Charles P. Ordahl

Early stages of chick somite development

We report on the formation and early differentiation of the somites in the avian embryo. The somites are derived from the mesoderm which, in the body (excluding the head), is subdivided into four compartments: the axial, paraxial, intermediate and lateral plate mesoderm. Somites develop from the paraxial mesoderm and constitute the segmental pattern of the body. They are formed in pairs by epithelialization, first at the cranial end of the paraxial mesoderm, proceeding caudally, while new mesenchyme cells enter the paraxial mesoderm as a consequence of gastrulation. After their formation, which depends upon cell-cell and cell-matrix interactions, the somites impose segmental pattern upon peripheral nerves and vascular primordia. The newly formed somite consists of an epithelial ball of columnar cells enveloping mesenchymal cells within a central cavity, the somitocoel. Each somite is surrounded by extracellular matrix material connecting the somite with adjacent structures. The competence to form skeletal muscle is a unique property of the somites and becomes realized during compartmentalization, under control of signals emanating from surrounding tissues. Compartmentalization is accompanied by altered patterns of expression of Pax genes within the somite. These are believed to be involved in the specification of somite cell lineages. Somites are also regionally specified, giving rise to particular skeletal structures at different axial levels. This axial specification appears to be reflected in Hox gene expression. MyoD is first expressed in the dorsomedial quadrant of the still epithelial somite whose cells are not yet definitely committed. During early maturation, the ventral wall of the somite undergoes an epithelio-mesenchymal transition forming the sclerotome. The sclerotome later becomes subdivided into rostral and caudal halves which are separated laterally by von Ebners fissure. The lateral part of the caudal half of the sclerotome mainly forms the ribs, neural arches and pedicles of vertebrae, whereas within the lateral part of the rostral half the spinal nerve develops. The medially migrating sclerotomal cells form the peri-notochordal sheath, and later give rise to the vertebral bodies and intervertebral discs. The somitocoel cells also contribute to the sclerotome. The dorsal half of the somite remains epithelial and is referred to as the dermomyotome because it gives rise to the dermis of the back and the skeletal musculature. The cells located within the lateral half of the dermomyotome are the precursors of the muscles of the hypaxial domain of the body, whereas those in the medial half are precursors of the epaxial (back) muscles. Single epithelial cells at the cranio-medial edge of the dermomyotome elongate in a caudal direction, beneath the dermomyotome, and become anchored at its caudal margin. These post-mitotic and muscle protein-expressing cells form the myotome. At limb levels, the precursors of hypaxial muscles undergo an epithelio-mesenchymal transition and migrate into the somatic mesoderm, where they replicate and later differentiate. These cells express the Pax-3 gene prior to, during and after this migration. All compartments of the somite contribute endothelial cells to the formation of vascular primordia. These cells, unlike all other cells of the somite, occasionally cross the midline of the developing embryo. We also suggest a method for staging somites according to their developmental age.

Poly(ADP-Ribose) Polymerase Binds with Transcription Enhancer Factor 1 to MCAT1 Elements To Regulate Muscle-Specific Transcription

ABSTRACT Striated muscle-specific expression of the cardiac troponin T (cTNT) gene is mediated through two MCAT elements that act via binding of transcription enhancer factor 1 (TEF-1) to the MCAT core motifs and binding of an auxiliary protein to nucleotides flanking the 5′ side of the core motif. Using DNA-protein and protein-protein binding experiments, we identified a 140-kDa polypeptide that bound both the muscle-specific flanking sequences of the most distal MCAT1 element and TEF-1. Screening of an expression library with the MCAT1 element yielded a cDNA encoding a truncated form of poly(ADP-ribose) polymerase (PARP). Endogenous PARP from embryonic tissue nuclear extracts migrated as a 140-kDa protein. Recombinant full-length PARP preferentially bound the wild-type MCAT1 element and was shown to physically interact with TEF-1. In addition, endogenous TEF-1 could be coimmunoprecipitated with PARP from extracts of primary skeletal muscle cells. Recombinant PARP was able to ADP-ribosylate TEF-1 in vitro. Inhibition of the enzymatic activity of PARP repressed expression of an MCAT1-dependent reporter in transiently transfected primary muscle cells. Together, these data implicate PARP as the auxiliary protein that binds with TEF-1 to the MCAT1 element to provide muscle-specific gene transcription.

