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Developmental and Tissue-Specific Regulation of the Murine Cardiac Actin Genein VivoDepends on Distinct Skeletal and Cardiac Muscle-Specific Enhancer Elements in Addition to the Proximal Promoter
Abstract
Cardiac actin is an early marker of cardiac and skeletal muscle lineages in the mouse. After birth, the gene is down-regulated in skeletal muscle. High-level expression of the murine cardiac actin gene in skeletal myotubes in vitro involves distal (-7.8/-7.0 kb) and proximal (-5.4/-3.5 kb) enhancer sequences as well as the proximal promoter (-0.7/+0.1 kb). Transgenic mice carrying an nlacZ reporter gene under the control of different fragments of the upstream region of the cardiac actin gene were generated. This analysis led to the conclusions that (1) the proximal promoter is a weak but tissue specific element in vivo, (2) consistent high-level expression in skeletal muscle depends on the presence of at least one of the enhancers, (3) expression in adult cardiac muscle requires a cardiac enhancer located in the (-5.4/-0.7 kb) region, and (4) a construct containing these three elements gives a strong specific expression of the transgene in the heart throughout the life of the animal and in embryonic skeletal muscle. All transgenes tested reproduce the down-regulation observed in adult skeletal muscle for the cardiac actin gene. Nonuniform expression of these transgenes in the heart may mark cardiomyocytes derived from different cardiac progenitors.
... For example, myosin heavy chain (MHC)  transcripts become restricted to the ventricles by E10.5 and myosin light chain (MLC) 1A and MLC2A transcripts to the atria/inflow region by birth (Lyons et al., 1990;Kubalak et al., 1994). Further regionalisation of transcriptional potential in the heart has been documented for a number of transgenes which are ex- pressed in either the right or left ventricular or atrial myocardium: reporter genes controlled by MLC3F or ␣-cardiac actin regulatory sequences are expressed in the LV and right atrium (RA) (Kelly et al., 1995;Biben et al., 1996), whereas MLC2V and desmin regulatory elements confer RV and OFT expression (Ross et al., 1996;Kuisk et al., 1996). Such asymmetric expression has not been documented for most endogenous cardiac genes, although some, such as M-creatine phosphokinase (MCK), connexin 40, and MLC3F show left/right differences in the ventricles as they are being activated or downregulated (Lyons, 1994;Delorme et al., 1997;Kelly et al., 1998). ...
... The left/right regionalised expression patterns of transgenes, in contrast, tend to extend to both the compact and trabeculated zones of either the left or right ventricle (Kelly et al., 1999). Transcriptional differences between left and right atria have also been described for certain transgenes (Biben et al., 1996;Kelly et al., 1998); MLC1A, ␣-MHC, and MLC2A, however, were expressed equivalently in both atria at all stages examined. No left/right asymmetry has been reported during the early downregulation of -MHC and MLC1V transcription in the atria (Lyons et al., 1990), although such differences may be masked by the relatively late development of the left and right atrial chambers. ...
... In contrast to the transiently regionalised expression profiles of the endogenous genes discussed above, transgenes which display right or left chamber-specific expression patterns, such as those containing regulatory sequences from the MLC3F, ␣-cardiac actin, or MLC2V genes, tend to be expressed in a regionalised manner throughout development and in the adult heart (Kelly et al., 1995;Biben et al., 1996;Ross et al., 1996). In the case of MLC3F the endogenous gene shows transitory left/right ventricular differences during development which may be stably retained by MLC3F transgenes (Kelly et al., 1998), suggesting that cis-acting modules absent from the transgene may mediate the temporal control of MLC3F transcription. ...
Many cardiac genes are broadly expressed in the early heart and become restricted to the atria or ventricles as development proceeds. Additional transcriptional differences between left and right compartments of the embryonic heart have been described recently, in particular for a number of transgenes containing cardiac regulatory elements. We now demonstrate that three myosin genes which become transcriptionally restricted to the atria between embryonic day (E) 12.5 and birth, α-myosin heavy chain (MHC), myosin light chain (MLC) 1A and MLC2A, are coordinately downregulated in the compact myocardium of the left ventricle before that of the right ventricle. α-MHC protein also accumulates in the right, but not left, compact ventricular myocardium during this period, suggesting that this transient regionalisation contributes to fktal heart function. dHAND and eHAND, basic helix-loop-helix transcription factors known to be expressed in the right and left ventricles respectively at E10.5, remain regionalised between E12.5 and E14.5. Downregulation of α-MHC, MLC1A, and MLC2A in iv/iv embryos, which have defective left/right patterning, initiates in the morphological left (systemic) ventricle regardless of its anatomical position on the right or left hand side of the heart. This points to the importance of left/right ventricular differences in sarcomeric gene expression patterns during fktal cardiogenesis and indicates that these differences originate in the embryo in response to anterior-posterior patterning of the heart tube rather than as a result of cardiac looping. Dev Dyn 2000;217:75–85. © 2000 Wiley-Liss, Inc.
... (24). Twenty four hours after transfection, the medium was aspirated, and the cells were fixed for 5 min in 4% paraformaldehyde (PFA) and treated with X-gal solution as described (25). Intensive X-gal staining confined primarily to the cytoplasm was observed microscopically within 1-2 h. ...
... Analysis of Embryos and X-Gal Staining-For timed pregnancy and embryo staging, the mornings of vaginal plug observations were considered as E0.5. Pregnant females were sacrificed by cervical dislocation, the embryos were retrieved and fixed in 4% PFA for 30 min to 2 h depending on the developmental stage as described (25). The embryos were washed several times in phosphate-buffered saline and treated overnight with X-gal solution as in Ref. 25. Stained embryos were photographed under a stereomicroscope (Leica). ...
... Pregnant females were sacrificed by cervical dislocation, the embryos were retrieved and fixed in 4% PFA for 30 min to 2 h depending on the developmental stage as described (25). The embryos were washed several times in phosphate-buffered saline and treated overnight with X-gal solution as in Ref. 25. Stained embryos were photographed under a stereomicroscope (Leica). ...
In human, germ line mutations in the tumor suppressor retinoblastoma (Rb) predispose individuals to retinoblastoma, whereas somatic inactivation of Rb contributes to the progression of a large spectrum of cancers. In mice, Rb is highly expressed in restricted cell lineages including the neurogenic, myogenic, and hematopoietic systems, and disruption of the gene leads to specific embryonic defects in these tissues. The symmetry between Rb expression and the defects in mutant mice suggest that transcriptional control of Rb during embryogenesis is pivotal for normal development. We have initiated studies to dissect the mechanisms of transcriptional regulation of Rb during development by promoter lacZ transgenic analysis. DNA sequences up to 6 kilobase pairs upstream of the mouse Rb promoter, isolated from two different genomic libraries, directed transgene expression exclusively to the developing nervous system, excluding skeletal muscles and liver. Expression of the transgene in the central and peripheral nervous systems, including the retina, recapitulated the expression of endogenous Rb during embryogenesis. A promoter region spanning approximately 250 base pairs upstream of the transcriptional starting site was sufficient to confer expression in the central and peripheral nervous systems. To determine whether this expression pattern was conserved, we isolated the human Rb 5' genomic region and generated transgenic mice expressing lacZ under control of a 1.6-kilobase pair human Rb promoter. The human Rb promoter lacZ mice also expressed the transgene primarily in the nervous system in several independent lines. Thus, transgene expression directed by both the human and mouse Rb promoters is restricted to a subset of tissues in which Rb is normally expressed during embryogenesis. Our findings demonstrate that regulatory elements directing Rb expression to the nervous system are delineated within a well defined core promoter and are regionally separated from elements, yet to be identified, that are required for expression of Rb in the developing hematopoietic and skeletal muscle systems.
... Current cardiac-specific promoters investigated include α-myosin heavy chain, myosin light chain, enhanced myosin light chain and cardiac troponin T promoters. [9][10][11][12][13][14] Among the promoters investigated, some have shown chamber specificity. Because the atrium and ventricle differ morphologically, functionally and are molecularly distinct, pathophysiology also varies in cardiac diseases. ...
... 7 Thus, cardiac-specific promoters have been identified to improve cardiac specificity. [9][10][11][12][13][14][15][16] Even within these promoters, chamber specificity has been noted. 14,15,23 Because the atrium and ventricle have individual functions, mechanisms and molecular signatures, pathophysiology varies in cardiac diseases as well. ...
Background
Cardiac gene therapy using the adeno‐associated virus serotype 9 vector is widely used due to its efficient transduction. However, the promoters used to drive expression often cause off‐target localization. To overcome this, former studies have applied cardiac specific promoters but the expression is debilitated compared to that of ubiquitous promoters. To address these issues in the context of atrial specific gene expression, an enhancer calsequestrin cis‐regulatory module 4 (CRM4) and the highly atrial specific promoter sarcolipin were combined to enhance expression and minimize off tissue expression.
Methods
To observe expression and bio‐distribution, constructs were generated using two different reporter genes: luciferase and EGFP. The ubiquitous cytomegalovirus (CMV), sarcolipin (SLN), and the CRM4 combined with sarcolipin (CRM4.SLN) were compared and analyzed by luciferase assay, western blot, qPCR, and fluorescent imaging.
Results
The cytomegalovirus promoter containing vectors showed the strongest expression in vitro and in vivo. However, the module sarcolipin combination showed enhanced atrial expression and minimized off target effect even when compared to the individual sarcolipin promoter.
Conclusion
For gene therapy involving atrial gene transfer, the CRM4.SLN combination is a promising alternative to the use of the CMV promoter. CRM4.SLN had significant atrial expression and minimized extra‐atrial expression.
... This question inherently posed a [40]. The HSA promoter is known to be skeletal muscle-specific and is expressed from prenatal myocyte differentiation [4]. From this mating, a conditional knockout (cKO) mouse will be created, in which we will confirm NR1 ablation with immunofluorescence and Western analysis. ...
... Although the HSA Cre mouse line was designed to be expressed in skeletal muscle during development and differentiation, it was designed neither to be specific for nor inclusive of synaptic nuclei [4]. Like ACh receptor subunits, NR1 may be expressed exclusively from synaptic nuclei. ...
... The Mlc1v-nlacZ-24 transgenic line (Kelly et al., 2001), the T4 transgenic line (Biben et al., 1996) and the c -actin nlaacZ1.1/+ mouse line (Meilhac et al., 2003) have been described previously. ...
... line (Meilhac et al., 2003). In this line, the reporter has been introduced into an allele of the cardiac actin gene, which is expressed throughout the myocardium (Fig. 1E) and also in all developing skeletal muscles ( Fig. 1F) (Biben et al., 1996;Sassoon et al., 1988). This gene therefore provides an appropriate endpoint for clonal analysis of these tissues. ...
Head muscle progenitors in pharyngeal mesoderm are present in close proximity to cells of the second heart field and show overlapping patterns of gene expression. However, it is not clear whether a single progenitor cell gives rise to both heart and head muscles. We now show that this is the case, using a retrospective clonal analysis in which an nlaacZ sequence, converted to functional nlacZ after a rare intragenic recombination event, is targeted to the alpha(c)-actin gene, expressed in all developing skeletal and cardiac muscle. We distinguish two branchiomeric head muscle lineages, which segregate early, both of which also contribute to myocardium. The first gives rise to the temporalis and masseter muscles, which derive from the first branchial arch, and also to the extraocular muscles, thus demonstrating a contribution from paraxial as well as prechordal mesoderm to this anterior muscle group. Unexpectedly, this first lineage also contributes to myocardium of the right ventricle. The second lineage gives rise to muscles of facial expression, which derive from mesoderm of the second branchial arch. It also contributes to outflow tract myocardium at the base of the arteries. Further sublineages distinguish myocardium at the base of the aorta or pulmonary trunk, with a clonal relationship to right or left head muscles, respectively. We thus establish a lineage tree, which we correlate with genetic regulation, and demonstrate a clonal relationship linking groups of head muscles to different parts of the heart, reflecting the posterior movement of the arterial pole during pharyngeal morphogenesis.
