Temporally and spatially restricted expression of a gland cell gene during regeneration and in vitro transdifferentiation in the hydrozoan Podocoryne carnea

An antiserum to transdifferentiated striated muscle cells from the medusa of Podocoryne carnea was prepared and used to screen a gt11-expression library prepared from gonozoids of P. carnea. We isolated a cDNA clone termed Pod-EPPT with at least 63 tandem repeats of the tetrapeptide-motive glu-pro-pro-thr, named Pod-EPPT. Using Pod-EPPT as a molecular marker for head quality the morphological relationship between the two metagenic life stages of this hydroid, the polyp and the medusa, was studied. In situ hybridization demonstrated that expression of the gene corresponding is restricted to secretory cells in the endoderm of the oral hypostome region of polyps and medusae and, presumably, to progenitor cells of this type. Cells expressing Pod-EPPT could not be observed in the larval stage. During head regeneration in polyps, Pod-EPPT expression is upregulated soon after head removal in previously non-expressing cells and in newly differentiating secretory cells. This activation of a head-specific gene precedes the morphologically obvious events of head regeneration. Pod-EPPT is one of the genes that are activated during manubrium (mouth) regeneration from experimentally combined subumbrellar plate endoderm and striated muscle of the medusa.     

Evolution of striated muscle: jellyfish and the origin of triploblasty

The larval and polyp stages of extant Cnidaria are bi-layered with an absence of mesoderm and its differentiation products. This anatomy originally prompted the diploblast classification of the cnidarian phylum. The medusa stage, or jellyfish, however, has a more complex anatomy characterized by a swimming bell with a well-developed striated muscle layer. Based on developmental histology of the hydrozoan medusa this muscle derives from the entocodon, a mesoderm-like third cell layer established at the onset of medusa formation. According to recent molecular studies cnidarian homologs to bilaterian mesoderm and myogenic regulators are expressed in the larval and polyp stages as well as in the entocodon and derived striated muscle. Moreover striated and smooth muscle cells may have evolved directly and independently from non-muscle cells as indicated by phylogenetic analysis of myosin heavy chain genes (MHC class II). To accommodate all evidences we propose that striated muscle-based locomotion coevolved with the nervous and digestive systems in a basic metazoan Bauplan from which the ancestors of the Ctenophora (comb jellyfish), Cnidaria (jellyfish and polyps), as well as the Bilateria are derived. We argue for a motile tri-layered cnidarian ancestor and a monophyletic descent of striated muscle in Cnidaria and Bilateria. As a consequence, diploblasty evolved secondarily in cnidarian larvae and polyps.     

Complete primary structure and tissue expression of chicken pectoralis M-protein.

M-Protein (165 kDa) is a structural constituent of myofibrillar M-band in striated muscle. We generated a monoclonal antibody which recognized a 165-kDa protein from chicken pectoralis muscle in immunoblot analysis and stained the M-band under immunofluorescence microscopy. By screening a lambda gt11 cDNA library from chicken embryonic pectoralis muscle with this antibody, we isolated a cDNA clone encoding the M-protein. Northern blot analysis showed that M-protein mRNA is expressed in pectoralis and cardiac muscle but not in gizzard smooth muscle or non-muscle tissues. Moreover, the anterior latissimus dorsi muscle, which consists almost exclusively of slow fiber types, contains no detectable levels of the mRNA. The full-length cDNA sequence predicted a 1,450-amino acid polypeptide with a calculated molecular weight of 163 x 10(3). The encoded protein contains several copies of two different repetitive motifs: five copies of fibronectin type III repeats are in the middle part of the predicted molecule, and two and four copies of the immunoglobulin C2-type repeats are located toward the NH2-terminal and COOH-terminal regions, respectively. This indicates that M-protein, along with other thick filament-associated proteins such as C-protein, twichin, and titin, belongs to the superfamily of cytoskeletal proteins with immunoglobulin/fibronectin repeats.     

