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.     

Integrin and talin in the jellyfish Podocoryne carnea

We have isolated an integrin-beta and -alpha subunit from Podocoryne carnea (Cnidaria, Hydrozoa) and studied their expression in the life-cycle and during cell migration, in vitro transdifferentiation and regeneration. Comparison of the integrin expression pattern with a Podocoryne talin homologue by RT-PCR demonstrates that all three genes are maternal messages and continuously expressed in the life-cycle, in medusa development and in all medusae tissues. In situ hybridisation experiments confirm co-expression of both integrin subunits in the different life-stages. Integrin expression was furthermore studied in isolated striated muscle induced to transdifferentiate to new cell types, or grafted on ECM where the muscle adheres and migrates. Integrin expression was maintained continuously throughout both processes. These results suggest that in Podocoryne carnea processes such as cell migration and differentiation are not controlled by up- or downregulation of alternative integrin subunits, but by a single integrin heterodimer which activates different downstream signalling cascades.     

Groger H, callaerts P, gehring walter Jurgen, schmid volker. 1999. Gene duplication and recruitment of a specific tropomyosin into striated muscle cells in the jellyfish podocoryne carnea. J exp zool (Mol dev evol) 285:378-386

The third author of the article was listed as Walter Jurgen Gehring. His correct name is Walter Jakob Gehring. The authors regret the error.     

Effects of tumor promoters and diacylglycerol on the transdifferentiation of striated muscle cells of the medusa Podocoryne carnea to RF-amide positive nerve cells

Artemia sp (Tuticorin strain) was cultured at a density of 250 individuals 1^–1 at 35, 45, 60, 75 salinity using five combinations of groundnut oil cake, decayed cabbage leaves, single superphosphate and Baker's yeast as feed. Effects on survival, growth, and fecundity were noted.     

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.     

Animal pole determinants define oral-aboral axis polarity and endodermal cell-fate in hydrozoan jellyfish Podocoryne carnea

Cnidarians, in contrast with bilaterians, are generally considered to exhibit radial symmetry around a single body axis (oral-aboral) throughout their life-cycles. We have investigated how the oral-aboral axis is established in the hydrozoan jellyfish Podocoryne carnea. Vital labeling experiments showed that the oral end of the blastula derives from the animal pole region of the egg as has been demonstrated for other cnidarian species. Gastrulation is restricted to the oral pole such that the oral 20% of blastula cells give rise to endoderm. Unexpectedly, bisection experiments at the 8-cell stage showed that animal regions are able to develop into normally polarized larvae, but that vegetal (aboral) blastomeres completely fail to develop endoderm or to elongate. These vegetal-derived larvae also failed to polarize, as indicated by a lack of oral-specific RFamide-positive nerve cells and a disorganized tyrosinated tubulin-positive nerve net. A different result was obtained following bisection of the late blastula stage: aboral halves still lacked the capacity to develop endoderm but retained features of axial polarity including elongation of the larva and directional swimming. These results demonstrate for the first time in a cnidarian the presence of localized determinants responsible for axis determination and endoderm formation at the animal pole of the egg. They also show that axial polarity and endoderm formation are controlled by separable pathways after the blastula stage.     

A toxin homology domain in an astacin-like metalloproteinase of the jellyfish Podocoryne carnea with a dual role in digestion and development

