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.     

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.     

Conservation of Brachyury, Mef2, and Snail in the myogenic lineage of jellyfish: a connection to the mesoderm of bilateria

One major difference between simple metazoans such as cnidarians and all the bilaterian animals is thought to involve the invention of mesoderm. The terms diploblasts and triploblasts are therefore, often used to group prebilaterian and bilaterian animals, respectively. However, jellyfish contain well developed striated and smooth muscle tissues that derive from the entocodon, a mesoderm-like tissue formed during medusa development. We investigated the hypothesis, that the entocodon could be homologous to the third germ layer of bilaterians by analyzing the structures and expression patterns of the homologues of Brachyury, Mef2, and Snail in the jellyfish Podocoryne carnea. These are regulatory genes from the T-box, MADS-box and zinc finger families known to play important roles in bilaterian mesoderm patterning and muscle differentiation. The sequence and expression data demonstrate that the genes are structurally and functionally conserved and even more similar to humans or other deuterostomes than to protostome model organisms such as Drosophila or Caenorhabditis elegans. Based on these data we conclude that the common ancestor of the cnidarians and bilaterians not only shared genes that play a role in regulating myogenesis but already used them to develop and differentiate muscle systems similar to those of triploblasts.     

Ubiquitin-proteasome-mediated degradation, intracellular localization, and protein synthesis of MyoD and Id1 during muscle differentiation

Mammalian skeletal myogenesis results in the differentiation of myoblasts to mature syncytial myotubes, a process regulated by an intricate genetic network of at least three protein families: muscle regulatory factors, E proteins, and Id proteins. MyoD, a key muscle regulatory factor, and its negative regulator Id1 have both been shown to be degraded by the ubiquitin-proteasome system. Using C2C12 cells and confocal fluorescence microscopy, we showed that MyoD and Id1 co-localize within the nucleus in proliferating myoblasts. In mature myotubes, in contrast, they reside in distinctive subcellular compartments, with MyoD within the nucleus and Id1 exclusively in the cytoplasm. Cellular abundance of Id1 was markedly diminished from the very onset of muscle differentiation, whereas MyoD abundance was reduced to a much lesser extent and only at the later stages of differentiation. These reductions in MyoD and Id1 protein levels seem to result from a change in the rate of protein synthesis rather than the rate of degradation. In vivo protein stability studies revealed that the rates of ubiquitin-proteasome-mediated MyoD and Id1 degradation are independent of myogenic differentiation state. Id1 and MyoD were both rapidly degraded, each with a t 1/2 approximately = 1 h in myoblasts and in myotubes. Furthermore, relative protein synthesis rates for MyoD and Id1 were significantly diminished during myoblast to myotube differentiation. These results provide insight as to the interaction between MyoD and Id1 in the process of muscle differentiation and have implications for the involvement of the ubiquitin-proteasome-mediated protein degradation and protein synthesis in muscle differentiation and metabolism under abnormal and pathological conditions.     

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.     

Inhibition of muscle-specific gene expression by Id3: requirement of the C-terminal region of the protein for stable expression and function.

We have examined the role of an Id-like protein, Id3 (also known as HLH462), in the regulation of muscle-specific gene expression. Id proteins are believed to block expression of muscle-specific genes by preventing the dimerization between ubiquitous bHLH proteins (E proteins) and myogenic bHLH proteins such as MyoD. Consistent with its putative role as an inhibitor of differentiation, Id3 mRNA was detected in proliferating skeletal muscle cells, was further induced by basic fibroblast growth factor (bFGF) and was down-regulated in differentiated muscle cultures. Overexpression of Id3 efficiently inhibited the MyoD-mediated activation of the muscle-specific creatine kinase (MCK) reporter gene. Deletion analysis indicated that the C-terminal 15 amino acids of Id3 are critical for the full inhibitory activity while deleting up to 42 residues from the C-terminus of the related protein, Id2, did not affect its ability to inhibit the MCK reporter gene. Chimeric protein containing the N-terminal region of Id3 and the C-terminus of Id2 was also non-functional in transfected cells. In contrast, wild-type Id3, the C-terminal mutants, and the Id3/Id2 chimera could all interact with the E-protein E47in vitro. Additional studies indicated that truncation of the Id3 C-terminus might have adversely affected the expression level of the mutant proteins but the Id3/Id2 chimera was stably expressed. Taken together, our results revealed a more complex requirement for the expression and proper function of the Id family proteins than was hitherto expected.     

The protein Id: a negative regulator of helix-loop-helix DNA binding proteins

We have isolated a cDNA clone encoding a novel helix-loop-helix (HLH) protein, Id. Id is missing the basic region adjacent to the HLH domain that is essential for specific DNA binding in another HLH protein, MyoD. An in vitro translation product of Id can associate specifically with at least three HLH proteins (MyoD, E12, and E47) and attenuate their ability to bind DNA as homodimeric or heterodimeric complexes. Id is expressed at varying levels in all cell lines tested. In three cell lines that can be induced to undergo terminal differentiation, Id RNA levels decrease upon induction. Transfection experiments indicate that over-expression of Id inhibits the trans-activation of the muscle creatine kinase enhancer by MyoD. Based on these findings, we propose that HLH proteins lacking a basic region may negatively regulate other HLH proteins through the formation of nonfunctional heterodimeric complexes.     

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.