Animal studies and tissue preparation All animal experiments were performed in accordance with the guidelines of the institutional committee for the use of animals for research. Mice were killed by a lethal dose of anaesthesia. In vivo luciferase imaging and doxycycline treatment were performed as previously described 11 . Animals were injected intraperitoneally (i.p.) with 20 ng human recombinant TNFa (Roche) and killed after 1 h for liver p65 immunostaining and RNA and protein extraction. For anti-TNFa treatment, mice were injected with 22 mg of goat anti-mouse TNFa neutralizing antibodies i.p. (R&D Systems; AF-410-NA) for 3 consecutive days, or with control goat IgG. All mice were injected i.p. with BrdU (Amersham; 100 ml per 10 g body weight) 3 and 24 h before they were killed. In some animals, liver samples were removed and snap-frozen for protein and RNA analyses. The animal was then perfused through the left ventricle with 10 ml of cold heparinized PBS, followed with 25 ml of 4% buffered formalin. Livers were removed, weighed, photographed and fixed in formalin over night. The next day the entire liver was submitted for paraffin embedding in three to five cassettes. 5-mM sections were stained with H&E and evaluated by a pathologist to whom the genetic makeup or treatment group were not known. MRI analysis MRI was performed on a horizontal 4.7T Biospec spectrometer (Bruker Medical), using a birdcage coil. Mice were anaesthetized (30 mg kg 21 pentobarbital, i.p.) and placed supine with the liver located at the centre of the coil. Liver and tumour volumes were determined from multi-slice coronal and axial T1 weighted fast spin echo images covering the entire liver both coronally and axially (repetition time, 400 ms; echo time, 17 ms; slice thickness, 1 mm; field of view, 5 cm (coronal) and 3.4 cm (axial) using a 256 £ 256 matrix). To estimate tumour and liver volumes, the liver and tumour boundaries visualized in each slice were outlined by using image processing software (NIH image), by an observer to whom the genetic makeup was not known. The number of pixels (for both liver and tumour) were converted to an area by multiplying by the factor ((field of view)2 £ (matrix)2). Immunohistochemistry For p65 immunostains, 5-mM sections, cut on the same day, were de-waxed and hydrated through graded ethanols, cooked in 25 mM citrate buffer pH 6.0 in a pressure cooker at 115 8C for 3 min (decloaking chamber, Biocare Medical), transferred into boiling deinoized water and left to cool down for 20 min. After 5 min treatment in 3% H 2 O 2 , slides were incubated with rabbit polyclonal p65 antibodies diluted 1:100 in CAS-Block (Zymed) for 3 h at room temperature, washed three times with Optimax (Biogenex), incubated for 30 min with anti rabbit Envision þ (DAKO) and developed with DAB for 15 min. Antigen retrieval for TNFa and p65 double immunostaining was performed in 100 mM glycine buffer pH 9.0. After 5 min treatment in 3% H 2 O 2 , slides were incubated with goat polyclonal anti-TNFa antibodies diluted 1:50 in CAS-Block for 1 h at room temperature, washed in Optimax, incubated with (1:100) rabbit anti-goat antibodies (Jackson Laboratories) for 30 min, washed again with Optimax and incubated with anti-rabbit Envision þ (DAKO) and developed with AEC for 15min at 37 8C. Following this, slides were reboiled for 7 min in 100 mM glycine buffer in a microwave oven and cooled down to room temperature. Slides were incubated with rabbit polyclonal anti-p65 antibodies diluted 1:100 in CAS-Block for 3 h at room temperature, washed three times with Optimax, post-fixed for 5min in 4% formaldehyde in Tris buffered saline, washed with Optimax, incubated for 30 min with alkaline phosphatase-conjugated polymer anti-rabbit IgG (Zymed) and developed with BCIP/NBT. 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This research was supported by grants from the Israel Science Foundation funded by the Israel Academy for Sciences and Humanities (Center of Excellence Program), Prostate Cancer Foundation Israel—Center of Excellence, German-Israeli Foundation for Scientific Research and Development (GIF, in collaboration with H. Bujard), a grant in memory of H. and F. Brody from H. M. Krueger as trustee of a charitable trust and the Israel Cancer Research Foundation (ICRF). I.S. is supported by the Lady Davis Fellowship Trust. Competing interests statement The authors declare that they have no competing financial interests. Correspondence and requests for materials should be addressed to E.P. (peli@hadassah.org.il) and Y.B.-N. (yinon@cc.huji.ac.il). .............................................................. Mrf4 determines skeletal muscle identity in Myf5:Myod double-mutant mice Lina Kassar-Duchossoy 1 , Barbara Gayraud-Morel 1 *, Danielle Gome `s 1 , Didier Rocancourt 2 , Margaret Buckingham 2 , Vasily Shinin 1 * & Shahragim Tajbakhsh 1 1 Stem Cells and Development, 2 Molecular Genetics of Development, Department of Developmental Biology, CNRS URA 2578, 25 rue du Dr Roux, 75724 Paris Cedex 15, France * These authors contributed equally to the work ............................................................................................................................................................................. In vertebrates, skeletal muscle is a model for the acquisition of cell fate from stem cells 1 . Two determination factors of the basic helix–loop–helix myogenic regulatory factor (MRF) family, Myf5 and Myod, are thought to direct this transition because double- mutant mice totally lack skeletal muscle fibres and myoblasts 2–4 . In the absence of these factors, progenitor cells remain multi- potent and can change their fate 5,6 . Gene targeting studies have revealed hierarchical relationships between these and the other MRF genes, Mrf4 and myogenin, where the latter are regarded as differentiation genes 7 . Here we show, using an allelic series of three Myf5 mutants that differentially affect the expression of the genetically linked Mrf4 gene, that skeletal muscle is present in the letters to nature NATURE | VOL 431 | 23 SEPTEMBER 2004 | www.nature.com/nature 466 ©2004 Nature Publishing Group