The cardiac beta-myosin heavy chain isogene is induced selectively in alpha 1-adrenergic receptor-stimulated hypertrophy of cultured rat heart myocytes.

Cardiac hypertrophy produced in vivo by pressure overload is characterized by selective up-regulation of the fetal/neonatal beta-cardiac myosin heavy chain (MHC) isogene. However, a molecular signal for beta-MHC isogene induction has not been identified. We examined cardiac MHC isogene expression in a cell culture model for hypertrophy. alpha-MHC and beta-MHC iso-protein and iso-mRNA levels in cultured cardiac myocytes were quantified during hypertrophy stimulated by the alpha 1-adrenergic agonist, norepinephrine (NE). beta-MHC iso-protein content was increased 3.2-fold vs. control (P less than 0.001), whereas alpha-MHC isoprotein content was not changed significantly (1.4-fold vs. control, P = NS). MHC iso-mRNA levels were quantified by nuclease S1 analysis, using a single oligonucleotide probe. NE increased beta-MHC iso-mRNA content by 3.9-fold vs. control (P less than 0.001), but there was no change in alpha-MHC iso-mRNA (1.1-fold vs. control, P = NS). The NE-stimulated increase in beta-MHC iso-mRNA preceded in time the increase in beta-MHC isoprotein accumulation. The EC50 for NE induction of beta-MHC was 40 nM, and pharmacologic experiments indicated alpha 1-adrenergic receptor specificity. alpha-MHC isogene expression was predominant in control myocytes (68% alpha-isoprotein and 60% alpha-iso-mRNA). In contrast, beta-MHC expression was equal to alpha-MHC or predominant after treatment with NE (51% beta-isoprotein and 69% beta-iso-mRNA). Thus, alpha 1-adrenergic receptor stimulation increases the cellular contents of beta-MHC iso-mRNA and beta-MHC isoprotein during hypertrophy of cultured neonatal rat cardiac myocytes, but does not change the levels of alpha-MHC iso-mRNA or isoprotein. The effect on beta-MHC is mediated primarily at the level of mRNA steady-state level (pretranslational). Activation of the alpha 1-adrenergic receptor is the first identified molecular signal for increased beta-MHC isogene expression in a model of cardiac hypertrophy.

Induction of the skeletal alpha-actin gene in alpha 1-adrenoceptor-mediated hypertrophy of rat cardiac myocytes.

Myocardial hypertrophy in vivo is associated with reexpression of contractile protein isogenes characteristic of fetal and neonatal development. The molecular signals for hypertrophy and isogene switching are unknown. We studied alpha (sarcomeric)-actin messenger RNA (mRNA) expression in cultured cardiac myocytes from the neonatal rat. In the cultured cells, as in the adult heart in vivo, expression of cardiac alpha-actin (cACT) predominated over that of skeletal alpha-actin (sACT) mRNA, the fetal/neonatal isoform. alpha 1-Adrenergic receptor stimulation induced hypertrophy of these cells, increasing total RNA and cytoskeletal actin mRNA by 1.8-fold over control, and total alpha-actin mRNA by 4.3 fold. This disproportionate increase in total alpha-actin mRNA was produced by a preferential induction of sACT mRNA, which increased by 10.6-fold over control versus only 2.6-fold for cACT mRNA. The alpha 1-adrenoceptor is the first identified molecular mediator of early developmental isogene reexpression in cardiac myocyte hypertrophy.

Isolation and characterization of an avian myogenic cell line.

Myogenic cell lines have proven extremely valuable for studying myogenesis in vitro. Although a number of mammalian muscle cell lines have been isolated, attempts to produce cell lines from other classes of animals have met with only limited success. We report here the isolation and characterization of seven avian myogenic cell lines (QM1-4 and QM6-8), derived from the quail fibrosarcoma cell line QT6. A differentiation incompetent QM cell derivative was also isolated (QM5DI). The major features of QM cell differentiation in vitro closely resemble those of their mammalian counterparts. Mononucleated QM cells replicate in medium containing high concentrations of serum components. Upon switching to medium containing low serum components, cells withdraw from the cell cycle and fuse to form elongated multinucleated myotubes. Cultures typically obtain fusion indices of 43-49%. Northern blot and immunoblot analyses demonstrate that each differentiated QM cell line expresses a wide variety of genes encoding muscle specific proteins: desmin, cardiac troponin T, skeletal troponin T, cardiac troponin C, skeletal troponin I, alpha-tropomyosin, muscle creatine kinase, myosin light chain 2, and a ventricular isoform of myosin heavy chain. While all QM lines analyzed to date express at least some myosin light chain 2, only one line, QM7, expresses this gene at high levels. Surprisingly, none of the QM lines reported here express any known form of alpha-actin. The absence of sarcomeric actin expression may explain the absence of myofibrils in QM myotubes. These novel features of muscle gene expression in QM cells may prove useful for studying the role of specific muscle proteins during myogenesis. More importantly, however, the isolation of QM cell lines indicates that it may be feasible to isolate other avian myogenic cell lines with general utility for the study of muscle development.

