Studies on hydroid differentiation. VII. The hydrozoan stolon

The stolon of the colonial marine hydroid Podocoryne carnea differentiates sequentially as a function of age, forming four distinguishable regions characterized by epidermal cell differentiation: The Tip, New Stolon, Cnidogenic Masses, Old Stolon. Radioautographs of sections of colonies exposed to tritiated thymidine show that although cells of the epidermis and gastrodermis of the stolon incorporate the nucleoside into acid stable polynucleotide, cells of the stolon tips do not. Stolon extension is not, therefore, the result of a localized meristem-like growth zone. Stolon branching and new polyp formation are, similarly, not signaled by increased thymidine incorporation. The initial event heralding these morphogenetic activities appears to be the reorientation of epidermal cells along a new axis, and the acquisition of perisarc dissolving ability. This evidence is contraindicative of direct dependence of colony form on colony growth. The larger part of stolon epidermal cells are organized into cnidogenic masses where cnidocytes and possibly other amoebocytic cells are produced. Although no mitotic figures have been observed in gastroderm cells of the stolon, thymidine incorporation in this tissue occurs with the same frequency as it does in epidermis. Considerable numbers of gastroderm cells can be found in the gastric cavity. Frequently these and gastroderm cells in the stolon and polyps contain more than one nucleus.     

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.     

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.     

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.     

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 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 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 fibrous system of the extracellular matrix of Podocoryne carnea and its degradation by the subumbrellar plate endoderm demonstrated by a monoclonal antibody

Homogenized fragments of crude umbrellar material of the hydromedusa Podocoryne carnea was injected into BALB/c mice. The immunization resulted in the isolation of a monoclonal antibody designated 3A1 which specifically binds to fibrils in the mesogloeal extracellular matrix (ECM) of hydromedusae. In vivo, the architecture and the ultrastructure of the fibrous system in the outer mesogloea (outer ECM) of Podocoryne carnea, and its degradation under in vitro conditions have been described by morphological and immunological criteria. In vivo, 120-150 A thick, striated fibrils (with periodicities up to 50 nm) form a threedimensional network which fills in the entire outer ECM. Vertical fibers (up to 150 nm in diameter) penetrate the three-dimensional network and branch at the subumbrellar and the exumbrellar side. The vertical fibers show uniform distribution over the entire outer ECM. The branches impinge on a dense matrix (about 30 nm in thickness) covering the exumbrellar and subumbrellar surface. In vitro, the fibrillar system does not alter in its basic pattern, neither in isolated outer ECM, nor in portions of outer ECM which is either covered by the exumbrella, or which is attached to both: the exumbrella at the outside, and the subumbrellar plate endoderm at the inner side. After removal of the exumbrellar cells in the latter portions, a characteristic pattern of selective degradation of the outer ECM by the endodermal cells is observed. This process involves three distinct steps: an initial extracellular condensation within the ECM fibrillar network, followed by intercellular internalization of the fibrillar elements and subsequent endocytosis of ECM material. The first step immediately follows the removeal procedure of the exumbrellar cells and is completed within minutes. This process cannot be interrupted by dihydrocytochalasin B (H(2)CB). The second step lasts 24-48 hr, is mediated by cell mechanisms, and can be stopped by H(2)CB. The third step is a slow process (of up to 14 days). It involves intercellular degradation of fibrillar material, endocytosis, and completion of digestion within lysosomes.     

Studies on submaxillary gland immunoreactive glucagon.

Biologically active immunoreactive glucagon is present in submaxillary gland of rat, mouse, guinea pig and human and can be extracted by saline adjusted to pH 2.8 with HCl. Chromatography on Sephadex G-150 indicates its molecular weight to be 29,000. It has similar immunologic characteristics as pancreatic glucagon. It is biologically active and elevates plasma glucose and insulin when injected intraperitoneally into rats. Compared to pancreatic glucagon, the hyperglycemic effect persists much longer. It competes with pancreatic glucagon for binding to specific glucagon receptors of rat liver plasma membranes. It is stable to pH changes, however, urea dissociates it into several smaller molecular weight fragments including that of 3500. It appears to be an aggregate of smaller glucagon molecules and is not responsible for immunoreactive glucagon in totally eviscerated rats. $\text{In vitro}$, the submaxillary gland does not release immunoreactive glucagon in response to arginine or glucose.