 Metalloproteinases of the astacin family such as tolloid play major roles in animal morphogenesis. Cnidarians are thought to be evolutionary simple organisms and, therefore, a metalloproteinase from the marine hydrozoan Podocoryne carnea was analysed to evaluate the role of this conserved gene familiy at the base of animal evolution. Surprisingly, the proteinase domain of Podocornyne PMP1 is more similar to human meprin than to HMP1 from another hydrozoan, the freshwater polyp Hydra vulgaris. However, PMP1 and HMP1 both contain a small C-terminal domain with six cysteines that distinguishes them from other astacin-like molecules. Similar domains have been described only recently from sea anemone toxins specific for potassium channels. This toxin homology (Tox1) domain is clearly distinct from epidermal growth factor (EGF)-like domains or other cysteine-rich modules and terminates with the characteristic pattern CXXXCXXC with three out of six cysteines in the last eight residues of the protein. PMP1 is transiently expressed at various sites of morphogenetic activity during medusa bud development. In the adult medusa, however, expression is concentrated to the manubrium, the feeding organ, where the PMP1 gene is highly induced upon feeding. These disparate expression patterns suggest a dual role of PMP1 comparable to tolloid in development and, like astacin in the crayfish, also for food digestion. The Tox1 domain of PMP1 could serve as a toxin to keep the pray paralysed after ingestion, but as a sequence module such Tox1 domains with six cysteines are neither restricted to cnidarians nor to toxins. Received: 13 February 1998 / Accepted: 6 April 1998     

Smooth muscle fibers of Podocoryne carnea (Hydrozoa) demonstrated by a specific monoclonal antibody and their association with neurons showing FMRFamide-like immunoreactivity

Summary A mouse monoclonal antibody was prepared by using homogenized fragments of crude umbrella material of the hydromedusa Podocoryne carnea as an antigen. The selected clone produced an IgG (mAb sm-1) which decorated smooth muscle cells of hydrozoans. Immunohistochemical testing of mAb sm-1 on whole-mount preparations revealed reactivity with a cytoplasmic, formaldehyde-resistant antigen present in the smooth muscle cells, but absent in all other cell-types. The antibody can therefore be used as a selective and highly sensitive marker to trace the pattern of the smooth muscle system in hydrozoans. The tight association between smooth muscle cells and nerve cells which show FMRFamide-like immunoreactivity can be demonstrated in whole-mount preparations of the hydromedusa Podocoryne carnea with a polyclonal anti-FMRFamide antiserum and in double-labelling experiments.     

The proportion altering factor (PAF) and the in vitro transdifferentiation of isolated striated muscle of jellyfish into nerve cells

The effect of proportion altering factor (PAF) on the transdifferentiation of isolated striated muscle into RFamide-positive nerve cells was investigated. The factor reduces incorporation of 3H-thymidine into replicating DNA; the effect is concentration-dependent and reversible. Transdifferentiation to nerve cells increases by up to 60% if PAF is applied shortly before or at the time of initiation of DNA synthesis. When treatment was terminated 4 h before the start of S-phase or when PAF was applied at the peak of S-phase no increase in nerve cell formation was observed.     

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.     

Functional importance of the carboxyl-terminal region of striated muscle tropomyosin

Striated muscle tropomyosin (TM) interacts with actin and the troponin complex to regulate calcium-mediated muscle contraction. Previous work by our laboratory established that alpha- and beta-TM isoforms elicit physiological differences in sarcomeric performance. Heart myofilaments containing beta-TM exhibit an increased sensitivity to calcium that is associated with a decrease in the rate of relaxation and a prolonged time of relaxation. To address whether the carboxyl-terminal, troponin T binding domain of beta-TM is responsible for these physiological alterations, we exchanged the 27 terminal amino acids of alpha-TM (amino acids 258 -284) for the corresponding region in beta-TM. Hearts of transgenic mice that express this chimeric TM protein exhibit significant decreases in their rates of contraction and relaxation when assessed by ex vivo work-performing cardiac analyses. There are increases in the time to peak pressure and a dramatic increase in end diastolic pressure. In myofilaments, this chimeric protein induces depression of maximum tension and ATPase rate, together with a significant decrease in sensitivity to calcium. Our data are the first to demonstrate that the TM isoform-specific carboxyl terminus is a critical determinant of sarcomere performance and calcium sensitivity in both the whole heart and in isolated myofilaments.     

