Nadia Rosenthal

Distinct gene expression patterns in skeletal and cardiac muscle are dependent on common regulatory sequences in the MLC1/3 locus

The myosin light-chain 1/3 locus (MLC1/3) is regulated by two promoters and a downstream enhancer element which produce two protein isoforms in fast skeletal muscle at distinct stages of mouse embryogenesis. We have analyzed the expression of transcripts from the internal MLC3 promoter and determined that it is also expressed in the atria of the heart. Expression from the MLC3 promoter in these striated muscle lineages is differentially regulated during development. In transgenic mice, the MLC3 promoter is responsible for cardiac-specific reporter gene expression while the downstream enhancer augments expression in skeletal muscle. Examination of the methylation status of endogenous and transgenic promoter and enhancer elements indicates that the internal promoter is not regulated in a manner similar to that of the MLC1 promoter or the downstream enhancer. A GATA protein consensus sequence in the proximal MLC3 promoter but not the MLC1 promoter binds with high affinity to GATA-4, a cardiac muscle- and gut-specific transcription factor. Mutation of either the MEF2 or GATA motifs in the MLC3 promoter attenuates its activity in both heart and skeletal muscles, demonstrating that MLC3 expression in these two diverse muscle types is dependent on common regulatory elements.

A transgene target for positional regulators marks early rostrocaudal specification of myogenic lineages

In transgenic mice, muscle-specific regulatory elements from the myosin light chain (MLC) 1/3 locus drive graded expression of a linked CAT reporter gene in selected fast muscles along the anteroposterior axis of the adult animal. The gradient of MLC-CAT transcripts is established early in development, during the generation of somites from the paraxial mesoderm and the activation of myogenic factor gene expression, and is not reflected in the expression of the endogenous MLC1 gene. At later embryonic stages, the gradient of MLC-CAT transcripts persists in intercostal and intervertebral muscles, but is not maintained in other axial muscles. Profiles of CAT transgene activity reveal that the gradient is generated during the maturation of increasingly caudal somites, opposite to the direction of somite development, and is retained in dissociated somite cultures. We propose that coexpression of myogenic factors is necessary but not sufficient to regulate expression of the MLC-CAT transgene, which is responsive to additional positional cues in the embryo.

Muscle-specific expression of human insulin receptor in transgenic mice

Variations in skeletal muscle insulin signaling are thought to have important effects on in vivo glucose homeostasis. To address the role of the insulin receptor in insulin action in muscle, we overexpressed human insulin receptors in the skeletal muscle of transgenic mice. A muscle-specific transgene (TMPE/HIR) was constructed by using promotor and enhancer elements derived from the rat MLC1/3 locus coupled to the intact protein-coding region of the human insulin-receptor cDNA. After testing the transgene for expression in cultured C2C12 myotubes, six founder mice transgenic for TMPE/HIR were generated. We determined that one line of mice had significant expression of human insulin-receptor mRNA in skeletal muscle. The analysisof several tissues from these mice by immunoprecipitation of labeled insulin receptors with a human-specific antireceptor antibody, revealed exclusive expression of human insulin receptors in skeletal muscle. Using both human-specific and non-species-specific anti-insulin receptor antibodies, we developed two immunoassays capable of quantitating the relative amounts of human and total insulin receptors in muscle. Compared with nontransgenic littermate controls, the total number ofinsulin receptors was increased by 30% in heterozygous transgenics and 68% in homozygotes. Human insulin-receptor protein contributed substantially to the total insulin-receptor pool present in transgenic muscle (42% for heterozygotes, 61% for homozygotes). Intraperitoneal glucose and insulin tolerance tests were performed with homozygous transgenic and nontransgenic littermate mice. Results with both approaches were significantly different for the two groups of mice, suggesting that the modest increase in insulin receptors in the muscle of transgenic mice causes a direct increase in insulin responsiveness. This study represents the first successful expression of human insulin receptors in transgenic mice. This model and others like it will provide valuable insights into the regulation of insulin-receptor expression and the role of insulin receptors in specific tissues or cell types in metabolism.

Expression of the IGF-II Gene in Brain and Muscle

The insulin-like growth factors (IGFs) have been implicated in many processes related to growth and development. Despite a growing interest by many investigators into the biological role of the insulin-like growth factors, their function remains poorly understood. Advances in molecular biological techniques have allowed for the characterization of the genes for these factors, and provided tools with which to study their expression. Using these tools, investigators have been elucidating the pattern of IGF gene expression in various tissues throughout a wide range of species. We have examined the expression of insulin-like growth factor II (IGF-II) during development in the nervous system and in muscle. IGF-II gene expression is developmentally regulated in both sites, suggesting that it plays an important role during the developmental program.