... In Xenopus, microinjected copies of the gene require the proximal CArG box and a distal myogenic factor binding site for expression in embryo explants that have been induced to form skeletal muscle (Mohun et al., 1989b). In transgenic mouse embryos, a proximal enhancer of the mouse gene, including the four CArG boxes, is sufficient for correct but weak expression of a transgene, while more distal sequences are required for appropriate expression in the adult (Biben et al., 1996). Since the cardiac-actin gene is activated during formation of both cardiac and skeletal muscle in vertebrates, its study may also shed light on the mechanisms that distinguish terminal differentiation in these two muscle types. ...
... In transgenic mice, the proximal portion of the cardiac-actin promoter is sufficient for proper expression in embryos but inadequate for appropriate expression in postnatal mice (Biben et al., 1996). Our results are strikingly similar. ...
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.
... Thus the mouse dystrophin muscle promoter contains the required cis-acting sequences to target restricted sections of the heart but requires the enhancer to target the skeletal muscle. Other muscle-specific genes such as cardiac actin (Biben et al., 1996), myosin light chain 3F (Alonso et al., 1995), and troponin C (Par- Fig. 4. Expression patterns in mice that express the BE3 transgene. In the BE3 construct, lacZ expression is driven by a cartridge that includes the mouse dystrophin muscle promoter (green box) and the 500-bp enhancer containing fragment (blue box) that is located upstream. ...
... Other muscle-specific enhancers that target skeletal muscle in transgenic mice include the myosin light chain (MLC) 1/3 (Rosenthal et al., 1989;Wenthworth et al., 1991), muscle creatine kinase (MCK) (Amacher et al., 1993;Shield et al., 1996;Donoviel et al., 1996), and the distal enhancer of the cardiac alpha actin (Biben et al., 1996). The MLC, MCK, and dystrophin enhancers feature an E-box consensus 5Ј-AACAc/g c/g TGC a/t that is paired to a second E-box consensus 5Ј-GG a/c CANGTGGc/gN a/g. ...
Duchenne muscular dystrophy is a muscle wasting disease that results from a dystrophin deficiency in skeletal and cardiac muscle. Studies concerning the regulatory elements that govern dystrophin gene expression in skeletal and/or cardiac muscle in both mouse and human have identified a promoter and an enhancer located in intron 1. In transgenic mice, the muscle promoter alone targets the expression of a lacZ reporter gene only to the right ventricle of the heart, suggesting the need for other regulatory elements to target skeletal muscle and the rest of the heart. Here we report that the mouse dystrophin enhancer from intron 1 can target the expression of a lacZ reporter gene in skeletal muscle as well as in other heart compartments of transgenic mice. Our results also suggest that sequences surrounding the mouse dystrophin enhancer may affect its function throughout mouse development.
... As a result of a genetic analysis of actin genes, a mutation in the cardiac actin locus of BALB/c mice led to new insights into an upstream regulatory region of the cardiac actin gene [16]. Later, we isolated distinct skeletal and cardiac muscle enhancer sequences located at the 5 ′ -end of the gene [17]. In the 1980s, initial studies on the transcriptional regulation of muscle genes depended on transcriptional "run on" experiments in the C2 cell line and then transfection of differentiating cultured cells with reporter plasmids controlled by candidate regulatory sequences. ...
I joined François Gros' laboratory as a postdoc at the end of 1971 and continued working with him as a research scientist until 1987, when I became an independent group leader at the Institut Pasteur. In the early 1970s, it was the beginning of research in his lab on muscle cell differentiation, as a model eukaryotic system for studying mRNAs and gene regulation. In this article, I recount our work on myogenesis and mention the other research themes in his lab and the people concerned. I remained in close contact with François and pay tribute to him as a major figure in French science and as my personal mentor who provided me with constant support.
... Mouse embryo culture E8.5 embryos from wild-type [Swiss] mice, or from the T4-nlacZ [Swiss] transgenic line (Biben et al., 1996) were collected, transferred to Hank's solution and labelled by injection of a lipophilic carbocyanine (Interchim, France) as described previously (Domínguez et al., 2012). Symmetrical dye injections were done at the right and left venous pole of the embryo using DiO and DiI to distinguish them. ...
How left-right patterning drives asymmetric morphogenesis is unclear. Here, we have quantified shape changes during mouse heart looping, from 3D reconstructions by HREM. In combination with cell labelling and computer simulations, we propose a novel model of heart looping. Buckling, when the cardiac tube grows between fixed poles, is modulated by the progressive breakdown of the dorsal mesocardium. We have identified sequential left-right asymmetries at the poles, which bias the buckling in opposite directions, thus leading to a helical shape. Our predictive model is useful to explore the parameter space generating shape variations. The role of the dorsal mesocardium was validated in Shh-/- mutants, which recapitulate heart shape changes expected from a persistent dorsal mesocardium. Our computer and quantitative tools provide novel insight into the mechanism of heart looping and the contribution of different factors, beyond the simple description of looping direction. This is relevant to congenital heart defects.
... The formation of cardiac muscle (23) depends on a combination of more widely expressed transcription factors-such as NK homeobox protein 2-5 (Nkx2-5), GATA DNA binding factor 4 (Gata4), and members of the Mef2 family-that activate downstream striated muscle genes, some of which encode proteins also present in skeletal muscle. In this case, regulation often depends on skeletal or cardiac-specific enhancers, as for the α-cardiac actin gene (24). Expression of cardiac or skeletal muscle-specific isoforms of the same gene family also distinguish the two striated muscles (11). ...
Skeletal muscle in vertebrates is formed by two major routes, as illustrated by the mouse embryo. Somites give rise to myogenic progenitors that form all of the muscles of the trunk and limbs. The behavior of these cells and their entry into the myogenic program is controlled by gene regulatory networks, where paired box gene 3 (Pax3) plays a predominant role. Head and some neck muscles do not derive from somites, but mainly form from mesoderm in the pharyngeal region. Entry into the myogenic program also depends on the myogenic determination factor (MyoD) family of genes, but Pax3 is not expressed in these myogenic progenitors, where different gene regulatory networks function, with T-box factor 1 (Tbx1) and paired-like homeodomain factor 2 (Pitx2) as key upstream genes. The regulatory genes that underlie the formation of these muscles are also important players in cardiogenesis, expressed in the second heart field, which is a major source of myocardium and of the pharyngeal arch mesoderm that gives rise to skeletal muscles. The demonstration that both types of striated muscle derive from common progenitors comes from clonal analyses that have established a lineage tree for parts of the myocardium and different head and neck muscles. Evolutionary conservation of the two routes to skeletal muscle in vertebrates extends to chordates, to trunk muscles in the cephlochordate Amphioxus and to muscles derived from cardiopharyngeal mesoderm in the urochordate Ciona, where a related gene regulatory network determines cardiac or skeletal muscle cell fates. In conclusion, Eric Davidson's visionary contribution to our understanding of gene regulatory networks and their evolution is acknowledged.
... Interestingly, transgenic experiments using different fragments of the promoter region of the mouse cardiac actin gene inserted usptream of a LacZ reporter gene showed a transgene expression in the trigeminal and facial acoustic ganglia at 10.5 d.p.c. (9). Yet, the endogenous α-skeletal or cardiac actin transcripts have not been detected suggesting that either the promoters of both cardiac and skeletal actin are subject to similar position effects or low level of transcripts of both genes may have been missed while detection of β-galactosidase activity is extremely sensitive. ...
Spatially and temporally regulated somatic mutations can be achieved by using the Cre/LoxP recombination system of bacteriophage
P1. In order to develop gene knockouts restricted to striated muscle, we generated a transgenic mouse line expressing Cre
recombinase under the control of the human α-skeletal actin promoter. Specific excision of a loxP-flanked gene was demonstrated
in striated muscle, heart and skeletal muscle, in a pattern very similar to the expression of the endogenous α-skeletal actin
gene. Therefore, the reported transgenic line can be used to target inactivation or activation of a given gene to the skeletal
muscle lineage.
... However, the quantitative ratio need not reflect the situation in vivo, because certain long range interactions may have been lost in the reporter constructs. The mouse actin gene has been shown to be regulated by cardiac-and smooth muscle-specific enhancer elements located 7-10 kb upstream of the transcription start (29). Moreover, the proximity of the 1a and 1c promoters may lead to cross-interaction of the regulatory factors, while in the constructs used in this work the two regions were separated. ...
The sodium-calcium exchange activity is mediated by proteins encoded in a small gene family, of which the geneNCX1 is ubiquitously expressed in mammalian tissues. In this study, the multipartite promoter of this gene was analyzed in the
human and rat genomes by means of DNA cloning, reverse transcriptase-polymerase chain reaction, and transient transfection
of fusion constructs with the firefly luciferase gene into cultured rat aortic smooth muscle cells. The gene-proximal promoter,
located 30 kilobase pairs (kb) away from the first coding exon 2, has features of a GC-rich housekeeping promoter and is apparently
always active; in specific tissues, however, it is augmented by one or two additional promoters, located either within 1.5
kb upstream of it, or 35 kb upstream. The gene proximal promoter shows the highest activity in aortic smooth muscle cells.
In mammalian species transcripts from all three promoters undergo splicing via an intermediate, containing two noncoding exons,
of which the downstream one is normally not present in the terminal splicing product.
... 21 The T4 mouse line expresses an nlacZ reporter under the regulation of 5′ sequences of the α-cardiacactin gene that lead to expression throughout the myocardium. 22 These mouse lines are all on a mixed genetic background (mainly C57B6/DBA2/129/SJL). Embryonic day (E) 0.5 was counted from the appearance of the vaginal plug. ...
Rationale:
Genetic tracing experiments and cell lineage analyses are complementary approaches that give information about the progenitor cells of a tissue. Approaches based on gene expression have led to conflicting views about the origin of the venous pole of the heart. Whereas the heart forms from 2 sources of progenitor cells, the first and second heart fields, genetic tracing has suggested a distinct origin for caval vein myocardium, from a proposed third heart field.
Objective:
To determine the cell lineage history of the myocardium at the venous pole of the heart.
Methods and results:
We used retrospective clonal analyses to investigate lineage segregation for myocardium at the venous pole of the mouse heart, independent of gene expression.
Conclusions:
Our lineage analysis unequivocally shows that caval vein and atrial myocardium share a common origin and demonstrates a clonal relationship between the pulmonary vein and progenitors of the left venous pole. Clonal characteristics give insight into the development of the veins. Unexpectedly, we found a lineage relationship between the venous pole and part of the arterial pole, which is derived exclusively from the second heart field. Integration of results from genetic tracing into the lineage tree adds a further temporal dimension to this reconstruction of the history of venous myocardium and the arterial pole.
... Interestingly, the P1 region is contained in a highly conserved region between OlMA1 and Fugu α-Sk1 (Fig. 6B, Venkatesh et al., 1996), suggesting that a mechanism for skeletal muscle-specific expression of skeletal actin gene is conserved in Fugu and medaka. Biben et al. (1996) have succeeded in roughly locating separate but partly overlapping enhancer regions for skeletal and cardiac muscle-specific expression of the murine cardiac actin gene during embryogenesis. The regions consist of two distal enhancer elements (0.8 and 1.9 kb long) and a proximal promoter (0.7 kb). ...