Alpha 1-adrenergic receptor stimulation of sarcomeric actin isogene transcription in hypertrophy of cultured rat heart muscle cells.

During pressure-load hypertrophy of the adult heart in vivo, there is up-regulation of the mRNA encoding skeletal alpha-actin, the sarcomeric actin iso-mRNA characteristic of mature skeletal muscle and the fetal/neonatal heart. We have shown previously that during alpha 1-adrenergic receptor-stimulated hypertrophy of cultured rat heart myocytes, the induction of skeletal alpha-actin mRNA is greater than that of the mRNA encoding cardiac alpha-actin, the sarcomeric actin iso-mRNA characteristic of the adult heart. To determine if this actin iso-mRNA switch during cardiac hypertrophy reflects changes in the transcriptional status of the myocyte nucleus, we quantified the rate of transcription of actin mRNAs and total RNA, using an in vitro run-on transcription assay with nuclei isolated from the cultured myocytes after stimulation with norepinephrine (NE). Transcription of skeletal alpha-actin was increased at 3 h after NE, reached a maximum 6.1-fold increase at 12 h, and returned to the control level at 24 h. The EC50 for NE was 200 nM, and pharmacologic studies indicated alpha 1-receptor specificity. Transcription of cardiac alpha-actin was also increased rapidly by NE (maximum 4.6-fold vs. control at 3 h). However, cardiac alpha-actin transcription had returned to the control level at 6 h, when NE-stimulated skeletal alpha-actin transcription was still increasing. Transcription of the cytoskeletal (beta) actin gene was not changed significantly by NE treatment. Total RNA transcription was not increased until 6 h after NE (1.5-fold vs. control) and remained elevated through 24 h. Inhibition of protein synthesis did not attenuate NE-stimulated actin gene transcription. Thus the alpha 1-adrenoceptor mediates a rapid, transient, and selective increase in transcription of the sarcomeric actin isogenes during cardiac myocyte hypertrophy. Skeletal alpha-actin, the fetal/neonatal isogene, is induced preferentially to cardiac alpha-actin, the adult isogene. The different kinetics of actin isogene and total RNA transcription and the independence of transcription from protein synthesis suggest that transcriptional induction via the alpha 1 receptor is complex and may involve preexisting regulatory factors. These results are the first to demonstrate that the alpha 1-adrenergic receptor is a molecular mediator of transcriptional changes underlying an isogene switch that is known to be associated with cardiac myocyte hypertrophy.

Transcription of early developmental isogenes in cardiac myocyte hypertrophy.

We have developed a cell culture system to study molecular mechanisms important in myocardial hypertrophy. alpha 1-Adrenergic receptor stimulation produces hypertrophy of neonatal rat cardiac myocytes. Myocyte hyperplasia is not induced by alpha 1 stimulation, although alpha 1-adrenergic receptor-mediated DNA synthesis and cell division have been observed in other types of cells. The myocyte hypertrophic response does not require contractile activity. Activation of the alpha 1 receptor also produces highly specific alterations in gene expression, as measured at the mRNA and protein levels. In particular, there is selective up-regulation of two contractile protein isogenes that are expressed in vivo during early development and in pressure-load hypertrophy, skeletal alpha-actin and beta-myosin heavy chain. Studies with an in vitro transcription assay indicate that stimulation of the alpha 1-adrenergic receptor leads to a distinctive temporal sequence of transcriptional activation. Transcription of the skeletal alpha-actin isogene is induced preferentially to that of cardiac alpha-actin. Thus, early developmental isogene induction in alpha 1-stimulated hypertrophy reflects a fundamental change in the transcriptional program of the cardiac myocyte nucleus. The goal now is to define an intracellular pathway connecting the alpha 1-adrenergic receptor in the plasma membrane to activation of RNA polymerase II on the skeletal alpha-actin gene in the cardiac myocyte nucleus. There is evidence that protein kinase C may be one component of this pathway. A model for alpha 1-mediated transcription is presented.