Immunochemical analysis of myosin heavy chains in the developing chicken heart

Monoclonal antibodies (McAb) against myosin from the pectoralis muscle of the adult chicken have been generated and shown to react specifically with the myosin heavy chain (MHC). The reactivities of two such McAbs with myosin from adult chicken atrial and ventricular myocardium were further analysed by immunoautoradiography, radioimmunoassay, and immunofluorescence microscopy. Monoclonal antibody MF 20 was found to bind both atrial and ventricular MHC and stain all striated muscle cells of the adult chicken heart. In contrast, McAb B1 bound specifically to atrial myocytes in immunofluorescence studies, while immunoautoradiography and radioimmunoassay demonstrated the specificity of this antibody for the atrial MHC. Upon reacting these McAbs with myosin isolated from embryonic hearts where definitive atria and ventricles were present, the same specificity of antibody binding was observed. Immunofluorescence studies demonstrated that all striated muscle cells of the embryonic heart contained MHCs recognized by MF 20, while only atrial muscle cells were bound by B1. When extracts of presumptive atrial and ventricular tissue were reacted with MF 20 and B1, significant reactivity of MF 20 was first observed at stage 10 in the presumptive ventricle and thereafter this McAb reacted with all regions of the developing myocardium. Binding of B1 was detected approximately 1 day later at stage 15 and was confined to atrial-forming tissues. These data demonstrate antigenic similarity between adult and embryonic MHC isolated from atrial myocardium and suggest the expression of an atrial-specific MHC early in the regional differentiation of the heart.     

Two distinct nonmuscle myosin-heavy-chain mRNAs are differentially expressed in various chicken tissues. Identification of a novel gene family of vertebrate non-sarcomeric myosin heavy chains

Two distinct cDNA clones for nonmuscle myosin heavy chain (MHC) were isolated from a chicken fibroblast cDNA library by cross-hydridization under a moderate stringency with chicken gizzard smooth muscle MHC cDNA. These two fibroblast MHC and the gizzard MHC are each encoded in different genes in the chicken genome. Northern blot analysis showed that both of the nonmuscle MHC mRNAs were expressed not only in fibroblasts but also in a variety of tissues including brain, lung, kidney, spleen, and skeletal, cardiac and smooth muscles. However, the relative contents of the two nonmuscle MHC mRNAs varied greatly among tissues. The encoded amino acid sequences of the nonmuscle MHCs were highly similar to each other (81% identity) and to the smooth muscle MHC (81-84%), but much less similar to vertebrate skeletal muscle MHCs (38-41%) or to protista nonmuscle MHCs (35-36%). A phylogenic tree of MHC isoforms was constructed by calculating the similarity scores between these MHC sequences. An examination of the tree showed that the vertebrate sarcomeric (skeletal and cardiac) MHC isoforms are encoded in a very closely related multigene family, and that the vertebrate non-sarcomeric (smooth muscle and nonmuscle) MHC isoforms define a distinct, less conserved MHC gene family.     

The transformational potential of striated muscle in hydromedusae.

Isolated striated muscle tissue of the Anthomedusa Podocoryne carnea participates in the regeneration of a functional manubrium (the feeding organ of medusae) when it is combined homoclonally with endodermal cells of the medusa umbrella. The morphogenetic potential of striated muscle cells in this regeneration process was evaluated by combining nuclear labeled striated muscle cells with some unlabeled endoderm cells. Histological and autoradiographical results demonstrate that transformation of striated muscle cells into smooth muscle cells of the ectoderm and also into endoderm cells must have occurred in the regenerate. The potential for cell transformation of isolated striated muscle cells of Podocoryne carnea is discussed and it is postulated that under appropriate conditions all cell types necessary for the regeneration of a manubrium can be formed from striated muscle cells.     

The homeobox gene Otx of the jellyfish Podocoryne carnea: role of a head gene in striated muscle and evolution

In many bilaterian animals members of the Otx gene family are expressed in head or brain structures. Cnidarians, however, have no clearly homologous head and no distinct brain; but an Otx homolog from the jellyfish Podocoryne carnea is highly conserved in sequence and domain structure. Sequence similarities extend well beyond the homeodomain and Podocoryne Otx can be aligned over its entire length to human OTX1, OTX2, and CRX. The overall structure of Otx is better conserved from Podocoryne to deuterostomes while protostomes appear to be more derived. In contrast, functions seem to be conserved from protostomes to vertebrates but not in Podocoryne or echinoderms. Podocoryne Otx is expressed only during medusa bud formation and becomes restricted to the striated muscle of medusae. Cnidaria are the most basal animals with striated muscle. Podocoryne polyps have no striated muscle and no Otx expression; both appear only during the asexual medusa budding process. The common ancestor of all animals that gave rise to cnidarians, protostomes, and deuterostomes already had an Otx gene more similar to today's Podocoryne and human homologs than to Drosophila otd, while the head-specific function appears to have evolved only later.     

