Targeted Deletion of the MLC1f/3f Downstream Enhancer Results in Precocious MLC Expression and Mesoderm Ablation

Abstract

The expression of skeletal muscle contractile proteins is tightly regulated during embryonic development. In the mouse, the myosin light chain (MLC) 1f/3f gene locus is not activated until E9.5, exclusively in skeletal muscle precursor cells. A potent enhancer downstream of the MLC1f/3f locus confers correct temporal and spatial activation of linked reporter gene in transgenic mouse embryos. To examine roles of the MLC downstream enhancer (MLCE) in its native context of the MLC1f/3f gene locus, we eliminated a 1.5-kb DNA segment containing the enhancer from the mouse genome by targeted deletion, leaving no exogenous sequences at the deletion site. Mouse embryos homozygous for the MLCE deletion were smaller and developmentally delayed, formed no mesoderm by E7.5, and were resorbed almost completely at E8.5. In situ hybridization and RT-PCR analyses of affected mutant embryos at E7.5 revealed ectopic MLC transcripts, whose products would be predicted to interfere with a variety of nonmuscle cell functions determining differentiation of mesoderm. These results suggest that the MLC downstream enhancer and its flanking sequences include negative regulatory elements which block precocious activation of MLC expression in mesodermal precursors during a critical window of development, as well as positive elements which subsequently permit tissue-restricted MLC transcription in differentiating skeletal muscles.

Myosin essential light chain in health and disease

“…These results suggest that precocious transcription activated from the MLC1f/3f locus may interfere with functions of cells in the mesodermal lineage during early embryogenesis (11). Since myosin plays an important role in all stages of cytokinesis (47), the early embryonic lethality observed in the homozygous mutant mice might be caused by defects in cytokinesis resulting in the failure of cell replicative capacity during gastrulation (36).…”

“…Elimination of a 1.5-kb DNA segment containing the enhancer sequence (MLCE) from the mouse genome resulted in precocious MLC expression and mesoderm ablation (36). Mouse embryos homozygous for the MLCE deletion were smaller and developmentally delayed, formed no mesoderm by embryonic (E) day 7.5 (E7.5) and were resorbed almost completely at E8.5.…”

“…Mouse embryos homozygous for the MLCE deletion were smaller and developmentally delayed, formed no mesoderm by embryonic (E) day 7.5 (E7.5) and were resorbed almost completely at E8.5. Heterozygotic embryos carrying only one MLC downstream enhancer-deleted locus developed normally but showed no MLC1f/3f transcripts at E7.5 (36). It is possible that normal mesodermal development was affected by low-level ectopic transcription of the MLC1f/3f locus generated by a single mutant allele.…”

Interplay of IKK/NF-κB signaling in macrophages and myofibers promotes muscle degeneration in Duchenne muscular dystrophy

“…For this analysis, mdx;IKK F/F mice were crossed with Cre knock-in mice under the regulation of the myosin light chain 1f promoter (MLC-Cre mice), whose expression has been shown to be highly restricted to skeletal muscles (32). Surprisingly, genotyping analysis from more than 100 progeny showed that MLC-Cre animals, in contrast to myeloid-depleted IKKβ mice, were not obtained in the expected Mendelian ratios, and more than 98% of the viable mdx;IKKβ F/F ;MLC-Cre litters were females, suggesting a sex bias in the segregation of these alleles Since expression of MLC-1f occurs early in skeletal muscle development (32,33), it is possible that the requirement of IKKβ during embryonic development might be differentially regulated in a sex-specific manner in a dystrophic background. Based on these considerations, mdx;IKKβ F/F ;MLC-Cre female mice were used for our analyses and were compared with age-matched mdx;IKKβ F/F female controls.…”

Transgenic mice that express Cre recombinase under control of a skeletal muscle-specific promoter frommef2c

“…However, these Cre lines have limited use for disrupting genes with shared expression in cardiac and skeletal muscle, particularly if the gene is required early in cardiac development. An additional skeletal muscle-specific Cre line has been generated by homologous recombination of Cre into the myosin light chain 1f (mlc) locus, but Cre expression in this line is restricted to fast fibers (Bothe et al, 2000) and is activated relatively late during myogenesis (Jiang et al, 2002).…”