the Ifng GOF mouse line. Murine Ifng is ectopically expressed in the Pax3-expressing domain after Cre-mediated excision of a LoxP-CAT-Stop cassette. B. The amount of Ifng in kidneys increases about 13 fold in mutant vs. normal kidneys, when Ifng expression is targeted to the MM. The amount of Ifng was measured by ELISA. C. Ifng expression (red) is elevated in the MM-derived areas. The UB is marked with Hoxb7/myr-venus (green). Scale bar = 20 μm. D. Ifng mRNA is elevated in kidneys from Ifng GOF mice. mRNA is measured by semi-quantitative RT-PCR. Ifngr1 and Ifngr2 are also detected in mouse embryonic kidneys. E. Stat1 protein is activated in mutant kidneys, as determined by phosphorylation of Y701 and S727 in immunoblots. As a loading control, β-Actin was used. All these experiments used E14.5 kidneys. nor—normal, mu—mutant.
Fig 2.
Overexpression of Ifng in MM progenitor cells results in renal agenesis or hypoplasia.
49% of mutant embryos have two small kidneys (mu-left), 30% of mutant embryos have one small kidney (mu-middle), and 21% of mutant embryos have no kidneys at E14.5 (mu-right). nor—normal, mu—mutant.
Fig 3.
Proliferation and cell death during embryonic kidney development.
A. Proliferating cells were detected with phosphohistone 3 antibody in kidneys at E14.5. Proliferation rates over comparable areas are similar between normal and mutant kidneys. B. TUNEL analysis of kidney at E14.5. The incidence of TUNEL-+ cells at this stage over comparable areas is similar between normal and mutant kidneys. C. immunostaining for anti-phosphohistone 3 in proliferating cells in kidneys at E11.5. The dotted line encircles comparable portions of metanephroi. D. TUNEL analysis of kidney at E10.5 (upper) and E11.5 (lower). The kidney is stained with a rabbit anti-Pax2 antibody to demarcate the MM/UB area. The epithelial structure is the nephric duct (ND), and the adjacent Pax2+ tissue delineates the MM region. The UB is also marked with Hoxb7/myr-venus (A, B, D). TUNEL+ cells are numerous in mutant MMs. nor—normal, mu—mutant.
Fig 4.
UB branching morphogenesis is retarded in Ifng GOF mutants.
A. WISH for c-Ret at E10.5 (upper) or at E11.5 (lower). UB tips are marked with black arrows. ND—nephric duct, CND—common nephric duct. B. Calbindin staining of kidney explants cultured for 24h. C. UB tip numbers are significantly reduced in mutants relative to normal embryos during development. Hoxb7/myr-venus containing kidneys were isolated at E11.5, E12.5 and E13.5 and UB tips were counted. D. Hoxb7/myr-venus-expressing cultured kidney explants after treatment with mouse recombinant Ifng (20ng/ml). The fluorescent (upper) or bright field (lower) images were taken from cultures at 5h, 24h and 48h. For explant cultures, E11.5 kidneys were used (B & D). nor—normal, mu—mutant.
Fig 5.
The expression of Sall1 and its potential targets are modulated in mutant kidneys.
E14.5 kidneys were used for all analyses. A. semi-quantitative RT-PCR for Sall1. The expression level of Sall1 mRNA is decreased in mutants. B. Immunostaining of Sall1. Sall1 protein expression is also decreased in mutants. Lower: higher magnification of areas shown in the white rectangles. C. Active β-Catenin is determined by immunoblotting with a β-Catenin antibody that detects unphosphorylated residues at Ser33/Ser37/Thr41, which target the molecule for ubiquitination. β-Catenin is activated in mutants. D. RT-PCR of Wnt9b and Axin2. The mRNA levels of Wnt9b and Axin2, a target gene of Wnt/β-Catenin signaling, are increased in mutants. E. semi-quantitative RT-PCR for Kif26b. The mRNA level of Kif26b, a target gene for Sall1, is reduced in mutants. nor—normal, mu—mutant.