We isolated two striated muscle actin genes from medaka Oryzias latipes. OIMA1 is a skeletal muscle actin gene expressed in somitic muscle and head muscle and OIMA2 is probably a cardiac muscle actin gene expressed in both somitic and cardiac muscle. The differential transcription mechanisms for these two genes were examined in embryos by introducing fusion genes in which the OIMA1 or OIMA2 upstream region was connected to the green fluorescent protein gene. Embryos were injected with these fusion genes at the 2-cell stage. A fusion gene containing the region up to -949 of OIMA1 exhibited strong expression in somitic muscle. The coexistence of two regions, -949/-662 and -421/-201, is necessary for skeletal muscle-specific expression of OIMA1. Two E boxes and other unidentified sequences cooperatively function to achieve the full activity of the enhancer -949/-662. As for OIMA2, the region up to -520 is sufficient for strong muscle-specific expression. The region between -520 and -174 of OIMA2 is necessary for specific expression in both skeletal and cardiac muscles. In addition to the CArG box located at -140, an E-box at -430 is important for the expression in cardiac muscle as well as skeletal muscle. When the enhancers for the two muscle actin genes were switched and combined with each other's promoter, they were able to upregulate tissue-specific expression according to their origin. These results suggest that distinct expression patterns of OIMA land OIMA2 are regulated by combination of regulatory modules, each of which contains multiple regulatory elements.
... In developing muscle cells, aSMA, aCAA and aSKA are sequentially expressed in a coordinated fashion [Cox et al., 1990;Biben et al., 1996;Lancioni et al., 2007]. This actin isoform switch has been well studied in ex vivo differentiating myoblasts. ...
The dynamic actin cytoskeleton, consisting of six actin isoforms in mammals and a variety of actin binding proteins is essential for all developmental processes and for the viability of the adult organism. Actin isoform specific functions have been proposed for muscle contraction, cell migration, endo- and exocytosis and maintaining cell shape. However, these specific functions for each of the actin isoforms during development are not well understood. Based on transgenic mouse models, we will discuss the expression patterns of the six conventional actin isoforms in mammals during development and adult life. Ablation of actin genes usually leads to lethality and affects expression of other actin isoforms at the cell or tissue level. A good knowledge of their expression and functions will contribute to fully understand severe phenotypes or diseases caused by mutations in actin isoforms.
... This switch in striated actin isoforms is mediated during development by control regions present in transcriptional regulatory sequences (2). When linked to a -galactosidase reporter, the proximal promoter of the mouse cardiac actin gene produces low level but specific in vivo expression that mimics the developmental down-regulation of the endogenous cardiac actin gene (3). ...
Cardiac alpha-actin is activated early during the development of embryonic skeletal muscle and cardiac myocytes. The gene product remains highly expressed in adult striated cardiac muscle yet is dramatically reduced in skeletal muscle. Activation and repression of cardiac alpha-actin gene activity in developing skeletal muscle correlates with changes in the relative content of the four myogenic regulatory factors. Cardiac alpha-actin promoter activity, assessed in primary chick myogenic cultures, was activated by endogenous myogenic regulatory factors but was inhibited in the presence of co-expressed MRF4. By exchanging N- and C-terminal domains of MRF4 and MyoD, the N terminus of MRF4 was identified as the mediator of repressive activity, revealing a novel negative regulatory role for MRF4. The relative ratios of myogenic regulatory factors may have fundamental roles in selecting specific muscle genes for activation and/or repression.
Somatic gene therapy represents a promising approach for the treatment of inherited and acquired heart diseases. In contrast to final stadium cancer or rare inherited diseases, established pharmacological, catheter-based or surgical alternatives for treatment of cardiac disorders frequently exist. Therefore, the safety of the vector system and its application requires careful consideration in cardiac gene therapy approaches. An ideal vector system should be immunologically tolerated, allow gene expression specifically in the myocardium, and should be administered with high efficacy as specific as possible.
Actin is the central building block of the actin cytoskeleton, a highly regulated filamentous network enabling dynamic processes of cells and simultaneously providing structure. Mammals have six actin isoforms that are very conserved and thus share common functions. Tissue-specific expression in part underlies their differential roles, but actin isoforms also coexist in various cell types and tissues, suggesting specific functions and preferential interaction partners. Gene deletion models, antibody-based staining patterns, gene silencing effects, and the occurrence of isoform-specific mutations in certain diseases have provided clues for specificity on the subcellular level and its consequences on the organism level. Yet, the differential actin isoform functions are still far from understood in detail. Biochemical studies on the different isoforms in pure form are just emerging, and investigations in cells have to deal with a complex and regulated system, including compensatory actin isoform expression.
Different types of regulatory genes are involved in cardiac muscle development and cardiac gene regulation, including ubiquitous factors, such as SRF, SP1 and TEF-1, and genes coding for specific transcription factors: MADS-box transcription factor genes, such as the MEF2 genes which are also involved in the specification of skeletal and smooth muscle, homeobox genes, such as Nkx2.5, zinc-finger genes of the GATA family, such as GATA 4–6, and bHLH genes, such as dHAND and eHAND [1,2]. Gene regulation seems to require combinatorial interactions between cardiac-specific and ubiquitous factors: for example a physical interaction between Nkx2.5 and SRF is involved in the activation of the cardiac a-actin gene [3]. The study of cardiac gene regulation is complicated by the specific pattern of transcription of each gene, both with respect to temporal specificity during development and spatial specificity in the various heart chambers, presumably reflecting a modular regulation via multiple cis-acting elements [4]. Multiple approaches, including promoter analyses in cultured cells, in adult heart and in transgenic mice, are required to dissect the activity in time and space of cardiac regulatory genes and their combinatorial interactions.
In this essay I trace my own research experience as a developmental biologist, from the study of cell differentiation in vitro to tissue formation and regeneration in vivo. Beginning with a thesis on histone modifications, I went on to study gene regulation during myogenesis, first in muscle cells in culture and then in the mouse embryo. Later, we also worked on muscle regeneration in the adult. Our work on striated muscle genes also led us into the field of cardiogenesis and characterization of cardiac progenitor cells that form the heart. Comments on the state of the art—changing concepts and the technological advances that underlie scientific progress—accompany this account, with concluding remarks about future directions.
The vertebrate heart is composed of a series of distinct morphological compartments which are established in the early embryo and remodelled during subsequent cardiogenesis. Transgenic mouse studies have shown that these compartments, including right and left cardiac chambers, correspond to a series of independant transcriptional units. These results, supported by the transiently compartmentalised expression profiles of a subset of endogenous cardiac genes, reveal a modular basis to transcription in the myocardium. Regionalised expression domains provide informative markers with which the contribution of different regions of the embroyonic heart to particular structures of the adult heart can be followed during normal cardiogenesis, and, using mouse models of cardiac malformations, during abnormal heart development. Congenital heart disease in man is frequently the result of abberant interactions between different cardiac compartments. Regionalisation within the myocardium becomes evident at the time of cardiac looping, and is the product of two patterning inputs: firstly position along the anterior-posterior axis of the early heart tube, and secondly embryonic laterality signals which dictate cardiac asymmetry. Recent experiments have begun to elucidate the role of different cardiac transcription factors in directing regionalised expression domains in the embryonic heart.
This chapter summarizes the current understanding of the role of three GATA family transcription factors, GATA-4,-5, and -6, in cardiovascular development. It is found that GATA family members expressed in temporally distinct patterns in the developing mammalian heart. In addition, GATA-6 is expressed in both arterial and venous SMCs, suggesting a potential role for this protein in the development of the vasculature. GATA-4,-5, and -6 can each bind to the transcriptional regulatory regions of multiple cardiac promoters and enhancers, and transactivate these transcriptional regulatory elements in nonmuscle cells. Each of these factors share two unique and independent transcriptional activation domains. Gene targeting experiments have demonstrated a necessary role for GATA-4 in regulating the second stage of cardiac development in the migration of specified procardiomyocytes from the dorsolateral region of the embryo to form the ventral linear heart tube. Chimera experiments suggest that GATA-4 is required to initiate and/or maintain the ventral morphogenic signal rather than to act as a regulator of cardiomyocyte responsiveness to this signal. GATA-6 is also required for early embryonic viability and its precise role in cardiovascular development is currently under investigation. In contrast, GATA-5 does not appear to be required for the development of the heart and vasculature. Finally, preliminary evidence suggests that GATA-4 and GATA-6 may belong to a common developmental pathway in which GATA-4 normally down regulates the expression of GATA-6 in the heart and extra embryonic membranes.
The congenital myopathies are a clinically, genetically, and pathologically heterogeneous group of muscle disorders defined by muscle weakness usually present at birth and characteristic morphological features on muscle biopsy. The advent of histochemistry and electron microscopy in the 1960s led to the emergence of the congenital myopathies as a group, distinct from other causes of early-onset muscle weaknesses such as the congenital muscular dystrophies (CMD). The clinical features vary, but “typical” features of the early onset forms are hypotonia, generalized muscle weakness, poor muscle bulk, feeding difficulties, and skeletal abnormalities developing secondary to the muscle weakness, such as a high-arched palate, pectus excavatum, kyphoscoliosis, and hip dysplasia. Congenital myopathies can be inherited as autosomal dominant (AD), autosomal recessive (AR), or X-linked disorders, or may arise through de novo mutations. The identification of gene defects for the congenital myopathies allows exploring the molecular pathogenesis in multiple model systems. Despite the genetic advances in the congenital myopathies, as with the genetic advances in the muscular dystrophies, there are still no curative treatments. Developing effective treatments must be a major research focus for the future. Advances in the supportive treatment of congenital myopathy patients have nevertheless been considerable, especially in the area of assisted ventilation.
The D-1A dopamine receptor gene consists of a short, noncoding exon 1 separated from a longer coding exon 2 by a small intron, Recently, we found that in addition to its original TATA-less promoter located upstream of exon 1, the human D-1A dopamine receptor gene is transcribed in neural cells from a second strong promoter located In its intron, In the present study, we addressed the possibility that these two promoters are used for the tissue-specific regulation of the D-1A gene in neuronal and renal cells, Reverse transcription polymerase chain reaction revealed that D-1A transcripts in the kidneys of humans and rats lack exon 1, Transient transfection analysis of these two promoters in D-1A-expressing cells indicated that the upstream promoter has no detectable activity in the opossum kidney (OK) cell line, in contrast to its strong activity in two neuronal cell lines, SK-N-MC and NS20Y, On the other hand, the D-1A intron promoter showed transcriptional activity both in OK cells and in neuronal cells, The activator sequence AR1, which enhances transcription from the upstream promoter in SK-N-MC and NS20Y cells, could not activate this promoter in OK cells, In addition, no protein binding to AR1 could be detected by gel mobility shift assay using nuclear extracts from either OK cells or from rat kidney tissue, These findings indicate that the differential expression of short and long D-1A transcripts is due, at least in part, to the tissue-specific expression of the activator protein binding to AR1 driving transcription from the upstream promoter, Absence of this activator protein accounts for the nonfunctional D-1A upstream promoter in the kidney.
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To investigate seven congenital myopathy patients from six families: one French Gypsy, one Spanish Gypsy, four British Pakistanis, and one British Indian. Three patients required mechanical ventilation from birth, five died before 22 months, one is ventilator-dependent, but one, at 30 months, is sitting with minimal support. All parents were unaffected.
