Know Your Neighbors: Three Phenotypes in Null Mutants of the Myogenic bHLH Gene MRF4

skeletal muscle and skeletal muscle precursors, and on cell culture experiments, all of which pointed to functions in specification of myoblasts and execution of muscle differentiation. The major muscle phenotypes The University of Texas Southwestern Medical Center at Dallas described thus far are the failure of muscle differentiation in mice homozygous for myogenin null mutations Dallas, Texas 75235–9148 † Technische Universitat Braunschweig (Hasty et al., 1993; Nabeshima et al., 1993) and failure to produce the myogenic precursor cell population in Institut fur Biochemie und Biotechnologie 3300 Braunschweig mice deficient in both MyoD and Myf5 (Rudnicki et al., 1993). Unlike muscle deficits, the skeletal phenotypes Federal Republic of Germany ‡ Division of Eukaryotic Molecular Genetics were not expected, although they can account for the lethality of several MRF mutations. (Rhodes and Konieczny, 1989) is the final myo-genic bHLH gene to be targeted in mice. It is located United Kingdom § Division of Biology 156–29 approximately 8 kb 5Ј of Myf5 on mouse chromosome 10. The proximity of MRF4 and Myf5 to each other raises the possibility of cis-regulatory interactions within the locus, and prior observations that MRFs can cross-regulate each other in cell culture raise the possibility of trans-regulatory interactions between MRF4 and Myf5 The most widely used strategy for gene disruption in or other MRF gene products. During embryogenesis, the mouse involves the deletion of part or all of the MRF4 is expressed transiently in the somite myotome target gene together with concomitant insertion of a between embryonic days 9.0 (E9.0) and 11.5 and it sub-drug selection cassette, the goal being efficient creation sequently reappears in differentiated muscle fibers be-of definitive null alleles. However, such manipulations ginning around E16. MRF4 is further upregulated in may disrupt the expression of other genes located near newborns relative to other MRFs and becomes the pre-the intended target and therefore confound the interpre-dominant myogenic bHLH gene expressed in adult skel-tation of phenotypes from alleles designed to be simple etal muscle (Rhodes and Konieczny, 1989). null mutations. A major unknown has been the frequency The three mutations we introduced into MRF4 were and severity of possible neighborhood phenotypic ef-each designed to eliminate MRF4 function by deleting fects in mouse knockouts. This will presumably depend part of the MRF4 protein coding sequence, and each on specific aspects of long-range gene organization and mutation concomitantly inserted the same PGKneo se-cis-regulatory element function that are not known for …