Distinct enhancers regulate skeletal and cardiac muscle-specific expression programs of the cardiac alpha-actin gene in Xenopus embryos

During vertebrate embryonic development, cardiac and skeletal muscle originates from distinct precursor populations. Despite the profound structural and functional differences in the striated muscle tissue they eventually form, such progenitors share many features such as components of contractile apparatus. In vertebrate embryos, the alpha-cardiac actin gene encodes a major component of the myofibril in both skeletal and cardiac muscle. Here, we show that expression of Xenopus cardiac alpha-actin in the myotomes and developing heart tube of the tadpole requires distinct enhancers within its proximal promoter. Using transgenic embryos, we find that mutations in the promoter-proximal CArG box and 5 bp downstream of it specifically eliminate expression of a GFP transgene within the developing heart, while high levels of expression in somitic muscle are maintained. This sequence is insufficient on its own to limit expression solely to the myocardium, such restriction requiring multiple elements within the proximal promoter. Two additional enhancers are active in skeletal muscle of the embryo, either one of which has to interact with the proximal CArG box for correct expression to be established. Transgenic reporters containing multimerised copies of CArG box 1 faithfully detect most sites of SRF expression in the developing embryo as do equivalent reporters containing the SRF binding site from the c-fos promoter. Significantly, while these motifs possess a different A/T core within the CC(A/T)(6)GG consensus and show no similarity in flanking sequence, each can interact with a myotome-specific distal enhancer of cardiac alpha-actin promoter, to confer appropriate cardiac alpha-actin-specific regulation of transgene expression. Together, these results suggest that the role of CArG box 1 in the cardiac alpha-actin gene promoter is to act solely as a high-affinity SRF binding site.     

Analysis of muscle creatine kinase gene regulatory elements in skeletal and cardiac muscles of transgenic mice.

Regulatory regions of the mouse muscle creatine kinase (MCK) gene, previously discovered by analysis in cultured muscle cells, were analyzed in transgenic mice. The 206-bp MCK enhancer at nt-1256 was required for high-level expression of MCK-chloramphenicol acetyltransferase fusion genes in skeletal and cardiac muscle; however, unlike its behavior in cell culture, inclusion of the 1-kb region of DNA between the enhancer and the basal promoter produced a 100-fold increase in skeletal muscle activity. Analysis of enhancer control elements also indicated major differences between their properties in transgenic muscles and in cultured muscle cells. Transgenes in which the enhancer right E box or CArG element were mutated exhibited expression levels that were indistinguishable from the wild-type transgene. Mutation of three conserved E boxes in the MCK 1,256-bp 5' region also had no effect on transgene expression in thigh skeletal muscle expression. All these mutations significantly reduced activity in cultured skeletal myocytes. However, the enhancer AT-rich element at nt - 1195 was critical for expression in transgenic skeletal muscle. Mutation of this site reduced skeletal muscle expression to the same level as transgenes lacking the 206-bp enhancer, although mutation of the AT-rich site did not affect cardiac muscle expression. These results demonstrate clear differences between the activity of MCK regulatory regions in cultured muscles cells and in whole adult transgenic muscle. This suggests that there are alternative mechanism of regulating the MCK gene in skeletal and cardiac muscle under different physiological states.     

A retinoic acid responsive Hoxa3 transgene expressed in embryonic pharyngeal endoderm, cardiac neural crest and a subdomain of the second heart field

A transgenic mouse line harbouring a β-galacdosidase reporter gene controlled by the proximal 2 kb promoter of Hoxa3 was previously generated to investigate the regulatory cues governing Hoxa3 expression in the mouse. Examination of transgenic embryos from embryonic day (E) 8.0 to E15.5 revealed regionally restricted reporter activity in the developing heart. Indeed, transgene expression specifically delineated cells from three distinct lineages: a subpopulation of the second heart field contributing to outflow tract myocardium, the cardiac neural crest cells and the pharyngeal endoderm. Manipulation of the Retinoic Acid (RA) signaling pathway showed that RA is required for correct expression of the transgene. Therefore, this transgenic line may serve as a cardiosensor line of particular interest for further analysis of outflow tract development.     

Functional conservation between rodents and chicken of regulatory sequences driving skeletal muscle gene expression in transgenic chickens

Background  

Regulatory elements that control expression of specific genes during development have been shown in many cases to contain functionally-conserved modules that can be transferred between species and direct gene expression in a comparable developmental pattern. An example of such a module has been identified at the rat myosin light chain (MLC) 1/3 locus, which has been well characterised in transgenic mouse studies. This locus contains two promoters encoding two alternatively spliced isoforms of alkali myosin light chain. These promoters are differentially regulated during development through the activity of two enhancer elements. The MLC3 promoter alone has been shown to confer expression of a reporter gene in skeletal and cardiac muscle in transgenic mice and the addition of the downstream MLC enhancer increased expression levels in skeletal muscle. We asked whether this regulatory module, sufficient for striated muscle gene expression in the mouse, would drive expression in similar domains in the chicken.     