The alpha-skeletal muscle actin gene (ACTA1) was sequenced. Available muscle biopsies were investigated by standard histological and electron microscopic techniques. The expression of various proteins was determined by immunohistochemistry, western blotting, or both.
Three homozygous ACTA1 null mutations were identified: p.Arg41X in the French patient, p.Tyr364fsX in the Spanish patient, and p.Asp181fsX10 in all five British patients. An absence of alpha-skeletal muscle actin protein but presence of alpha-cardiac actin was shown in all muscle biopsies examined, with more alpha-cardiac actin in the biopsy from the child with the greatest muscle function. Muscle biopsies from all patients exhibited nemaline bodies whereas three also contained zebra bodies.
The seven patients have recessive nemaline myopathy caused by absence of alpha-skeletal muscle actin. The level of retention of alpha-cardiac actin, the skeletal muscle fetal actin isoform, may determine alpha-skeletal muscle actin disease severity. This has implications for possible future therapy.
Myocardial genes tend to be coexpressed throughout the embryonic heart at the earliest stages of heart tube formation. Atrial myosin heavy chain (MHC) transcripts are expressed predominantly in the posterior heart tube as the cardiac primordia fuse. In the mouse, significant regionalization of gene expression within the developing heart emerges from the time of the cardiac looping. The temporal profile of chamber-specific restriction of cardiac genes is highly variable: Some markers are restricted early in development, for example, the regulatory myosin light chain 2V gene, which is predominantly expressed in the primitive ventricle. Other myosin genes encoding alkali MLCs continue to be expressed throughout the myocardium after looping. A subset of markers appear to be initially coexpressed in a graded manner; the MHC genes, for example are expressed in inverse gradients in the embryonic heart, with αMHC being expressed at a higher level at the arterial pole and βMHC at the venous pole of the tubular heart. The βMHC gene shows relatively early restriction of transcription, becoming confined predominantly to the ventricular compartment by E10.5 in the mouse. MLC1V transcripts, in contrast, continue to be detectable in atrial cells until E15 and MLC1A transcripts remain detectable in the ventricles even after birth. Changes in myocardial gene expression do not necessarily occur in a synchronized fashion even within a cardiac compartment.
The mouse Cer1 (mCer1, Cer-l, Cerr1) gene encodes one member of a family of cytokines structurally and functionally related to the Xenopus head-inducing factor, Cerberus (xCer). We generated a mouse line in which the Cer1 gene was inactivated by replacing the first coding exon with a lacZ reporter gene. Mice homozygous for this allele (Cer1lacZ) showed no apparent perturbation of embryogenesis or later development. However, the lacZ reporter revealed a number of hitherto uncharacterised sites of Cer1 expression in late fetal and adult tissues. Preliminary analysis suggests that Cer1 is not essential for their morphogenesis, differentiation, or homeostasis. genesis 26:259–264, 2000. © 2000 Wiley-Liss, Inc.
The embryonic heart consists of five segments comprising the fast-conducting atrial and ventricular segments flanked by slow-conducting segments, i.e. inflow tract, atrioventricular canal and outflow tract. Although the incorporation of the flanking segments into the definitive atrial and ventricular chambers with development is generally accepted now, the contribution of the outflow tract myocardium to the definitive ventricles remained controversial mainly due to the lack of appropriate markers. For that reason we performed a detailed study of the pattern of expression of myosin light chain (MLC) 2a and 2v by in situ hybridization and immunohistochemistry during rat and mouse heart development. Expression of MLC2a mRNA displays a postero-anterior gradient in the tubular heart. In the embryonic heart it is down-regulated in the ventricular compartment and remains high in the outflow tract, atrioventricular canal, atria and inflow tract myocardium. MLC2v is strongly expressed in the ventricular myocardium and distinctly lower in the outflow tract and atrioventricular canal. The co-expression of MLC2a and MLC2v in the outflow tract and atrioventricular canal, together with the single expression in the atrial (MLC2a) and ventricular (MLC2v) myocardium, permits the delineation of their boundaries. With development, myocardial cells are observed in the lower endocardial ridges that share MLC2a and MLC2v expression with the myocardial cells of the outflow tract. In neonates, MLC2a continues to be expressed around both right and left semilunar valves, the outlet septum and the non-trabeculated right ventricular outlet. These findings demonstrate the contribution of the outflow tract to the definitive ventricles and demonstrate that the outlet septum is derived from outflow tract myocardium. Anat Rec 254:135–146, 1999. © 1999 Wiley-Liss, Inc.
One of the earliest, and most crucial, steps in cardiac morphogenesis is looping of the primitive heart tube. This process provides the spatial context for all future steps in heart development, and defects in cardiac loop formation result in complex structural heart disease. Loop formation is also one of the first visible asymmetries along the left-right body axis, and as such is dependent upon the development of the entire embryonic left-right axis. Formation of the left-right body axis is a developmental process, that, like the formation of the anteroposterior and dorsoventral axes, is programmed by multiple genetic steps. It is also a process that is essential and unique to vertebrate development. Therefore, an understanding of the development of left-right asymmetry at the genetic and molecular level will greatly aid in the understanding of the etiology of this complex group of congenital heart defects.
Transgenesis provides a means to modify the mammalian genome. By directing expression of a engineered protein to the heart, one now is able to remodel effectively the cardiac protein profile and study the consequences of a single genetic manipulation at the molecular, biochemical, cytological, and physiologic levels. Often, a particular pathology or even a global remodeling process such as hypertrophy is accompanied by the upregulation or downregulation of a gene or set(s) of genes. What is not known is whether these changes represent a beneficial compensatory response or contribute to the continued degeneration of normal heart function. The ability to perform genetic manipulations on cardiac gene expression via transgenesis offers one a rapid and effective means of extending the correlations noted to the mechanistic level. Now, one can, in theory, express a candidate protein at a particular developmental time and determine the direct consequences of its appearance. Similarly, one can explore structure-function relationships, both between different forms of a protein family and in terms of active domains within a protein, by expressing a transgene that encodes a suitable mutation or ectopic protein isoform. This review explores the practical considerations of the transgenic approach in terms of what is important for a successful experiment from the necessary animal husbandry to designing constructs that will express at appropriate levels in the heart. (Trends Cardiovasc Med 1997;7:185–191). © 1997, Elsevier Science Inc.
This study demonstrates a novel method by which multiple separate plasmids can be stably integrated into the genome using single antibiotic resistance for selection. This method was used to integrate three different cardiac-specific promoters driving different fluorophores into murine embryonic stem cells, allowing sequential visualization of various stages of cardiac differentiation. This method is broadly applicable to the study of cell lineage of different stem/progenitor cells.
Application of molecular genetic tools to inherited cardiovascular disorders has provided important insights into the molecular mechanisms underlying cardiomyopathies, arrhythmias, blood pressure regulation, and atherosclerosis. In addition, alteration of gene expression has been observed under common cardiovascular conditions such as cardiac hypertrophy and heart failure. Recent advances in transgenic and gene-targeting approaches allow a sophisticated manipulation of the mouse genome by gene addition, gene deletion, or gene modifications. These transgenic models enable the dissection of in vivo pathways responsible for these complex disease phenotypes. This review describes tissue-specific promoters suitable for targeting candidate genes to the cardiovascular system as well as a number of valuable transgenic animal models of blood pressure regulation, atherogenesis, defects in the coagulation system, cardiac hypertrophy, myocarditis, cardiomyopathies, and heart failure. Limitations and difficulties associated with these transgenic approaches are discussed. Animal models which may provide a basis for future gene therapy of cardiovascular diseases are introduced. Finally, methods are described to regulate the spatial and temporal expression level of a transgene, to inactivate a target gene in a tissue-specific manner, and to introduce specific mutations into the genome. These recent advances in transgenic technology are expected to have a considerable impact on cardiovascular research in the near future.
Carbonic anhydrase II localization was studied in mouse embryonic and fetal hearts for better understanding of the functions of this enzyme during cardiac organogenesis. Immunocytolabelling was performed on serial sections of frozen hearts after one night's fixation in 4% paraformaldehyde. In the earliest stages studied, 10, 11 and 12 ed (ed = embryonic day; vaginal plug = day 1), a sharp decrease of labelled cells was observed in the endocardium form which cushion-tissue mesenchyme is derived. During the same period, differences in the decreasing frequencies of labelled cells were also observed between three different cushion-tissue mesenchyme localizations: immunostained cells were abundant in the atrioventricular cushions, less numerous in the proximal part of the conotruncal ridges and rare in their distal part. From 13 ed their repartition was more regular along the conotruncus. From 13 to 16 ed the signal was also present in a peculiar region of the myocardium: the anterior and left walls of the left ventricle. At the 18 and 20 ed labelling was found only in some endothelial cells of coronary vessels, particularly in the interventricular septum. The pattern of expression of carbonic anhydrase II in activated endothelial cells and endothelial-derived mesenchyme cells of the cardiac cushion tissue, strongly suggests that this isoenzyme can be a useful marker for a subpopulation of endothelial cells and cells derived from this endothelium that morphologically express signs of active cell behavior (e.g., invasion, migration, proliferation).
Looping of the primitive heart tube is one of the earliest and most crucial steps in cardiac morphogenesis. Cardiac looping is dependent on normal left-right development, and defects in left-right development result in both heterotaxia and complex congenital heart disease. Single gene defects result in the wide spectrum of heterotaxy phenotypes, and conversely, different gene defects result in similar heterotaxy phenotypes. Elucidation of the molecular-genetic mechanisms of left-right development will greatly increase our understanding of the etiology of this complex group of congenital heart defects.
Within the embryonic heart, five segments can be distinguished: two fast-conducting atrial and ventricular compartments flanked by slow-conducting segments, the inflow tract, the atrioventricular canal, and the outflow tract. These compartments assume morphological identity as a result of looping of the linear heart tube. Subsequently, the formation of interatrial, interventricular, and outflow tract septa generates a four-chambered heart. The lack of markers that distinguish right and left compartments within the heart has prevented a precise understanding of these processes. Transgenic mice carrying an nlacZ reporter gene under transcriptional control of regulatory sequences from the MLC1F/3F gene provide specific markers to investigate such regionalization. Our results show that transgene expression is restricted to distinct regions of the myocardium: beta-galactosidase activity in 3F-nlacZ-2E mice is confined predominantly to the embryonic right atrium, atrioventricular canal, and left ventricle, whereas, in 3F-nlacZ-9 mice, the transgene is expressed in both atrial and ventricular segments (right/left) and in the atrioventricular canal, but not in the inflow and outflow tracts. These lines of mice illustrate that distinct embryonic cardiac regions have different transcriptional specificities and provide early markers of myocardial subdivisions. Regional differences in transgene expression are not detected in the linear heart tube but become apparent as the heart begins to loop. Subsequent regionalization of transgene expression provides new insights into later morphogenetic events, including the development of the atrioventricular canal and the fate of the outflow tract.
Skeletal, cardiac and smooth muscle cells express overlapping sets of muscle-specific genes, such that some muscle genes are expressed in only a single muscle cell lineages. Recent studies in transgenic mice have revealed that, in many cases, multiple, independent cis-regulatory regions, or modules, are required to direct the complete developmental pattern of expression of individual muscle-specific genes, even within a single muscle cell type. The temporospatial specificity of these myogenic regulatory modules is established by unique combinations of transcription factors and has revealed unanticipated diversity in the regulatory programs that control muscle gene expression. This type of composite regulation of muscle gene expression appears to reflect a general strategy for the control of cell-specific gene expression.