The germ line and somatic stem cell gene Cniwi in the jellyfish Podocoryne carnea

In most animal phyla from insects to mammals, there is a clear division of somatic and germ line cells. This is however not the case in plants and some animal phyla including tunicates, flatworms and the basal phylum Cnidaria, where germ stem cells arise de novo from somatic cells. Piwi-like genes represent essential stem cell genes in diverse multicellular organisms. The cnidarian Piwihomolog Cniwiwas cloned from Podocoryne carnea, a hydrozoan with a full life cycle. CniwiRNA is present in all developmental stages with highest levels in the egg and the medusa. In the adult medusa, Cniwi expression is prominent in the gonads where it likely functions as a germ stem cell gene. The gene is also expressed, albeit at low levels, in differentiated somatic cells like the striated muscle of the medusa. Isolated striated muscle cells can be induced to transdifferentiate into smooth muscle cells which proliferate and differentiate into nerve cells. Cniwi expression is upregulated transiently after induction of transdifferentiation and again when the emerging smooth muscle cells proliferate and differentiate. The continuous low-level expression of an inducible stem cell gene in differentiated somatic cells may underlie the ability to form medusa buds from polyp cells and explain the extraordinary transdifferentation and regeneration potential of Podocoryne carnea.     

The homeobox gene Msx in development and transdifferentiation of jellyfish striated muscle

Bilaterian Msx homeobox genes are generally expressed in areas of cell proliferation and in association with multipotent progenitor cells. Likewise, jellyfish Msx is expressed in progenitor cells of the developing entocodon, a cell layer giving rise to the striated and smooth muscles of the medusa. However, in contrast to the bilaterian homologs, Msx gene expression is maintained at high levels in the differentiated striated muscle of the medusa in vivo and in vitro. This tissue exhibits reprogramming competence. Upon induction, the Msx gene is immediately switched off in the isolated striated muscle undergoing transdifferentiation, to be upregulated again in the emerging smooth muscle cells which, in a stem cell like manner, undergo quantal cell divisions producing two cell types, a proliferating smooth muscle cell and a differentiating nerve cell. This study indicates that the Msx protein may be a key component of the reprogramming machinery responsible for the extraordinary transdifferentation and regeneration potential of striated muscle in the hydrozoan jellyfish.     

Complete primary structure of a scallop striated muscle myosin heavy chain. Sequence comparison with other heavy chains reveals regions that might be critical for regulation

We have determined the primary structure of the myosin heavy chain (MHC) of the striated adductor muscle of the scallop Aequipecten irradians by cloning and sequencing its cDNA. It is the first heavy chain sequence obtained in a directly Ca(2+)-regulated myosin. The 1938-amino acid sequence has an overall structure similar to other MHCs. The subfragment-1 region of the scallop MHC has a 59-62% sequence identity with sarcomeric and a 52-53% identity with nonsarcomeric (smooth and metazoan nonmuscle) MHCs. The heavy chain component of the regulatory domain (Kwon, H., Goodwin, E. B., Nyitray, L., Berliner, E., O'Neall-Hennessey, E., Melandri, F. D., and Szent-Gy?rgyi, A. G. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4771-4775) starts at either Leu-755 or Val-760. Ca(2+)-sensitive Trp residues (Wells, C., Warriner, K. E., and Bagshaw, C. R. (1985) Biochem. J. 231, 31-38) are located near the C-terminal end of this segment (residues 818-827). More detailed sequence comparison with other MHCs reveals that the 50-kDa domain and the N-terminal two-thirds of the 20-kDa domain differ substantially between sarcomeric and nonsarcomeric myosins. In contrast, in the light chain binding region of the regulatory domain (residues 784-844) the scallop sequence shows greater homology with regulated myosins (smooth muscle, nonmuscle, and invertebrate striated muscles) than with unregulated ones (vertebrate skeletal and heart muscles). The N-terminal 25-kDa domain also contains several residues which are preserved only in regulated myosins. These results indicate that certain heavy chain sites might be critical for regulation. The rod has features typical of sarcomeric myosins. It is 52-60% and 30-33% homologous with sarcomeric and nonsarcomeric MHCs, respectively. A Ser-rich tailpiece (residues 1918-1938) is apparently nonhelical.     