Coenzymatic activity of randomly broken or intact double-stranded DNAs in auto and histone H1 trans-poly(ADP-ribosylation), catalyzed by poly(ADP-ribose) polymerase (PARP I).

The enzymatic transfer of ADP-ribose from NAD to histone H1 (defined as trans-poly(ADP-ribosylation)) or to PARP I (defined as auto-poly(ADP-ribosylation)) was studied with respect to the nature of the DNA required as a coenzyme. Linear double-stranded DNA (dsDNA) containing the MCAT core motif was compared with DNA containing random nicks (discontinuous or dcDNA). The dsDNAs activated trans-poly(ADP-ribosylation) about 5 times more effectively than dcDNA as measured by V max. Activation of auto-poly(ADP-ribosylation) by dcDNA was 10 times greater than by dsDNA. The affinity of PARP I toward dcDNA or dsDNA in the auto-poly(ADP-ribosylation) was at least 100-fold lower than in trans-poly(ADP-ribosylation) (K a = 1400versus 3–15, respectively). Mg2+ inhibited trans-poly(ADP-ribosylation) and so did dcDNA at concentrations required to maximally activate auto-poly(ADP-ribosylation). Mg2+ activated auto-poly(ADP-ribosylation) of PARP I. These results for the first time demonstrate that physiologically occurring dsDNAs can serve as coenzymes for PARP I and catalyze preferentially trans-poly(ADP- ribosylation), thereby opening the possibility to study the physiologic function of PARP I.

9 Determination and Morphogenesis in Myogenic Progenitor Cells: An Experimental Embryological Approach

Publisher Summary This chapter reviews the recent and historical experiments indicating that the molecular events underlying myogenic determination occur within discrete regions of the somite and that they may be coupled to the morphogenetic movements of muscle precursor cells during somitogenesis. The notochord challenge analysis of the somite dorsomedial quadrant have revealed unexpected and potentially important information about the onset of appearance of myogenic progenitor cells possessing different types of myogenic memory. Two observations suggested that myogenic stem cells with increasing mitotic capacity progressively appear in a somite-stage-dependent fashion. First, although the sizes of transplanted dorsomedial quadrant of the somite (DMQ) fragments were similar, the mass of myogenic tissue obtained using later-staged DMQ fragments was far greater than that from earlystaged DMQs. Second, DNA labeling showed that myogenic precursor cells within the DMQ had passed through the S phase of the cell cycle prior to differentiating in the presence of the notochord challenge. A third observation suggests that these stem cells also had morphogenetic capacity characteristic of progenitor stem cells because myogenic cells from early staged somites formed myoclusters that consisted of 50 or fewer disorganized, mononucleated myocytes while later somites gave rise to multinucleated myofibers that were highly organized into parallel fiber bundles.

The skeletal and cardiac α-action genes are coexpressed in early embryonic striated muscle

Abstract The relative steady-state abundance of cardiac and skeletal α-actin mRNAs at different stages of embryonic skeletal and cardiac (striated) muscle development was determined by a reverse transcriptase extension assay employing an single oligonucleotide primer complementary to a perfectly conserved region near the 5′ end of both mRNAs. Both mRNAs were found to be present at every stage of embryonic striated muscle development tested, including the earliest assayable stages of limb muscle and cardiac muscle development. At early stages of skeletal muscle development the two mRNAs are present at similar levels while at later stages the abundance of the skeletal α-actin mRNA far exceeds that of the cardiac α-actin mRNA. Both mRNAs are also present at similar levels throughout embryonic cardiac muscle development while in adult cardiac muscle the cardiac α-actin mRNA predominates over the skeletal α-actin mRNA. These results for early embryonic striated muscle, in combination with previous results with late embryonic and adult striated muscle, indicate that both genes are coexpressed throughout striated muscle ontogeny. These two genes may not, therefore, be regulated under unique tissue-specific regulatory programs but each may have acquired regulatory elements which confer important quantitative differences in their level of expression in mature striated muscle cells.