Recent discoveries have led to a greater appreciation of the diverse mechanisms that underlie cardiac morphogenesis. Genetic strategies (primarily gene targeting approaches in mice) have significantly broadened research in cardiovascular developmental biology by illuminating new pathways involved in heart development and by allowing the genetic evaluation of pathways that have previously been implicated in these events. Advances have also been made using biochemical and cell- and tissue-based approaches. This review summarizes the author's interpretation of current trends in the effort to understand the molecular basis of cardiac-development, with an emphasis on insights obtained from genetic models.
The heart is the first embryonic organ to function. Early in development, the heart shows autorhythmycity and peristaltoid contraction waves [1, 2]. Contraction requires the expression of a specific set of proteins that form the contractile apparatus, i.e. the sarcomere. The contraction–relaxation cycle of the sarcomeric apparatus is mediated by changing local concentrations of free calcium. This function is achieved by another set of specific proteins, located in the sarcoplasmic reticulum and in the sarcolemma.
Fascinating questions that are still poorly understood are how the cardiogenic lineage becomes established to form the peristaltoid contracting tube without valves and how this tube becomes transformed into the synchronous-contracting four-chambered heart with unidirectional valves. It is well documented that the expression of the different isoforms of contractile proteins changes considerably during these stages (for a review see [3]). However, a detailed analysis of the changes in the patterns of gene expression in relation to cardiac morphogenesis is lacking. In the present review we try to fill this gap. We have centred our attention on gene products (mRNA and protein) expressed in the working myocardium of mammals and birds. No distinction has been made when mRNA and protein display the same pattern of expression, however we have highlighted those cases where the pattern of expression differs between mRNA and protein. The development and expression pattern of genes of the conduction system of the heart merits an independent review [4](Moorman et al., Circ. Res., in press). Data referring to other experimental models as Drosophila , Xenopus or zebrafish ( Danio rerio ) are included only if they are helpful for our general understanding. Often only gene expression in the presumptive atria and/or presumptive ventricles is mentioned, whereas understanding the functional significance of the patterns of gene expression requires knowledge of the entire pattern including the …
* Corresponding author. Tel.: +31 (20) 5664928; fax: +31 (20) 6976177; e-mail: a.f.moorman@amc.uva.nl
It has recently emerged that transcriptional differences exist between left and right cardiac chambers. An example is provided by transgenic mice with an nlacZ reporter gene under transcriptional control of the fast skeletal muscle alkali myosin light chain (MLC) 3 promoter and 3' enhancer, which express beta-galactosidase in a left ventricular-right atrial dominant pattern in the developing and adult heart. Here, we demonstrate that endogenous MLC3F transcripts are also left/right regionalised in the mouse heart during embryonic development. Regionalisation is observed as early as embryonic day (E) 8.5, and by E10.5 MLC3F transcripts are present predominantly in the future left ventricle and right atrium, and to a lesser extent in the left atrium. Subsequently, MLC3F transcripts are down-regulated in the left ventricle, and by E12.5 expression is restricted to both atria and left-ventricular trabeculae. No MLC3F protein can be detected in the adult or embryonic mouse heart, suggesting that post-transcriptional regulation prevents this fast myosin isoform contributing to myocardial contraction. Left ventricular-right atrial dominant MLC3F transgenes therefore reflect transitory left/right regionalisation of the endogenous gene, unlike other reported cases of transgene regionalisation. MLC3F transgenes, however, maintain an embryonic-like distribution throughout development suggesting that myocardial gene expression is controlled by distinct temporal, as well as spatial, regulatory modules.
The embryonic heart consists of five segments comprising the fast-conducting atrial and ventricular segments flanked by slow-conducting segments, i.e. inflow tract, atrioventricular canal and outflow tract. Although the incorporation of the flanking segments into the definitive atrial and ventricular chambers with development is generally accepted now, the contribution of the outflow tract myocardium to the definitive ventricles remained controversial mainly due to the lack of appropriate markers. For that reason we performed a detailed study of the pattern of expression of myosin light chain (MLC) 2a and 2v by in situ hybridization and immunohistochemistry during rat and mouse heart development. Expression of MLC2a mRNA displays a postero-anterior gradient in the tubular heart. In the embryonic heart it is down-regulated in the ventricular compartment and remains high in the outflow tract, atrioventricular canal, atria and inflow tract myocardium. MLC2v is strongly expressed in the ventricular myocardium and distinctly lower in the outflow tract and atrioventricular canal. The co-expression of MLC2a and MLC2v in the outflow tract and atrioventricular canal, together with the single expression in the atrial (MLC2a) and ventricular (MLC2v) myocardium, permits the delineation of their boundaries. With development, myocardial cells are observed in the lower endocardial ridges that share MLC2a and MLC2v expression with the myocardial cells of the outflow tract. In neonates, MLC2a continues to be expressed around both right and left semilunar valves, the outlet septum and the non-trabeculated right ventricular outlet. These findings demonstrate the contribution of the outflow tract to the definitive ventricles and demonstrate that the outlet septum is derived from outflow tract myocardium.
Differential regulation of cardiac gene expression in vertebrates has been extensively documented in the context of atrial and ventricular morphogenesis. Recent data, largely from the analysis of transgene and endogenous gene expression patterns, have revealed transcriptional differences between left and right cardiac chambers which suggest that the heart is composed of a series of distinct transcriptional domains. Such phenomena provide regional markers (cardiosensors) for the fine analysis of normal and abnormal heart development. Regional subdivisions and transcriptional heterogeneity within the myocardium emerge in response to patterning of the precardiac mesoderm and early heart tube on the anterior-posterior axis, and to embryonic left-right signals which dictate the direction of cardiac looping. Several families of transcription factors have been characterized which may be implicated in the regionalization of myocardial gene expression.
The detailed anatomy of muscle progenitor cells in mouse—accompanied by the expression patterns of key genes in normal and mutant mice—has led to changes in the conceptual framework that influences the current thinking about myogenesis. In particular, the importance of the localization of progenitor cell populations in relation to signaling molecules produced by surrounding tissues has become a major issue. The role of other gene families in this context has already modified our view of the genetic hierarchy that regulates myogenesis. These considerations are focus of this chapter, which deals primarily with myogenic progenitor cells in the mouse somite, with reference to other species where appropriate. The identification of the MyoD family of myogenic regulatory factors (MRFs), MyoD, Myf5, myogenin, and MRF4, their critical role in the formation of the muscle precursor cell population as well as in the differentiation of these cells in the embryo are demonstrated by gene knockout experiments. MyfS and MyoD were identified as upstream determination factors, whereas myogenin is required for most muscle cell differentiation.
Phenotypic modulation of smooth muscle cells (SMC) is a key event during the development of atherosclerotic and restenotic lesions. During this process, the composition of the cytoskeleton is substantially altered, with changes predominantly in actin expression reflecting a shift from smooth muscle alpha-actin to the non-muscle beta-isoform. We now demonstrate that yet another actin isoform, cardiac alpha-actin, is synthesized, de novo, in SMC of various atherosclerotic lesions. Using a highly specific monoclonal antibody against cardiac alpha-actin, we analyzed and compared the accumulation of this actin isoform in diverse SMC by immunofluorescence microscopy and immunoblotting. As expected, cardiac alpha-actin was present in human myocardium but not in healthy SMC of adult aorta, coronary arteries, trabeculae of the spleen, colon, stomach or skeletal muscle. Interestingly, the presence of cardiac alpha-actin was detected in umbilical cord vessels, human myometrium, in atherosclerotic coronary lesions and atherosclerotic lesions from peripheral vascular disease. The distribution of cardiac alpha-actin often paralleled that of cytokeratins 8 and 18, intermediate filament proteins typically found in dedifferentiated SMC. Taken together, the data presented here illustrate the expression of cardiac alpha-actin to be limited to either fetal vessels or those vessels or tissue having suffered damage or atrophy, outside its 'native' environment in the heart. The demonstration of cardiac alpha-actin in SMC of umbilical cord vessels and in atherosclerotic lesions but not in apparently healthy vessels supports the notion that SMC in atherosclerotic lesions exhibit a dedifferentiated phenotype.
An underpinning of basic physiology and clinical medicine is that specific protein complements underlie cell and organ function. In the heart, contractile protein changes correlating with functional alterations occur during both normal development and the development of numerous pathologies. What has been lacking for the majority of these observations is an extension of correlation to causative proof. More specifically, different congenital heart diseases are characterized by shifts in the motor proteins, and the genetic etiologies of a number of different dilated and hypertrophic cardiomyopathies have been established as residing at loci encoding the contractile proteins. To establish cause, or to understand development of the pathophysiology over an animal's life span, it is necessary to direct the heart to synthesize, in the absence of other pleiotropic changes, the candidate protein. Subsequently one can determine whether or how the protein's presence causes the effects either directly or indirectly. By affecting the heart's protein complement in a defined manner, the potential to establish the function of different proteins and protein isoforms exists. Transgenesis provides a means of stably modifying the mammalian genome. By directing expression of engineered proteins to the heart, cardiac contractile protein profiles can be effectively remodeled and the resultant animal used to study the consequences of a single, genetic manipulation at the molecular, biochemical, cytological, and physiological levels.
Isoform diversity in striated muscle is largely controlled at the level of transcription. In this review we will concentrate on studies concerning transcriptional regulation of the alkali myosin light chain 1F/3F gene. Uncoupled activity of the MLC1F and 3F promoters, together with complex patterns of transcription in developing skeletal and cardiac muscle, combine to make analysis of this gene particularly intriguing. In vitro and transgenic studies of MLC1F/3F regulatory elements have revealed an array of cis-acting modules that each drive a subset of the expression pattern of the two promoters. These cis-acting regulatory modules, including the MLC1F and 3F promoter regions and two skeletal muscle enhancers, control tissue-specificity, cell or fibre-type specificity, and the spatiotemporal regulation of gene expression, including positional information. How each of these regulatory modules acts and how their individual activites are integrated to coordinate transcription at this locus are discussed.
Cardiovascular disease and particularly ischemic disorders of the heart are the leading causes of death in Western countries. In 1996 the total mortality in Germany was 882,843; 9.7% died of acute myocardial infarction and 10.7% of congestive heart failure, which is mainly related to ischemic cardiomyopathy occurring after myocardial infarction. Depending on the severity of the disease (New York Heart Association classification) current treatment strategies include bypass operation and percutaneous transluminal coronary angioplasty (PTCA) and stunt implantation. Progressive heart failure related to ischemic cardiomyopathies can ultimately only be treated by transplantation.
See article by Gao et al. [1] (pages 197–204) in this issue.
Manipulation of the mouse genome by transgenic approaches is a powerful tool to examine gene function as well as interaction of gene products in the intact animal [2–4]. In the past, gain-in-function models were the most frequently used transgenic animals to define the physiological relevance of gene products. In these animals, transgene expression is controlled by well characterized promoter elements that drive transgene expression by their cardio-specificity, while the onset of transgene expression depends on the temporal activation of the promotor employed [5–11]. However, the temporal regulation of transgene expression might prevent investigation of a number of important questions. In its worst case, the inappropriate onset of transgene expression can interfere with proper embryonal development resulting in early lethal phenotypes [12–14]. Less dramatically but more commonly, constitutive transcriptional activity of the promoter does not provide any information on developmental aspects of transgene expression [15–17] and prevents examination of the gene dosis and phenotype [18,19]. These limitations are overcome by conditional transgenic systems allowing control of the timing as well as the spatial pattern of gene expression (for reviews, see Refs. [20,21]).