Transdifferentiation and regeneration in vitro

Striated muscle tissue and endoderm can be isolated from the anthomedusa Podocoryne carnea. The isolates are uncontaminated by other cell types and can be cultivated in artificial seawater for months without undergoing autonomous regeneration. However, if the endoderm is combined with collagenase-treated striated muscle, a regeneration process is initiated which leads to the formation of the sexual and feeding organ (manubrium) of the medusa. The original endoderm and striated muscle are replaced in the regenerate by at least seven new cell types, including gametes. Labeling experiments with [³H]thymidine and experiments in which mitosis is inhibited in either the striated muscle or the endoderm with mitomycin C demonstrate that the striated muscle is able to transdifferentiate into all the cell types found in the regenerate. With the possible exception of ectodermal smooth muscle this statement is also valid for the endoderm.     

Characterization of diverse forms of myosin heavy chain expressed in adult human skeletal muscle.

In an attempt to define myosin heavy chain (MHC) gene organization and expression in adult human skeletal muscle, we have isolated and characterized genomic sequences corresponding to different human sarcomeric MHC genes (1). In this report, we present the complete DNA sequence of two different adult human skeletal muscle MHC cDNA clones, one of which encodes the entire light meromyosin (LMM) segment of MHC and represents the longest described MHC cDNA sequence. Additionally, both clones provide new sequence data from a 228 amino acid segment of the MHC tail for which no protein or DNA sequence has been previously available. One clone encodes a "fast" form of skeletal muscle MHC while the other clone most closely resembles a MHC form described in rat cardiac ventricles. We show that the 3' untranslated region of skeletal MHC cDNAs are homologous from widely separated species as are cardiac MHC cDNAs. However, there is no homology between the 3' untranslated region of cardiac and skeletal muscle MHCs. Isotype-specific preservation of MHC 3' untranslated sequences during evolution suggests a functional role for these regions.     

Characterization of myosin heavy chains in cultured aorta smooth muscle cells. A comparative study

Myosin heavy chains (MHCs) from rat aorta smooth muscle cells were analyzed prior to and after these cells were placed into cell culture using sodium dodecyl sulfate-5% polyacrylamide gels, immunoblots, and two-dimensional peptide maps of tryptic digests. Rat aorta smooth muscle cells prior to culture were found to contain two MHCs (mass = 204 and 200 kDa) which cross-reacted with antibodies raised to smooth muscle myosin, but not with antibodies raised to platelet myosin. Tryptic peptide maps of these two MHCs showed no major differences when compared to each other and to maps of vas deferens and uterus smooth muscle MHCs. When rat aorta smooth muscle cells were placed into culture, the MHCs isolated from the cell extracts differed, depending on whether the cells were rapidly growing or postconfluent. Extracts from log-phase cultures contained predominantly MHCs that migrated more rapidly than smooth muscle myosin in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (mass = 196 kDa) and cross-reacted with antibodies raised to platelet myosin, but not to smooth muscle myosin. Tryptic peptide maps of this MHC were very similar to those obtained with MHCs from non-muscle sources such as platelets and fibroblasts. In contrast, extracts from postconfluent rat aorta cell cultures contained three MHCs (mass = 204, 200, and 196 kDa). Using immunoblots and peptide maps, the fastest migrating MHC was found to be identical to the 196-kDa non-muscle MHC, while the two slower migrating MHCs had the same properties as aorta smooth muscle MHCs prior to culture. These results suggest that smooth muscle cells grown in primary culture contain predominantly (greater than 80%) non-muscle myosin while actively growing, but at a postconfluent stage, contain more equivalent amounts of smooth muscle and non-muscle myosins.     

Immunolocalization of myotonic dystrophy protein kinase in corbular and junctional sarcoplasmic reticulum of human cardiac muscle

The subcellular localization of myotonic dystrophy protein kinase has been examined in human cardiac muscles with confocal laser-scanning microscopy and electron microscopy. A polyclonal antibody was produced against the synthesized peptide from a human kinase cDNA clone. We checked the antibody specificity for cardiac myotonic dystrophy protein kinase using an immunoblotting technique. Immunoblotting of extract from human cardiac muscles showed mainly 70 kDa and 55 kDa molecular weight bands. Confocal images of the protein kinase immunostaining showed striated banding patterns similar to those of skeletal muscles. In addition, the kinase was strongly detected around the intercalated disc. Immunoelectron microscopy showed that the kinase was mainly expressed in both corbular and junctional sarcoplasmic reticulum, but not in network sarcoplasmic reticulum. These results suggest that myotonic dystrophy protein kinase may be involved in the modulation of Ca2+ homeostasis in cardiac myofibres. © 1998 Chapman & Hall     

