MYOTUBE DRIVEN MYOGENIC RECRUITMENT OF CELLS DURING IN-VITRO MYOGENESIS

Muscular dysgenesis (mdg) is a recessive lethal mutation in the mouse which drastically affects skeletal muscle development during embryonic life. Physiologically, the disease is characterized by a complete par alysis resulting from a lack of excitation-contraction coupling. Exist ing electrophysiological, biochemical, and genetic evidence shows that mdg/mdg mice express a basic alteration of L-type voltage-sensitive C a2+ channels in skeletal muscle. Studies on mdg/mdg myotubes in primar y culture have shown that +/+ fibroblasts or +/+ Schwann cells may fus e with them and correct their functional deficiency by genetic complem entation. As the spontaneous formation of heterocaryons is thought to be an exclusive property of myoblasts, we asked whether fibroblasts ma y have changed their properties before fusion occurred. We used primar y cells issued from sciatic nerves dissected from newborn transgenic m ice carrying the pHuDes1-nls-LacZ transgene (Des-LacZ cells) as non-mu scle cells. These cells were mainly fibroblasts (80%) positive for Thy 1.1 and Schwann cells positive for S100. The cultures were negative fo r myogenic markers (desmin, troponin T), did not form myotubes long-te rm, and did not display significant activation of the muscle reporter gene (pHuDes1-nls-LacZ). After a few days in coculture with dysgenic o r normal myotubes, the muscle reporter gene (beta-galactosidase) was d etected both within dysgenic myotubes, correlating with the restoratio n of normal contractile activity, and normal myotubes. As well as conf irming that fusion takes place, this shows that Des-LacZ cells nuclei incorporated into recipient myotubes express their own myogenic genes. Moreover, individual mononucleated Des-LacZ cells expressing beta-gal actosidase were observed, indicating that myogenic genes were being ex pressed before fusion. This suggests a mechanism of myotube driven myo genic recruitment of cells during the in vitro myogenesis. Analysis of the distribution of the induced Des-LacZ cells (positive for beta-gal actosidase) in compartmentalized muscle cocultures showed that in the presence of dysgenic myotubes, these cells were equally distributed in both myotube free and enriched areas, whereas in the presence of norm al myotubes, the positive cells remained in close vicinity of the myot ubes. This difference could be explained by the fact that the dysgenic phenotype might include release of the induction process from its nor mal controls. Our results are consistent with the idea of a transcellu lar mechanism triggering myogenic differentiation in non-muscle cells, and that myotubes themselves are able to drive myogenic recruitment o f cells during the in vitro myogenesis. This phenomenon could be the r esult of either a myogenic induction in non-muscle cells, imposing a p henotypic change, or the activation of pre-myoblastic quiescent cells by the myotubes themselves. (C) 1995 Wiley-Liss, Inc.