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• W2065926472 abstract "Targeted mutations of the Myf-5/MRF4 gene locus in mice have led to defects in myotome formation and to severe malformations of the ribs. We have generated two new mouse strains in which the coding region of Myf-5 was replaced with the cDNA for FGF-6 and exon I of Myf-5 gene was deleted by Cre-recombinase, respectively. Both strains do not show malformations of the ribs despite a loss of early myotome formation in homozygous mice. We conclude that the rib phenotype in previously described Myf-5 and MRF4 mutant mice is caused by long-distance effects on a yet unknown gene. The myogenic program in mammals is dependent on a family of myogenic regulatory factors (MRFs) comprising Myf-5, MyoD, Myogenin, and MRF4 (also independently isolated as Myf-6 and Herculin; 13Weintraub H. Davis R. Tapscott S. Thayer M. Krause M. Benezra R. Blackwell T.K. Turner D. Rupp R. Hollenberg S. et al.Science. 1991; 251: 761-766Crossref PubMed Scopus (1205) Google Scholar). Gene knockout experiments of each MRF confirmed the essential role of MRFs for muscle cell determination and differentiation in mice (for a recent review see 1Arnold H.H. Braun T Curr. Top. Dev. Biol. 2000; 48: 129-164Crossref PubMed Scopus (112) Google Scholar). Since the activity of MRFs is restricted to striated skeletal muscle, the presence of skeletal abnormalities specific to the ribs in homozygous Myf-5 mutant mice came as a surprise. Homozygous Myf-5 mutant mice lack the distal parts of the ribs, which leads to perinatal death (3Braun T. Rudnicki M.A. Arnold H.-H. Jaenisch R Cell. 1992; 71: 369-382Abstract Full Text PDF PubMed Scopus (535) Google Scholar). This striking phenotype was also observed in a second Myf-5 mutation in which the lacZ gene, together with the PGK-neo selection cassette, was inserted at the same position into the Myf-5 locus (11Tajbakhsh S. Rocancourt D. Buckingham M Nature. 1996; 384: 266-270Crossref PubMed Scopus (239) Google Scholar). To explain the Myf-5 rib phenotype it has been suggested that the early myotome might be the source of inductive, trophic, and/or patterning interactions required to form the ribs, which arise from a subset of sclerotomal cells immediately adjacent to the myotome (3Braun T. Rudnicki M.A. Arnold H.-H. Jaenisch R Cell. 1992; 71: 369-382Abstract Full Text PDF PubMed Scopus (535) Google Scholar). Since the formation of the myotome is dependent on Myf-5 and a number of potential inductive signals expressed in myotome like FGFs and PDGF are absent in Myf-5 mutant mice (4Grass S. Arnold H.H. Braun T Development. 1996; 122: 141-150PubMed Google Scholar), this hypothesis appeared plausible and logical. The view that the rib phenotype is caused by secondary events due to myotome defects was further supported by the generation of three different alleles of the MRF4 gene, which is located 8 kb upstream of the Myf-5 gene on mouse chromosome 10 (2Braun T. Arnold H.H EMBO J. 1995; 14: 1176-1186Crossref PubMed Scopus (149) Google Scholar, 9Patapoutian A. Yoon J.K. Miner J.H. Wang S. Stark K. Wold B Development. 1995; 121: 3347-3358Crossref PubMed Google Scholar, 14Zhang W. Behringer R.R. Olson E.N Genes Dev. 1995; 9: 1388-1399Crossref PubMed Scopus (240) Google Scholar). All three MRF4 knockout alleles were characterized by a variable intensity of rib defects that correlated with the severity of myotomal defects. The differing effects of MRF4 mutations on rib formation were believed to result from cis-effects of induced MRF4 mutations on the expression of the neighboring Myf-5 gene. We wanted to prove our initial hypothesis that the lack of trophic signals from the myotome may account for the rib defects by replacing the coding region of Myf-5 with the cDNA for FGF-6 yielding Myf-5FGF6-ki mice. The mutant allele does not contain a PGK-neo-TK selection cassette since the latter was removed by Cre-recombinase-mediated excision nor does it show an alteration of exon–intron boundaries (Figure 1). We also generated a conditionally active Myf-5 allele by insertion of a loxP site upstream of exon I of the Myf-5 gene and a loxP-flanked PGK-neo-TK cassette into the first intron of the Myf-5 gene. The selection cassette was enzymatically removed by Cre-recombinase. Subsequently, mouse strains were created from these cells and mice were bred with the deleter strain mice (10Schwenk F. Baron U. Rajewsky K Nucleic Acids Res. 1995; 23: 5080-5081Crossref PubMed Scopus (935) Google Scholar), which carry a ubiquitously active Cre-recombinase gene driven by the human cytomegalovirus minimal promotor (PBi-2) generating Myf-5ΔloxP mice. The Cre-mediated deletion removes a 3 kb fragment encompassing exon I, which contains most of the coding region including the bHLH DNA binding region and 2 kb of upstream sequences (Figure 1). As expected the Myf-5loxP conditional allele is functional prior to recombination while Myf-5 mRNA was fully absent from Myf-5ΔloxP/Myf-5ΔloxP mice. Heterozygous Myf-5FGF6-ki and Myf-5ΔloxP mice were mated, and the genotype of the offspring was determined by Southern Blot analysis at birth. Newborn mice including homozygous mutants appeared clinically normal at birth. This observation is in marked contrast to the situation caused by the original Myf-5m1 mutation and in Myf-5lacZ and Myf-6m1 homozygous mutants, which die without exception a few minutes after delivery due to the lack of the distal parts of the ribs. Homozygous E18.5 Myf-5FGF6-ki and Myf-5ΔloxP embryos were subjected to whole mount bone and cartilage staining; no alterations of the ribs or other skeletal elements were discernible (Figure 2). Most Myf-5ΔloxP/Myf-5ΔloxP mice reached adulthood and