Regellalll;oll 177S I I I I I I I I I I I I I I I I I I I I I I I I I I I I I MYOSIN HEAVY CHAIN GENE IN DUGESIA (G,) TIGRINA: A TOOL FOR STUDYING MUSCLE REGENERATION IN PLANARIANS Francese CEBRIA, Marcelo VISPO, David BUENO, Salvador CARRANZA, Phillip NEWMARK and Rafael ROMERO Dept. Gen/3/ica. Fac.Biologia. U.Barcelona. Avda. Diagonal, 645, 08071 Barcelona, Calalonia (Spain) In most organisms the main body axes are specified during embryonic development. However, in freshwater planarian Dugesia (G,) ligrina (Platyhelminlhes, Turbellaria) the body axes musl be continuously re-specified in the adult period (Bueno et ai" 1996a) due to the 9reat morphol09ical plasticity shown by these organisms. Planarians can grow or degrow in a continuous manner depending on food availability and temperature, and they can regenerate a new organism from a small piece of its body (Romero & Baguna, 1988). During regeneration three basic processes must occur: a) a redefinition of the body pattern to establish the territories where the missing structures will appear; b) stem cells called neoblasts (Brondsted, 1969) proliferate and differentiate into all the cellular types necessary to form the missing structures; and c) these "de novo" cells become properly arranged in time and space to restitute the original body pattern. One possible way to study how these processes take place is to consider the differentiation and restitution of the pattern of individual tissues and cell types and to examine possible relationships with the general mechanisms of regeneration. One of the planarian cell types of particularinterestis the muscle cells. These cells respond early in regeneration by closing the wound (contracting the body wall) in the zone where the regeneration blastema will appear, and together constitute what may be considered the skeleton that holds together all the other cell types. The complex and highly organized muscle net is arranged in several subepidermal layers of muscle fibers (Bag una, 1973) and may be distinguished using fluorescein-labeled phalloidin (a phallotoxin that binds specifically to F-actin; Wulf, 1979) as a marker and visualization with conventional epifluorescence or confocal microscopy. There is an outer layer of circular fibers that runs below the basal lamina and over an intermediate layer of diagonal fibers. Below these diagonal fibers one may observe the longitudinal fibers. This pattern of fibers from the outer surface to the inside of the body is observed in both dorsal and ventral surfaces. These surfaces are themseives connected through dorsoventral fibers that show their highest densities in the head, tail and lateral regions of the body. Careful examination of dorsal longitudinal fibers shows that they seem to converge upon a zone near the anterior border of the organism. In contrast. ventral longitudinal fibers diverge in a fan-shaped pattern as they approach the anterior border. During the regeneration process a regeneration blastema is formed in the wound region. Within this blastema, the new structures, including the new muscle fibers, will appear. Using fluorescein-labeled phalloidin one may distinguish the disorganized and thin fibers within the 3 day regeneration blastema. After 3 days of regeneration the muscle fibers become organized and by the fourth day of regeneration the re-establishment of the original muscle pattern can be observed. At 6 days of regeneration the muscle pattern is completely restored with the number of fibers increasing progressively in each of the following days. However, because phalloidin binds to differentiated muscle 'cells, we are unable to study the origin of these cells within the regeneration blastema. To gain insight into the differentiation of the muscle cells within the blastema, we have used a monoclonal antibody (MAb) called TMUS-13 that immunoreacts not only with all the muscle fibers (fig.1) but also with cells that appear to be neoblasts (myoblasts) committed to a differentiation pathway towards muscle cell identity (Bueno et al., 1996b). To characterise at the molecular level the antigen recognised by this MAb, a cDNA expression library constructed in Lambda Zap vector was screened with TMUS- 13 MAb using standard procedures. We isolated several positive clones that, by PCR and digestion analysis, seemed to contain an equally sized cDNA insert. Sequence anaiysis (BLAST WWW server) demonstrates that the TMUS-13 antigen is a myosin II heavy chain (MHC) gene (fig.2), the first identified from planarians. This MHC shows a high degree of sequence similarity with the MHC genes from Schistosoma mansoni (Platyhelminthes, Trematoda) and other species throughout phylogeny, from C.elegans to human. Myosin IIis a hexameric proteinwithtwo heavy and four light chains. Each heavy chain has an amino-terminal domain that folds into a globular head. The remainder of the myosin 11heavy chain dimerizes to form an alpha-helical coiled coil tail (Kiehart, 1990). The isolated cDNA fragment corresponds to this last domain. To isolate the Imus.13 gene, we have used the 1293 bp long cDNA to screen a D.(G.)/igrina genomic library and we are in the process of sequencing the complete gene. - - --- - --- ------------- Figure 1. Muscle net of the body wall of Dugesia (G) tigrina. immunostained with TMUS-13. Note the circular (from left to right). longitudinal (from up to dovvn) and diagonal muscle fibers.