To date a number of conditional gene expression systems have been introduced [22–26]. Among them the tetracycline-regulated binary system based on the tetracycline (tet) resistance operon of E. coli has emerged as the present system …
* Corresponding author. Tel.: +49-622-156-8670; fax: +49-622-156-5516. hugo_katus{at}med.uni-heidelberg.de
The reverse genetics technologies that have recently been developed for mice have provided new tools to probe gene function in vivo. Unfortunately these powerful systems often require the analysis of large numbers of DNA samples. The gene-targeting technology requires screening of embryonic stem-cell clones and later of the mice themselves, the latter also being the case for standard transgenic technology. It is not always possible or desirable to rely on PCR analyses, necessitating the isolation of large numbers of DNA samples of sufficient quality for Southern blot analysis. We have simplified the standard mammalian DNA isolation procedure with the aim of minimizing the number of manipulations required for each sample. The basic procedure applied to cultured cells does not require any centrifugation steps or organic solvent extractions.
The cardiac alpha-actin gene is expressed in both heart and skeletal muscle. In skeletal myogenic cells, the 177-base-pair promoter of the human cardiac alpha-actin (HCA) gene requires three transcription factors for activation: Sp1, serum response factor (SRF), and MyoD. However, MyoD is undetectable in heart. To search for a functional equivalent of MyoD, we analyzed the transcriptional regulation of the HCA promoter in primary cultures of rat cardiac myocytes. The same DNA sequence elements recognized by SRF, Sp1, and MyoD and required for HCA transcription in skeletal muscle cells were also found to be necessary for expression in cardiomyocytes. Overexpression of Id, a negative regulator of basic helix-loop-helix proteins, selectively attenuated expression of the HCA promoter. Cardiomyocyte nuclei contain a protein complex that specifically interacts with the same required sequence (E box) in the HCA promoter that is bound by MyoD in skeletal myogenic cells. Furthermore, these complexes contain a peptide that is a member of the E2A family of basic helix-loop-helix proteins. Cardiomyocyte nuclei appear to be enriched for a protein that can bind to the E-box site as dimers with the E12 protein. These results suggest that a member of the basic helix-loop-helix family, together with SRF and Sp1, activates the HCA promoter in heart. Alternative strategies for myocardial transcription of HCA are discussed.
Expression of the human cardiac alpha-actin gene (HCA) depends on the interactions of multiple transcriptional regulators with promoter elements. We report here that the tissue-specific expression of this promoter is determined by the simultaneous interaction of at least three specific protein-DNA complexes. The myogenic determinant gene MyoD1 activated the transcription of transfected HCA-CAT promoter constructs in nonmuscle cells, including CV-1 and HeLa cells. Gel mobility-shift and footprinting assays revealed that MyoD1 specifically interacted with a single consensus core sequence, CANNTG, at -50. Previously characterized sites interact with a protein identical with or related to the serum response factor (SRF) at -100 and Sp1 at -70. All three elements must be intact to support transcription in muscle cells: site-specific mutation within any one of these three elements eliminated transcriptional expression by the promoter. Furthermore, expression of the promoter in embryonic Drosophila melanogaster cells that lack MyoD1 and Sp1 is strictly dependent on all three sites remaining intact and on the presence of exogenously supplied Sp1 and MyoD1. These experiments suggest that the presence of three sequence-specific binding proteins, including MyoD1, and their intact target DNA sequences are minimal requirements for muscle-specific expression of the HCA gene.
Proximal upstream flanking sequences of the mouse myosin alkali light chain gene encoding MLC1F and MLC3F, the mouse α-cardiac actin gene and the chicken gene for the α-subunit of the acetylcholine receptor were linked to the bacterial
chioramphenicol acetyl transferase (CAT) gene and transfected into primary cultures derived from mouse skeletal muscle or
into myogenic cell lines. We demonstrate that the mouse MLC1F/MLC3F gene has two functional promoters. In primary muscle cultures, a 1200 bp sequence flanking exon 1 (MLC1F) and a 438 bp sequence flanking exon 2 (MLC3F) direct CAT activity in myotubes, but not in myoblasts or in non myogenic 3T6 and CV1 cells. Developmentally regulated expression
is also seen with the α-cardiac actin (320 bp) and acetylcholine receptor α-subunit (850 bp) upstream sequences in the primary
culture system. Transfection experiments with myogenic cell lines show different results with a given promoter construct,
reflecting possible differences in the levels of regulatory factors between lines. Different muscle gene promoters behave
differently in a given cell line, suggesting different regulatory factor requirements between these promoters.
We describe the structure and transcriptional activity of the 5' portion of the alpha-cardiac actin gene of BALB/c mice. Southern blotting and DNA sequencing reveal that the promoter and first three exons of the gene are present as perfect repeats in a direct duplication of 9.5 kbp situated immediately upstream of the gene. Both promoters are active in adult cardiac tissue. Transcripts from the partial gene duplication give rise to novel RNAs that are spliced correctly in the actin region and polyadenylated. The level of mature alpha-cardiac actin mRNA is only 16.5% that found in mice that do not possess the duplication. This is due, at least in part, to interference at the transcriptional level. Transcripts from the alpha-skeletal actin gene accumulate to abnormally high levels in the hearts of such mutant mice. This result suggests tight regulatory coupling for this actin gene pair.
A short sequence of amino acids including Lys-128 is required for the normal nuclear accumulation of wild-type and deleted forms of SV40 large T antigen. A cytoplasmic large T mutant that lacks sequences from around Lys-128 localizes to the nucleus if the missing sequence is attached to its amino terminus. The implication that the sequence element around Lys-128 acts as an autonomous signal capable of specifying nuclear location was tested directly by transferring it to the amino termini of beta-galactosidase and of pyruvate kinase, normally a cytoplasmic protein. Sequences that included the putative signal induced each of the fusion proteins to accumulate completely in the nucleus but had no discernible effect when Lys-128 was replaced by Thr. By reducing the size of the transposed sequence we conclude that Pro-Lys-Lys-Lys-Arg-Lys-Val can act as a nuclear location signal. The sequence may represent a prototype of similar sequences in other nuclear proteins.
The murine homeo box gene Nkx2-5 is expressed in precardiac mesoderm and in the myocardium of embryonic and fetal hearts. Targeted interruption of Nkx2-5 resulted in abnormal heart morphogenesis, growth retardation and embryonic lethality at approximately 9-10 days postcoitum (p.c.). Heart tube formation occurred normally in mutant embryos, but looping morphogenesis, a critical determinant of heart form, was not initiated at the linear heart tube stage (8.25-8.5 days p.c.). Commitment to the cardiac muscle lineage, expression of most myofilament genes and myofibrillogenesis were not compromised. However, the myosin light-chain 2V gene (MLC2V) was not expressed in mutant hearts nor in mutant ES cell-derived cardiocytes. MLC2V expression normally occurs only in ventricular cells and is the earliest known molecular marker of ventricular differentiation. The regional expression in mutant hearts of two other ventricular markers, myosin heavy-chain beta and cyclin D2, indicated that not all ventricle-specific gene expression is dependent on Nkx2-5. The data demonstrate that Nkx2-5 is essential for normal heart morphogenesis, myogenesis, and function. Furthermore, this gene is a component of a genetic pathway required for myogenic specialization of the ventricles.
The molecular control of the differentiation process depends in part on lineage-restricted transcription factors that regulate expression of tissue-specific genes. Although significant progress has been made in molecular understanding of skeletal muscle differentiation, no information is available concerning the genes involved in development of the heart, the first organ to form in vertebrate embryos. Many vertebrate homeobox-containing genes have been shown to be expressed in broad regions of the mouse embryo, but no expression of a homeobox gene has been found in the most anterior region of the early embryo, the heart primordium. We report here on the cloning of a murine homeobox cDNA, Csx (cardiac-specific homeobox). The Csx homeodomain sequence is divergent from those of the Hox class genes but is related to that of Drosophila msh-2 (NK-4), which plays a key role in Drosophila heart formation. Csx is conserved in evolution and Csx homologs exist in all vertebrates examined. Transcripts of Csx are detected from the presomite stage (7.5 days postcoitum), when mesoderm differentiates into promyocardium. Csx expression is restricted in the myocardial cells from 8.5 days postcoitum through adult. Csx is not expressed in skeletal or smooth muscle or any other tissues examined. Expression of Csx precedes that of cardiac-specific genes in embryonic stem cells differentiating into beating myocardial cells in vitro. Although physiological function of Csx is yet to be determined, the temporal and spacial pattern of Csx expression raises a possibility that Csx may play a critical role in the differentiation of cardiac cells.
Members of the myocyte enhancer binding factor-2 (MEF2) family of MADS (MCM1, agamous, deficiens, and serum response factor)
box transcription factors are expressed in the skeletal, cardiac, and smooth muscle lineages of vertebrate and Drosophila
embryos. These factors bind an adenine-thymidine-rich DNA sequence associated with muscle-specific genes. The function of
MEF2 was determined by generating a loss-of-function of the single mef2 gene in Drosophila (D-mef2). In loss-of-function embryos,
somatic, cardiac, and visceral muscle cells did not differentiate, but myoblasts were normally specified and positioned. These
results demonstrate that different muscle cell types share a common myogenic differentiation program controlled by MEF2.
The alpha-myosin heavy-chain (alpha-MHC) gene is the major structural protein in the adult rodent myocardium. Its expression is restricted to the heart by a complex interplay of trans-acting factors and their cis-acting sites. However, to date, the factors that have been shown to regulate expression of this gene have also been found in skeletal muscle cells. Recently, transcription factor GATA-4, which has a tissue distribution limited to the heart and endodermally derived tissues, was identified. We recently found two putative GATA-binding sites within the proximal enhancer of the alpha-MHC gene, suggesting that GATA-4 might regulate its expression. In this study, we establish that GATA-4 interacts with the alpha-MHC GATA sites to stimulate cardiac muscle-specific expression. Mutation of the GATA-4-binding sites either individually or together decreased activity by 50 and 88% in the adult myocardium, respectively. GATA-4-dependent enhancement of activity from a heterologous promoter was mediated through the alpha-MHC GATA sites. Coinjection of an alpha-MHC promoter construct with a GATA-4 expression vector permitted ectopic expression in skeletal muscle but not in fibroblasts. Thus, the lack of alpha-MHC expression in skeletal muscle correlates with a lack of GATA-4. GATA-4 DNA binding activity was significantly up-regulated in triiodothyronine- or retinoic acid-treated cardiomyocytes. Putative GATA-4-binding sites are also found in the regulatory regions of other cardiac muscle-expressed structural genes. This indicates a mechanism whereby triiodothyronine and retinoic acid can exert coordinate control of the cardiac phenotype through a trans-acting regulatory factor.
In order to elucidate mechanisms involved in striated muscle contractile protein isoform expression, we have defined regulatory elements in the cardiac actin gene necessary for postnatal expression at the level of transcript accumulation in the heart and hindlimb muscles of transgenic mice. During this developmental period in the rodent, cardiac actin expression essentially remains constant in the heart, but declines significantly in skeletal muscle. We determined that a 13-kilobase human cardiac actin gene fragment contains sufficient information to direct this maturation-based developmental expression, as well as striated muscle-specific and high level expression. We localized an element responsible for maturation-based down-regulation in the 3' flank of the gene between approximately 950 and 2120 base pairs downstream of the polyadenylation site. Furthermore, we determined that -800 base pairs of 5'-flanking DNA, which contains multiple MyoD1 binding sites, as well as serum response element and AP1 binding sites, can account for striated muscle-specific expression, but not high level expression. Findings indicate that sequence(s) responsible for high level expression of the gene must be located within the body of the gene. We conclude that the human cardiac actin gene contains distinct sequences which confer developmental, tissue-specific, and high level expression.