were fertile. The absence of any rib or other skeletal abnormalities was corroborated by analysis of a larger group of Myf-5ΔloxP/Myf-5ΔloxP mice in the F2 and F3 generation. From this set of data we conclude that the absence of Myf-5 mRNA is not sufficient to cause the rib phenotype observed in homozygous mutant Myf-5m1 mice. The unexpected phenotype of homozygous Myf-5ΔloxP and Myf-5FGF6-ki mice led us to reinvestigate the absence of myotome formation in Myf-5 mutant mice. At E9.75 we performed an in situ analysis of myogenin and MRF4 mRNA and MyHC, titin, nebulin, and desmin protein expression, respectively. While the different molecules were easily detectable in wild-type and in heterozygous mutant Myf-5ΔloxP embryos, homozygous Myf-5ΔloxP embryos failed to show any signals in somites (Figure 2). Later on during development, MyoD expression started and apparently rescued the early defect in myogenesis leading to grossly normal muscle tissue at birth (data not shown). Further breeding with Myf-5ΔloxP with MyoD mutant mice revealed that double homozygous MyoD/Myf-5ΔloxP mice lack all skeletal myotubes as their MyoD/Myf-5m1 counterparts. The Myf-5/MRF4 locus has been targeted several times. Most alleles published so far generated significant, though different, rib pattern formation deficits. The explanation for the rib phenotype concentrated on defects in myotome formation caused by a lack or a massive reduction of Myf-5 expression. The results of three different mutations introduced into the MRF4 gene, which is located approximately 8 kb upstream of Myf-5 on mouse chromosome 10 seemed to support this view (8Olson E.N. Arnold H.H. Rigby P.W. Wold B.J Cell. 1996; 85: 1-4Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar). The generation of the two new Myf-5 alleles reported here, which do not show any malformations of the ribs or other skeletal elements despite the absence of Myf-5 mRNA in somites and the complete loss of early myotome formation, exclude this explanation. We can rule out a direct involvement of the Myf-5 and/or MRF4 proteins in the generation of the short rib phenotype. Neither the Myf-5ΔloxP nor the “weakest” MRF4 mutations, which are true null-alleles of the Myf-5 and the MRF4 gene, respectively, nor Myf-5ΔloxP/Myf-6m1 mice and Myf-5ΔloxP/Myf-5m1 compound heterozygous mice show any rib truncations. These findings now strongly suggest that an alteration of the activity of a third or more unknown gene(s) is the real cause for the skeletal malformations seen in Myf-5m1, Myf-5lacZ, and the various MRF4 knockout mouse strains. This does not necessarily mean that no cross-talk between different somitic layers is necessary for rib formation. A number of separate experiments indicate that the dermomyotome but not necessarily the myotomal cell layer is involved in the morphogenesis of the distal parts of the ribs (6Henderson D.J. Conway S.J. Copp A.J Dev. Biol. 1999; 209: 143-158Crossref PubMed Scopus (65) Google Scholar, 7Huang R. Zhi Q. Schmidt C. Wilting J. Brand-Saberi B. Christ B Development. 2000; 127: 527-532PubMed Google Scholar). It is still feasible that inductive influences from the dermomyotome are necessary for early specification of lateral sclerotome. Alteration of a gene that is normally active in the dermomyotome by the Myf-5 mutation may therefore be causative for sclerotomal malformations. How does the phenotype of the knockout of the Myogenin gene that resides on a differerent chromosome but also results in a rib phenotype (5Hasty P. Bradley A. Morris J.H. Edmundson D.G. Venuti J. Olson E.N. Klein W.H Nature. 1993; 364: 501-506Crossref PubMed Scopus (964) Google Scholar) fit into this picture? (1) The myogenin phenotype is rather different from the Myf-5 phenotype. (2) It was never very likely to depend on the same mechanism, since the lack of Myogenin did not result in a lack of myotome formation and the development of primary muscle cells in Myogenin mutant embryos was not severely affected (12Venuti J.M. Morris J.H. Vivian J.L. Olson E.N. Klein W.H J. Cell. Biol. 1995; 128: 563-576Crossref PubMed Scopus (205) Google Scholar). It appears most likely that either the increase of the relative numbers of myoblasts during primary or secondary myogenesis affects the delicate balance between dermomyotome and sclerotome or at a later stage the interaction of the rib blastema with surrounding tissues. This may lead to a phenocopy of some aspects of the Myf-5 phenotype without affection of the gene that causes the phenotype in Myf-5 mutants. Sequence analysis of the greater Myf-5 locus is in progress. Expression analysis of potential open reading frames from this region during embryonic development in wild-type, Myf-5ΔloxP, and Myf-5m1 mice will allow us to determine whether these genes might be involved in formation and growth of the sclerotome, rib primordia, or chondrocytes. Final proof of the role of these genes will await genetic manipulation of their expression in transgenic mice. The Myf-5 example emphasizes the fact that our current ability to build valid models explaining the phenotype of targeted mutations is strongly biased by our knowledge of neighboring genes. Although a number of cases have been described where genes of the same multigene group in a cluster were affected by a targeted mutation, no examples have been described where an unrelated gene was influenced in cis by gene targeting experiments. Additional data related to this article can be found online (http://www.cell.com/cgi/content/full/102/1/17/DC1). Acknowledgments: work in the authors' laboratory was supported by the DFG, FCI, and the Volkswagen Foundation." @default.
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