Gene duplication and recruitment of a specific tropomyosin into striated muscle cells in the jellyfish Podocoryne carnea

Cnidaria are the most basal animal phylum in which smooth and striated muscle cells have evolved. Since the ultrastructure of the mononucleated striated muscle is similar to that of higher animals, it is of interest to compare the striated muscle of Cnidaria at the molecular level to that of triploblastic phyla. We have used tropomyosins, a family of actin binding proteins to address this question. Throughout the animal kingdom, a great diversity of tropomyosin isoforms is found in non-muscle cells but only a few conserved tropomyosins are expressed in muscle cells. Muscle tropomyosins are all similar in length and share conserved termini. Two cnidarian tropomyosins have been described previously but neither of them is expressed in striated muscle cells. Here, we have characterized a new tropomyosin gene Tpm2 from the hydrozoan Podocoryne carnea. Expression analysis by RT-PCR and by whole mount in situ hybridization demonstrate that Tpm2 is exclusively expressed in striated muscle cells of the medusa. The Tpm2 protein is shorter in length than its counterparts from higher animals and differs at both amino and carboxy termini from striated muscle isoforms of higher animals. Interestingly, Tpm2 differs considerably from Tpm1 (only 19% identity) which was described previously in Podocoryne carnea. This divergence indicates a functional separation of cytoskeletal and striated muscle tropomyosins in cnidarians. These data contribute to our understanding of the evolution of the tropomyosin gene family and demonstrate the recruitment of tropomyosin into hydrozoan striated muscles during metazoan evolution. J. Exp. Zool. (Mol. Dev. Evol.) 285:378-386, 1999.     

Diversity of fast myosin heavy chain expression during development of gastrocnemius, bicep brachii, and posterior latissimus dorsi muscles in normal and dystrophic chickens

The expression of fast myosin heavy chain (MHC) isoforms was examined in developing bicep brachii, lateral gastrocnemius, and posterior latissimus dorsi (PLD) muscles of inbred normal White Leghorn chickens (Line 03) and genetically related inbred dystrophic White Leghorn chickens (Line 433). Utilizing a highly characterized monoclonal antibody library we employed ELISA, Western blot, immunocytochemical, and MHC epitope mapping techniques to determine which MHCs were present in the fibers of these muscles at different stages of development. The developmental pattern of MHC expression in the normal bicep brachii was uniform with all fibers initially accumulating embryonic MHC similar to that of the pectoralis muscle. At hatching the neonatal isoform was expressed in all fibers; however, unlike in the pectoralis muscle the embryonic MHC isoform did not disappear. With increasing age the neonatal MHC was repressed leaving the embryonic MHC as the only detectable isoform present in the adult bicep brachii muscle. While initially expressing embryonic MHC in ovo, the post-hatch normal gastrocnemius expressed both embryonic and neonatal MHCs. However, unlike the bicep brachii muscle, this pattern of expression continued in the adult muscle. The adult normal gastrocnemius stained heterogeneously with anti-embryonic and anti-neonatal antibodies indicating that mature fibers could contain either isoform or both. Neither the bicep brachii muscle nor the lateral gastrocnemius muscle reacted with the adult specific antibody at any stage of development. In the developing posterior latissimus dorsi muscle (PLD), embryonic, neonatal, and adult isoforms sequentially appeared; however, expression of the embryonic isoform continued throughout development. In the adult PLD, both embryonic and adult MHCs were expressed, with most fibers expressing both isoforms. In dystrophic neonates and adults virtually all fibers of the bicep brachii, gastrocnemius, and PLD muscles were identical and contained embryonic and neonatal MHCs. These results corroborate previous observations that there are alternative programs of fast MHC expression to that found in the pectoralis muscle of the chicken (M.T. Crow and F.E. Stockdale, 1986, Dev. Biol. 118, 333-342), and that diversification into fibers containing specific MHCs fails to occur in the fast muscle fibers of the dystrophic chicken. These results are consistent with the hypothesis that avian muscular dystrophy is a developmental disorder that is associated with alterations in isoform switching during muscle maturation.