The human aldolase A gene is transcribed from three different promoters, pN, pM, and pH, all of which are clustered within a small 1.6-kbp DNA domain. pM, which is highly specific to adult skeletal muscle, lies in between pN and pH, which are ubiquitous but particularly active in heart and skeletal muscle. A ubiquitous enhancer, located just upstream of pH start sites, is necessary for the activity of both pH and pN in transient transfection assays. Using transgenic mice, we studied the sequence controlling the muscle-specific promoter pM and the relations between the three promoters and the ubiquitous enhancer. A 4.3-kbp fragment containing the three promoters and the ubiquitous enhancer showed an expression pattern consistent with that known in humans. In addition, while pH was active in both fast and slow skeletal muscles, pM was active only in fast muscle. pM activity was unaltered by the deletion of a 1.8-kbp region containing the ubiquitous enhancer and the pH promoter, whereas pN remained active only in fast skeletal muscle. These findings suggest that in fast skeletal muscle, a tissue-specific enhancer was acting on both pN and pM, whereas in other tissues, the ubiquitous enhancer was necessary for pN activity. Finally, a 2.6-kbp region containing the ubiquitous enhancer and only the pH promoter was sufficient to bring about high-level expression of pH in cardiac and skeletal muscle. Thus, while pH and pM function independently of each other, pN, remarkably, shares regulatory elements with each of them, depending on the tissue. Importantly, expression of the transgenes was independent of the integration site, as originally described for transgenes containing the beta-globin locus control region.
Regulatory sequences of the M isozyme of the creatine kinase (MCK) gene have been extensively mapped in skeletal muscle, but
little is known about the sequences that control cardiac-specific expression. The promoter and enhancer sequences required
for MCK gene expression were assayed by the direct injection of plasmid DNA constructs into adult rat cardiac and skeletal
muscle. A 700-nucleotide fragment containing the enhancer and promoter of the rabbit MCK gene activated the expression of
a downstream reporter gene in both muscle tissues. Deletion of the enhancer significantly decreased expression in skeletal
muscle but had no detectable effect on expression in cardiac muscle. Further deletions revealed a CArG sequence motif at position
-179 within the promoter that was essential for cardiac-specific expression. The CArG element of the MCK promoter bound to
the recombinant serum response factor and YY1, transcription factors which control expression from structurally similar elements
in the skeletal actin and c-fos promoters. MCK-CArG-binding activities that were similar or identical to serum response factor
and YY1 were also detected in extracts from adult cardiac muscle. These data suggest that the MCK gene is controlled by different
regulatory programs in adult cardiac and skeletal muscle.
We report the cDNA cloning and characterization of mouse GATA-4, a new member of the family of zinc finger transcription factors
that bind a core GATA motif. GATA-4 cDNA was identified by screening a 6.5-day mouse embryo library with oligonucleotide probes
corresponding to a highly conserved region of the finger domains. Like other proteins of the family, GATA-4 is approximately
50 kDa in size and contains two zinc finger domains of the form C-X-N-C-(X17)-C-N-X-C. Cotransfection assays in heterologous
cells demonstrate that GATA-4 trans activates reporter constructs containing GATA promoter elements. Northern (RNA) analysis
and in situ hybridization show that GATA-4 mRNA is expressed in the heart, intestinal epithelium, primitive endoderm, and
gonads. Retinoic acid-induced differentiation of mouse F9 cells into visceral or parietal endoderm is accompanied by increased
expression of GATA-4 mRNA and protein. In vitro differentiation of embryonic stem cells into embryoid bodies is also associated
with increased GATA-4 expression. We conclude that GATA-4 is a tissue-specific, retinoic acid-inducible, and developmentally
regulated transcription factor. On the basis of its tissue distribution, we speculate that GATA-4 plays a role in gene expression
in the heart, intestinal epithelium, primitive endoderm, and gonads.
AbstractDuring terminal differentiation of skeletal muscle cells in vitro there is a transition from a predominantly nonmuscle contractile protein phenotype to a sarcomeric contractile protein phenotype. In order to investigate whether this transition and subsequent changes in expression are primarily transcriptionally regulated, we have analysed the rate of transcription and level of corresponding RNA accumulation of actin and myosin light chain genes during differentiation of a mouse muscle cell line under different culture conditions (low-serum and serum-free). We have found by ‘nuclear run-on’ analysis, that the α-cardiac actin, α-skeletal actin, myosin light chain 1F/3F and embryonic myosin light chain genes are transcriptionally activated as myoblasts begin to fuse to form myotubes. In contrast the nonsarcomeric β-actin gene is transcribed at high levels in myoblasts and is transcriptionally down-regulated during differentiation. There is a sequential transition in transcription and RNA accumulation from predominantly α-cardiac to predominantly α-skeletal actin during subsequent myotube maturation, which reflects the pattern of expression found during development in vivo. A similar transition from embryonic to adult patterns of myosin light chain expression does not occur. RNA accumulation of actin and myosin light chains is regulated at both transcriptional and post-transcriptional levels. In our culture system the expression of myosin light chains 1F and 3F, which are encoded by a single gene, is uncoupled, 3F predominating. These data are discussed in the context of gene regulation mechanisms.
During primary and secondary myotube formation in utero and subsequent maturation of muscle fibers after birth there are complex changes in the pattern of contractile protein gene expression at the RNA and protein levels. In order to determine the degree of transcriptional regulation of actin and myosin genes we have carried out “nuclear run-on” experiments using nuclei prepared from the limb muscle of mice at 14.5, 15.5, 17.5, and 18.5 days in utero and at 10–12 and 12.5 days after birth. We show that transitions in the expression of these genes in vivo are regulated transcriptionally. Transcription of the sarcomeric α-actins changes from cardiac to predominantly skeletal actin over this time period; transcription of the β-actin gene is repressed. The myosin heavy chain and myosin light chain genes also undergo transcriptional transitions during muscle development. Notably, transcription from the MLC3F promoter is activated after that of the MLC1F promoter, which is part of the same gene. These results are discussed in the context of published RNA data.
This article reviews what is known about the earliest stages of heart development focusing on the periods of commitment and differentiation of cardiac progenitor cells and their molecular regulation. The pathway from precursor to differentiated cardiac myocyte is crucial to forming a normal, functional heart. Congenital cardiac abnormalities are some of the most common, estimated at 5-8 per 1000 live births worldwide. These conditions affect mortality and morbidity of patients as infants, children, and adults. Knowledge of what steps are critical to normal heart development would lead to earlier diagnosis and possibly repair of these defects.
A clonal cell line exhibiting many of the properties of skeletal muscle has been derived from embryonic BDIX rat heart tissue. Multinucleated myotubes, formed by fusion of mononucleated myoblasts, simultaneously contract and produce regenerative action potentials in response to electrical stimulation or iontophoretic application of acetylcholine. The acetylcholine response is inhibited by 1−3 × 10−7 M d-tubocurarine, 10−7 g/ml α-neurotoxin or 1 × 10−4 M atropine. The specific activities of the enzymes myokinase and creatine phosphokinase (CPK) increase 3-fold and 20-fold, respectively, after myotube formation, but only CPK activity parallels the extent of fusion. Exponentially dividing myoblasts synthesize a predominantly brain-type CPK isoenzyme while fused myotubes synthesize a muscle type CPK isoenzyme. Electron microscopic analysis reveals that the myotubes contain elaborately branched tubular systems and numerous bundles of thick filaments with distinct M-bands. Some of the thick filament bundles are associated with thin filaments and organized into sarcomere-like structures with faint Z-regions, but no distinct Z-bands are observed.
The failure of denervated muscle to undergo effective regeneration, despite reported increases in the number of muscle satellite cells, warranted an investigation of the viability and myoblastic capacity of these cells present in denervated muscle. Four types of satellite cells present in muscle denervated for three weeks are described, based on their ultrastructure and relationship to their principal fiber. The increased number of ribosomes, including helically arranged polysomes; the number of Golgi complexes; the presence of microtubules; the branching subsarcolemmal tubular system; and the appearance of regularly arranged 96 A microfilaments with diffuse electron dense areas are structural features of satellite cells that are similar to those of developing myoblasts in growing and regenerating muscle. The electron microscopic observations suggest that "activated" satellite cells do have myoblastic potential. Possible explanations for the ultimate failure of denervated muscle to regenerate include: 1) the inability of the muscle to produce satellite cells rapidly enough to keep pace with muscle degeneration; 2) a cytotoxic effect produced by the degenerating muscle fiber on the satellite cell; and 3) the inability of satellite cells to form stable, mature multinucleated fibers in the absence of the trophic effect of the nerve.
The myoD gene converts many differentiated cell types into muscle. MyoD is a member of the basic-helix-loop-helix family of
proteins; this 68-amino acid domain in MyoD is necessary and sufficient for myogenesis. MyoD binds cooperatively to muscle-specific
enhancers and activates transcription. The helix-loop-helix motif is responsible for dimerization, and, depending on its dimerization
partner, MyoD activity can be controlled. MyoD senses and integrates many facets of cell state. MyoD is expressed only in
skeletal muscle and its precursors; in nonmuscle cells myoD is repressed by specific genes. MyoD activates its own transcription;
this may stabilize commitment to myogenesis.
The alpha-actins are among the earliest muscle-specific mRNAs to appear in developing cardiac and skeletal muscle. To determine if there is coexpression of the alpha-actin proteins at early stages of myogenesis, we have used an alpha-actin-specific polyclonal antibody and in situ hybridization with specific cRNA probes to cardiac and skeletal alpha-actin transcripts on serial slides of mouse embryo sections. As soon as we can detect alpha-actin mRNAs in embryonic striated muscle, we also detect the protein suggesting that alpha-actin transcripts are translated very rapidly after transcription during myogenesis. In skeletal muscle, this colocalization of alpha-actin mRNA and protein was observed both in the myotomes of somites and in developing muscles in the limbs. In cardiac muscle, alpha-actin transcripts and proteins are abundantly expressed as soon as a cardiac tube forms.
BALB/c mice possess a 5' duplication of the alpha-cardiac actin gene which is associated with abnormal levels of alpha-cardiac and alpha-skeletal actin mRNAs in adult cardiac tissue. This mutation therefore provides a potential tool for the study of the inter-relationship between the striated muscle actins. We have examined the expression of this actin gene pair throughout the development of skeletal and cardiac muscle in BALB/c mice. During embryonic and fetal development, the expression of these two genes is indistinguishable from that in normal mice, as determined by in situ hybridization. A quantitative postnatal study demonstrates that in the hearts of normal mice the level of alpha-cardiac actin mRNA declines, whereas that of alpha-skeletal actin increases. In mutant mice, these trends are exaggerated so that whereas normal mice have 95.8% alpha-cardiac mRNA and 4.2% alpha-skeletal mRNA in the adult heart, BALB/c mice have 52.4 and 47.6% of these mRNAs, respectively. This difference is also reflected at the protein level. In developing skeletal muscle, the expression of these genes follows kinetics similar to that observed in the heart with a decrease in the relative level of alpha-cardiac mRNA as the muscle matures. Cardiac actin mRNA levels are again lower in the mutant mouse, but here the effect is less striking because skeletal actin is the predominant isoform. These results are discussed in the context of the interaction between this actin gene pair in developing and adult striated muscle.