The HOX-like gene Cnox2-Pc is expressed at the anterior region in all life cycle stages of the jellyfish Podocoryne carnea

The marine jellyfish Podocoryne carnea (Cnidaria, Hydrozoa) has a metagenic life cycle consisting of a larva, a colonial polyp and a free-swimming jellyfish (medusa). To study the function of HOX genes in primitive diploblastic animals we screened a library of P. carnea cDNA using PCR primers derived from the most conserved regions in helix 1 and helix 3 of the homeobox. A novel gene, Cnox2-Pc, has been isolated and characterized. Cnox2-Pc is a HOX cluster-like gene, and its homeodomain shows similarity to the Deformed subfamily of HOM-C/HOX genes. In situ hybridization revealed that Cnox2-Pc is expressed in the anterior region of the larva, the polyp head, and the most apical ectoderm of the differentiating bud during medusa development. In adult medusa expression is restricted to the gastrovascular entoderm. The results suggest that Cnox2-Pc is involved in establishment of an anterior-posterior axis during development in primitive metazoans. Received: 23 February 1999 / Accepted: 27 July 1999     

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.     

Physiological significance of troponin T binding domains in striated muscle tropomyosin

Striated muscle tropomyosin (TM) plays an essential role in sarcomeric contraction and relaxation through its regulated movement on the thin filament. Previous work in our laboratory established that alpha- and beta-TM isoforms elicit physiological differences in sarcomeric performance. To address the significance of isoform-specific troponin T binding regions in TM, in this present work we replaced alpha-TM amino acids 175-190 and 258-284 with the beta-TM regions and expressed this chimeric protein in the hearts of transgenic mice. Hearts that express this chimeric protein exhibit significant decreases in rates of contraction and relaxation when assessed by ex vivo work-performing cardiac analyses. There are increases in time to peak pressure and in half-time to relaxation. These hearts respond appropriately to beta-adrenergic stimulation but do not attain control rates of contraction or relaxation. With increased expression of the transgene, 70% of the mice die by 5 mo of age without exhibiting gross pathological changes in the heart. Myofilaments from these mice have no differences in Ca(2+) sensitivity of percent maximum force, but there is a decrease in maximum tension development. Our data are the first to demonstrate that the troponin T binding regions of specific TM isoforms can alter sarcomeric performance without changing the Ca(2+) sensitivity of the myofilaments.     

Incorporation of fluorescently labeled actin and tropomyosin into muscle cells

The two major proteins in the I-bands of skeletal muscle, actin and tropomyosin, were each labeled with fluorescent dyes and microinjected into cultured cardiac myocytes and skeletal muscle myotubes. Actin was incorporated along the entire length of the I-band in both types of muscle cells. In the myotubes, the incorporation was uniform, whereas in cardiac myocytes twice as much actin was incorporated in the Z-bands as in any other area of the I-band. Labeled tropomyosin that had been prepared from skeletal or smooth muscle was incorporated in a doublet in the I-band with an absence of incorporation in the Z-band. Tropomyosin prepared from brain was incorporated in a similar pattern in the I-bands of cardiac myocytes but was not incorporated in myotubes. These results in living muscle cells contrast with the patterns obtained when labeled actin and tropomyosin are added to isolated myofibrils. Labeled tropomyosins do not bind to any region of the isolated myofibrils, and labeled actin binds to A-bands. Thus, only living skeletal and cardiac muscle cells incorporate exogenous actin and tropomyosin in patterns expected from their known myofibrillar localization. These experiments demonstrate that in contrast to the isolated myofibrils, myofibrils in living cells are dynamic structures that are able to exchange actin and tropomyosin molecules for corresponding labeled molecules. The known overlap of actin filaments in cardiac Z-bands but not in skeletal muscle Z-bands accounts for the different patterns of actin incorporation in these cells. The ability of cardiac myocytes and non-muscle cells but not skeletal myotubes to incorporate brain tropomyosin may reflect differences in the relative actin-binding affinities of non-muscle tropomyosin and the respective native tropomyosins. The implications of these results for myofibrillogenesis are presented.