Myosin light chain-2 luciferase transgenic mice reveal distinct regulatory programs for cardiac and skeletal muscle-specific expression of a single contractile protein gene.

To examine the relationship between the cardiac and skeletal muscle gene programs, the current study employs the regulatory (phosphorylatable) myosin light chain (MLC-2) as a model system. Northern blotting, primer extension, and RNase protection studies documented the high level expression of the cardiac MLC-2 mRNA in both mouse cardiac and slow skeletal muscle (soleus). Transgenic mouse lines harboring a 2100- or a 250-base pair rat cardiac MLC-2 promoter/luciferase fusion gene were generated, demonstrating high levels of luciferase activity in cardiac muscle, and only background luminescence in slow skeletal muscle and non-muscle tissues. As assessed by in situ hybridization, immunofluorescence, and luminescence assays of luciferase reporter activity in various regions of the heart, both the endogenous MLC-2 gene and the MLC-2 luciferase fusion gene were expressed exclusively in the ventricular compartment, with expression in the atrium at background levels. Point mutations within the conserved regulatory sites HF-1a and HF-1b significantly cripple ventricular muscle specificity, while mutation of the single E-box site was without effect, suggesting that ventricular muscle-specific expression occurs through an E-box-independent pathway. This study provides direct evidence that the cis regulatory sequences in the cardiac/slow twitch MLC-2 gene which confer cardiac and skeletal muscle-specific expression can be clearly segregated, suggesting that distinct regulatory programs may have evolved to control the tissue-specific expression of this single contractile protein gene in cardiac and skeletal muscle.     

Expression patterns of FHL/SLIM family members suggest important functional roles in skeletal muscle and cardiovascular system

LIM domain containing proteins play critical roles in animal development and cellular differentiation. Here, we describe the cloning and expression patterns of three members of the four and a half LIM domain-only protein family, FHL1, 2, and 3, from mouse. A comparison of embryonic expression patterns of these three highly-related genes indicates that they are expressed in an overlapping pattern in the developing cardiovascular system, and skeletal muscle. In adult tissues, the three genes are expressed in a predominant and overlapping manner in cardiac and skeletal muscle. Of the three genes, FHL2 appears to have the most restricted expression pattern during development, in heart, blood vessels, and skeletal muscle. Expression in heart is highest in cardiac septa and in the region adjacent to the atrio-ventricular ring, suggesting a potential role in septation or conduction system development. In the heart, FHL1expression was observed strongly in developing outflow tract, and to a lesser extent in myocardium. FHL3 displays low and ubiquitous expression during mouse development. Cardiac ventricular expression of FHL1, but not FHL2 or FHL3, was upregulated in two mouse models of cardiac hypertrophic and dilated cardiomyopathy. Taken together, these data indicate the potential importance of this FHL family in the development and maintenance of the cardiovascular system and striated muscle, and suggest that FHL1 may play a role in the development of heart disease.     

Identification of a critical control element directing expression of the muscle-specific transcription factor MRF4 in the mouse embryo

Skeletal muscle development in the vertebrate embryo critically depends on the myogenic regulatory factors (MRFs) including MRF4 and Myf5. Both genes exhibit distinct expression patterns during mouse embryogenesis, although they are genetically closely linked with multiple regulatory elements dispersed throughout the common gene locus. MRF4 has a biphasic expression profile, first in somites and later in foetal skeletal muscles. Here, we demonstrate by transgenic analysis that elements within a 7.5-kb promoter fragment of the MRF4 gene are sufficient to drive the embryonic wave of expression very similar to the endogenous gene in somites of mouse embryos. In contrast, a 3-kb fragment of the proximal promoter fails to support expression in the myotome, suggesting that essential cis-acting elements are located between -7.5 and -3 kb upstream of MRF4. Further analysis of this sequence delimits an essential region between -6.6 and -5.6 kb that together with the 3-kb promoter fragment directs transgene expression in the epaxial myotome of all somites during the appropriate developmental period. These data provide evidence that the partly overlapping expression patterns of Mrf4 and Myf5 in somites are controlled by distinct regulatory elements. We also show that 11.4 kb sequence upstream of MRF4, including the promoter and the somitic control region identified in this study, is not sufficient to elicit target specificity towards the strong Myf5 (-58/-48 kb) enhancer, suggesting that additional yet unidentified elements are necessary to convey promoter selectivity and protect the MRF4 gene from this enhancer.