A total of 30 actins from various chordate and invertebrate muscle sources were either characterized by full amino acid sequence data or typed by those partial sequences in the NH2-terminal tryptic peptide which are known to be specific markers for different actin isoforms. The results show that most, if not all, invertebrate muscle actins are homologous to each other and to the isoforms recognized as vertebrate cytoplasmic actins. In contrast the actin forms typically found in muscle cells of warm-blooded vertebrates are noticeably different from invertebrate muscle actins and seem to have appeared in evolution already with the origin of chordates. During subsequent vertebrate evolution there has been a high degree of sequence conservation similar or stronger than that seen in histone H4. Urochordates, Cephalochordates and probably also Agnathes express only one type of muscle actin. Two types, a striated muscle-specific form and a smooth muscle form, are already observed in Chondrichthyes and Osteichthyes. Later in evolution, with the origin of reptiles, both muscle actins seem to have duplicated again; the striated muscle type branched into a skeletal- and cardiac-specific form, while the smooth muscle form duplicated into a vascular- and stomach-specific type. These findings support the hypothesis that each of the four muscle actins of warm-blooded vertebrates are coded for by a small number and possibly only one functional gene.
1. Isometric contractions of motor units, isolated functionally by ventral root splitting in vivo, were recorded from mouse soleus muscle. 2. Motor unit tensions varied over a narrow symmetrical range and averaged 4.7% of whole muscle tension, corresponding to twenty-one motor units per muscle. 3. There was considerable variation between muscles in isometric twitch times-to-peak and even greater variation for the motor units. The distribution of motor unit times-to-peak was apparently unimodal and could be fitted by a single normal population. A slightly better fit was, however, obtained with two normal populations, as suggested by the histochemistry. 4. Twitch time-to-peak decreased in proportion to axonal conduction velocity in individual animals. The whole population of motor units could be fitted by a linear relation between time-to-peak and the reciprocal of conduction time in the motor axon. Motor unit tension was also linearly related to the reciprocal of conduction time. 5. Histochemistry showed clear division between Type I and Type IIa fibres. Type I fibres reacted strongly with antibody against slow myosin of cat soleus muscle; Type IIa gave a reaction no stronger than the background. The division was as clear as in the cat or rat.
The homeobox-containing gene tinman (msh-2, Bodmer et al., 1990 Development 110, 661-669) is expressed in the mesoderm primordium, and this expression requires the function of the mesoderm determinant twist. Later in development, as the first mesodermal subdivisions are occurring, expression becomes limited to the visceral mesoderm and the heart. Here, I show that the function of tinman is required for visceral muscle and heart development. Embryos that are mutant for the tinman gene lack the appearance of visceral mesoderm and of heart primordia, and the fusion of the anterior and posterior endoderm is impaired. Even though tinman mutant embryos do not have a heart or visceral muscles, many of the somatic body wall muscles appear to develop although abnormally. When the tinman cDNA is ubiquitously expressed in tinman mutant embryos, via a heatshock promoter, formation of heart cells and visceral mesoderm is partially restored, tinman seems to be one of the earliest genes required for heart development and the first gene reported for which a crucial function in the early mesodermal subdivisions has been implicated.
Mice lacking myogenin have little skeletal muscle as fibres fail to differentiate. Lack of both MyoD and myf-5 results in no skeletal muscle and apparently no myoblasts, suggesting that these factors act earlier in muscle development.
The purpose of the present investigation was to study the distribution and subunit composition of type IIX fibers in mouse muscles. The existence of a population of type IIX fibers in fast-twitch muscles of the mouse was shown by mean of immunohistochemistry and gel electrophoresis. In the hindlimb muscles, tibialis anterior (TA) and extensor digitorum longus (EDL), type IIX fibers account for approximately one third of the total fiber number, with the superficial portion of the TA (TAS) being composed exclusively of type IIB and IIX fibers. A similar proportion of IIX fibers was found in diaphragm (DIA) while in tongue muscles approximately 40% of the fibers were IIX. Single fiber gel electrophoresis revealed a significant number of fibers in TAS that contain both IIB and IIX myosin heavy chain (MyHC). This was confirmed with immunohistochemistry, which revealed the presence of fibers with various degrees of staining intensity. This suggests that there may exist a degree of plasticity which results in the conversion of IIX fibers to IIB fibers and vice versa. Analysis of myosin light chain (MyLC) composition of type IIX fibers revealed that the ratio of MyLC3f to MyLC1f was significantly lower than in type IIB fibers.
Pgk-1 is an X-linked gene encoding 3-phosphoglycerate kinase, an enzyme necessary in every cell for glycolysis. The regulatory sequences of the Pgk-1 gene were used to drive the E. coli lacZ reporter gene and 2 strains of transgenic animals created with this Pgk-lacZ transgene carried on autosomes. The levels of expression of Pgk-1 varied from one adult tissue to another and the transgene was similarly regulated. However, in situ staining of the beta-galactosidase encoded by the transgene indicated extensive cell-to-cell variability in its level of expression. A reproducible subset of cells stained darkly for the transgene product. Some of these beta-galactosidase positive cells were rapidly proliferating while others appeared to be metabolically very active, suggesting that the Pgk-1 promoter is regulated so as to be more active in cells requiring high levels of glycolysis. Although Pgk-1 is X-linked and subject to X chromosome inactivation, the transgenes were not inactivated in either female somatic or male germ cells. Thus, the Pgk-1 promoter drives transgene expression in all tissues but the levels of expression are not uniform in each cell.
A DNase I-hypersensitive site analysis of the 5'-flanking region of the mouse alpha-cardiac actin gene with muscle cell lines derived from C3H mice shows the presence of two such sites, at about -5 and -7 kb. When tested for activity in cultured cells with homologous and heterologous promoters, both sequences act as muscle-specific enhancers. Transcription from the proximal promoter of the alpha-cardiac actin gene is increased 100-fold with either enhancer. The activity of the distal enhancer in C2/7 myotubes is confined to an 800-bp fragment, which contains multiple E boxes. In transfection assays, this sequence does not give detectable transactivation by any of the myogenic factors even though one of the E boxes is functionally important. Bandshift assays showed that MyoD and myogenin can bind to this E box. However, additional sequences are also required for activity. We conclude that in the case of this muscle enhancer, myogenic factors alone are not sufficient to activate transcription either directly via an E box or indirectly through activation of genes encoding other muscle factors. In BALB/c mice, in which cardiac actin mRNA levels are 8- to 10-fold lower, the alpha-cardiac actin locus is perturbed by a 9.5-kb insertion (I. Garner, A. J. Minty, S. Alonso, P. J. Barton, and M. E. Buckingham, EMBO J. 5:2559-2567, 1986). This is located at -6.5 kb, between the two enhancers. The insertion therefore distances the distal enhancer from the promoter and from the proximal enhancer of the bona fide cardiac actin gene, probably thus perturbing transcriptional activity.
The sequence CANNTG (E box) is frequently found in the promoters of muscle-specific genes and binds members of the basic helix-loop-helix (bHLH) family of transcription factors. We compared the need for the E box in the expression of a muscle-specific promoter normally expressed in both cardiac and skeletal muscle. The E box was mutated in a construct of the cardiac alpha-actin promoter driving the Escherichia coli lacZ gene. The wild-type and mutant constructs were transfected and stably integrated into the genomes of P19 embryonal carcinoma cells. The wild-type promoter was expressed in both cardiac and skeletal myocytes. The promoter lacking an E box was expressed in cardiac but not in skeletal muscle. Neither promoter was active in nonmuscle cells. Thus the E box is not necessary for the cardiac actin promoter activity in P19-derived cardiac muscle but is essential for its activity in skeletal muscle. This result is consistent with our inability to detect cardiac muscle-specific members of the MyoD family of bHLH transcription factors.
Prior studies using transient transfection assays in cultured avian and murine skeletal myotubes indicate that the proximal 2-kb segment of the 5' flanking region of the human myoglobin gene contains transcriptional control elements sufficient to direct muscle-specific and developmentally regulated expression of reporter genes. To examine the function of the human myoglobin gene promoter during development of skeletal and cardiac myocytes in the intact animal, a 2.0-kb myoglobin gene upstream fragment was fused to an Escherichia coli lacZ reporter gene and injected into fertilized mouse oocytes. beta-Galactosidase (beta-gal) activity was detected selectively in cardiac and skeletal myocytes of fetal and adult transgenic mice. A distinctive spatial pattern of myoglobin promoter activity was observed in fetal hearts: beta-gal staining was more pronounced within the left ventricular subendocardium than within the subepicardium and was essentially undetectable in the ventricular trabeculae or atria. Expression of endogenous myoglobin mRNA and protein, assessed by in situ hybridization and immunohistochemistry, demonstrated a similar spatial pattern. In contrast, hearts from adult transgenic mice demonstrated essentially homogeneous expression of beta-gal and of endogenous myoglobin mRNA and protein throughout the myocardium, including the trabeculae and atria. These data indicate that the 2.0-kb upstream region of the human myoglobin gene includes cis-acting regulatory elements sufficient to direct transgene expression during murine cardiac development that is myocyte-specific and responsive to positional cues in a similar manner to the endogenous myoglobin gene.
Muscle generegulation : Actin andmyosin sequences in the heart
- M Buckingham
Buckingham, M. (1990). Muscle generegulation : Actin andmyosin sequences in the heart. Heart Failure 6, 49±56
Functional activity of the two promoters Copyright ? 1996 by Academic Press, Inc. All rights of reproduction in any form reserved Isometric con-tractions of motor units and immunohistochemistry of mouse soleus muscle
- P Daubas
- A Klarsfeld
- I Garner
- C Pinset
- R Cox
- M E Buck-Ingham
Daubas, P., Klarsfeld, A., Garner, I., Pinset, C., Cox, R., and Buck-ingham, M. E. (1988). Functional activity of the two promoters Copyright ? 1996 by Academic Press, Inc. All rights of reproduction in any form reserved. r212 Biben et al. Lewis, D. M., Parry, D. J., and Rowlersen A. (1982). Isometric con-tractions of motor units and immunohistochemistry of mouse soleus muscle. J. Physiol. 325, 393±401. Li, Z., Marchand, P., Humbert, J., Babinet, C., andPaulin, D. (1993)
in the heart tubes of murine embryos lacking the homeobox gene
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in the heart tubes of murine embryos lacking the homeobox gene Vandekerckhove, J., Bugaisky, G., and Buckingham, M. (1986).
Upstream regions of the human Hollenberg, cardiac actin gene that modulate its transcription in muscle cells The MyoD gene family: Presence of an evolutionary conserved motif
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- M Thayer
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- A J Minty
- L Kedes
- R Benezra
- T K Blackwell
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- R Rupp
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tional regulation of actin and myosin genes during differentiation van
- R D Cox
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- K J Transcrip-Lee
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Cox, R. D., Garner, I., and Buckingham, M. E. (1990). Transcrip-Lee, K. J., Ross, R. S., Rockman, H. A., Harris, A., O'Brien, T. X., tional regulation of actin and myosin genes during differentiation van Bilsen, M., Shubeita, H., Kandolf, R., Brem, G., Price, J., of a mouse muscle cell line. Differentiation 43, 183– 191.
A skeletal muscle expression in directly injected skeletal and cardiac muscle. Mol. phenotype initiated by ectopic MyoD in the hearts of transgenic Cell
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The gene Tinman is required for specification
- R Bodmer
- R Kelly
- S Alonso
- S Tajbakhsh
- G Cossu
- Buckingham
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Which myogenic factors make muscle? expression in transgenic mice
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Developmental regula- Tran-tion of myogenesis in the mouse
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Contractile protein gene expres-Desmin sequence elements regulating skeletal muscle-specific sion in primary myotubes of embryonic mouse hindlimb mus-expression in transgenic mice
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