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.     

Ultrastructure of alimentary tract formation in embryos of two insect species: Melasoma saliceti and Chrysolina pardalina (Coleoptera, Chrysomelidae)

Embryogenesis of the alimentary tract in two chrysomelid species (Chrysolina pardalina and Melasoma saliceti) is described. The embryonic development of both species lasts 7days at room temperature. Stomodaeum and proctodaeum invaginate at the anterior and posterior ends of the germ band. Together with the ectodermal tissue the endoderm cells also enter into the embryo. The anterior and posterior parts of the alimentary tract wedge into the yolk in the form of conical structures. The endodermal cells remain at the yolk surface and start migration over the yolk mass as two lateral bands of cells. The endoderm is always accompanied by mesoderm. On the fifth day of development the endodermal cells together with the mesoderm layer spread over the ventral and dorsal sides of the yolk mass and form the single layered primordium of the midgut epithelium. On the sixth day of development a basal lamina appears between the endoderm and the mesoderm cells and differentiation of both tissues starts. The endodermal epithelium cells change shape from flat to cuboidal and eventually into columnar. Mesoderm cells differentiate into muscle and tracheae. On the 7thday of development stomodaeum and proctodaeum become lined with cuticle and the midgut becomes covered with microvilli. The yolk cells populating the yolk mass do not contribute to midgut formation in the species studied.     

Molecular cloning and characterization of Kremen, a novel kringle-containing transmembrane protein

Kringle domain, a triple-disulfide-linked domain, is conserved in diverse proteins which play important roles in various biological processes. We cloned Kremen, a novel member of kringle-containing proteins, using a newly developed unique strategy, 'Kringle-SAGE (serial analysis of gene expression)', which enables comprehensive analysis of kringle-containing proteins. Kremen is likely to be a type-I transmembrane protein composed of 473 amino acid residues. Kremen has a kringle domain, a WSC domain, and CUB domains in the extracellular region, while the intracellular region has no conserved motif involved in signal transduction. In the mouse embryo, the Kremen mRNA level, which was increased during embryonic development, was localized in the apical ectodermal ridge of limb buds, myotome, and sensory organs (e.g. optic vesicle, otic vesicle, nasal pit). In the adult mouse, Kremen mRNA was expressed in a variety of tissues with a relatively strong expression in the lung, heart, and skeletal muscle. Kremen mRNA expression in C2C12 and NIE-115 cells increased during respective differentiation into muscular and neural cells. These results suggest a potential role for Kremen in the regulation of cellular responses upon extracellular stimulus or cell-cell interaction in neuronal and/or muscle cells. Kringle-SAGE is expected to facilitate further elucidation of structure and functions of kringle proteins.     

Mef2s are required for thick filament formation in nascent muscle fibres

During skeletal muscle differentiation, the actomyosin motor is assembled into myofibrils, multiprotein machines that generate and transmit force to cell ends. How expression of muscle proteins is coordinated to build the myofibril is unknown. Here we show that zebrafish Mef2d and Mef2c proteins are required redundantly for assembly of myosin-containing thick filaments in nascent muscle fibres, but not for the earlier steps of skeletal muscle fibre differentiation, elongation, fusion or thin filament gene expression. mef2d mRNA and protein is present in myoblasts, whereas mef2c expression commences in muscle fibres. Knockdown of both Mef2s with antisense morpholino oligonucleotides or in mutant fish blocks muscle function and prevents sarcomere assembly. Cell transplantation and heat-shock-driven rescue reveal a cell-autonomous requirement for Mef2 within fibres. In nascent fibres, Mef2 drives expression of genes encoding thick, but not thin, filament proteins. Among genes analysed, myosin heavy and light chains and myosin-binding protein C require Mef2 for normal expression, whereas actin, tropomyosin and troponin do not. Our findings show that Mef2 controls skeletal muscle formation after terminal differentiation and define a new maturation step in vertebrate skeletal muscle development at which thick filament gene expression is controlled.