1.2.4.1 Transdifferentiation o f Skeletal Muscle into Smooth Muscle and Nerve Cells
The jellyfish Podocoryne camea is an interesting example of the role of cell- substrate interactions in maintaining the stabüity of the differentiated state. Jellyfish are m arine m edusoid forms composed of a swimrning organ (the bell), a feeding organ (or manubrium) and tentacles, w here gametogenesis and fertilization occur. The bell of the m edusa is composed of three tissue layers; the exumbreUar tissue, the subumbrellar plate endoderm and the innermost layer, which is composed of mononucleate-striated muscle cells that are not cycling. The tissue layers are separated by ECM (mesoglea), which gives the animal its consistency (Schmid, 1992; Schmid et al., 1999).
The striated muscle layer can be isolated in small patches and cultured, m aintaining the association between muscle and the ECM. In this case, there is stable m aintenance of the striated phenotype and of the post-mitotic state. However, if the tissue is isolated w ith protease treatment, disrupting the muscle- ECM association, the muscle cells shrink and form aggregates. After 24 hours, DNA synthesis is detected in the isolated muscle. Two days after isolation, the striated muscle gives rise to smooth muscle cells, and four to five days later, it gives rise to nerve cells. These smooth muscle cells are similar to the ones in the manubrium , and can be identified by flagella formation and antigenic markers. The nerve cells show flagella, and stain positive for FMRF-amide, typical of cells from the nervous system. The smooth muscle cells continue to divide and give rise to m ore muscle cells and nerve cells, but the nerve cells do not divide further (Alder and Schmid, 1987; Schmid, 1992; Schmid and Alder, 1984). Amazingly, a whole new m anubrium can occasionally be regenerated in vitro from the striated
muscle cells. In these cases, striated muscle cells transdifferentiate into several non-muscle types (Schmid, 1992).
The evidence for the occurrence of transdifferentiation from striated muscle to smooth muscle and nerve cells in these experiments was indisputable, since the initial striated muscle population was very homogeneous and the new smooth muscle and nerve cells had phenotypic remnants from the parental cell, such as bundles of striated myofibrils (Schmid and Alder, 1984). It is interesting that the phenotypic conversion to smooth muscle cells was independent of DNA synthesis, w hilst the generation of nerve cells w as completely prevented by inhibition of DNA synthesis (Schmid, 1992; Schmid and Alder, 1984).
1.2.4.2 Factors Involved in Destabilising the Differentiated State
Several experiments have shown the importance of die extracellular matrix and cellular shape in the maintenance of a stable differentiated state in striated muscle cells from the jellyfish. If the muscle is isolated w ithout enzyme treatment, and grafted on cell-free ECM (Fig. 1.2.3 (a)), 60-80% of the cells migrate from their native m atrix to the cell free one (Fig. 1.2.3 (d)). DNA synthesis and transdifferentiation are mostly inhibited in the peripheral portions of the grafted matrix, w here the cells are more stretched. The percentage of cells undergoing DNA synthesis and transdifferentiation is highly increased in the contact zone between the two matrices, called the gap zone. Cells in this zone are not stretched. In this region, proteolytic activity degrades both types of ECM underneath the cells. If the isolated muscle is grafted onto ECM, but not stretahed, then the muscle cells leave their native ECM and cover the grafted one, forming floating grafts. Both matrices are gradually degraded by the cells, and after three days, there is DNA replication and transdifferentiation of the skeletal muscle cells into nerve cells (Fig. 1.2.3 (b) and (c); Schmid et al., 1992).
If the muscle is isolated w ith enzyme treatment and grafted on top of large ECM that is perm anently stretched, DNA synthesis and transdifferentiation are
inhibited in comparison to muscle that was not grafted (Schmid et al., 1992). If cells are grafted after being activated, between the first and second cell cycles, subsequent cell cycles are reduced but the cells can stiU transdifferentiate into neurons (Schmid et al., 1992).
These results have shown that only stretched extracellular matrix can inhibit DNA synthesis. Stretched ECM did not inhibit transdifferentiation of the skeletal muscle cells into neurons (which requires DNA synthesis). These experiments established the im portant role of cell configuration and the ECM in maintaining the stability of the differentiated state in the skeletal muscle cells from the jellyfish.
The role of the ECM and cell shape in the regulation of the differentiated state was further investigated. First, oxidation of the carbohydrates of the ECM with sodium -m etaperiodate and the use of a monoclonal antibody that recognises a carbohydrate epitope in the medusa ECM, aboHshed cell-matrix adhesion of grafted isolates (Reber-MuUer et al., 1994; Schmid, 1992; Schmid et al., 1999). These experiments showed that carbohydrate m ediated cell-ECM interactions are necessary for cell adhesion and spreading, and consequently for the stability of the differentiated state. Second, according to the hypothesis that the shape of the cells is im portant for the stabüity of the differentiated state, that stability can be challenged by drugs that act upon the cytoskeleton. The events of transdifferentiation in mechanically isolated muscle could be triggered by activators of protein kinase C, as well as by disassembly of actin füaments induced by düiydrocytochalasin B (Schmid, 1992; Schmid and Reber-Muller, 1995).
PCS
T
Figure 1.2.3 Schematic drawing summarizing the combination of isolated ECMs with
isolated striated muscle tissue from Podocoryne camea. Isolated striated muscle tissue
was grafted onto cell-free ECMs, isolated from polyps and medusae, with the help of a piece of coverslip (a). For floating cultures, the ECMs together with the adherent and spreading tissue, were removed from the coverslip (b, c); cultures with permanent stretched ECMs remained unchanged (d). Arrowheads mark the position of gap cells. Stippled areas-grafted ECMs from medusae or polyp; hatched areas-inner ECM to which the muscle tissue adheres; Pcs-pieces of broken glass; gm-grafted ECM from polyp or medusae; st-striated muscle; im-inner ECM; f- flagellum. See text for explanations. Adapted from Schmid et al. (1992).