ROLE OF THE BRACHIAL SOMITES IN THE DEVELOPMENT OF THE APPENDICULAR MUSCULATURE IN RAT EMBRYOS

DiI, a fluorescent lipophilic dye, was micro-injected into the brachia l somites of 10.5 day rat embryos to determine whether these somites c an contribute cells to the development of the fore-limb bud. The injec ted embryos were cultured and harvested at the 20-25-somite stage. The dye did not interfere with somitogenesis because, at the injection si te, the DiI-labelled somites were able to differentiate into dermomyot ome and sclerotome. We have analyzed cryo-sections of 20-21-somite sta ge embryos and were unable detect the presence of DiI-labelled cells i n the fore-limb buds. However, at the 22-somite stage, a few DiI-posit ive cells were found in the proximal region of the limb bud. These lab elled cells had migrated into the limb from the lateral border of the dermomyotome. From the 23-somite stage onwards, there were even more D iI-positive cells inside the limb. We have performed an additional set of experiments to confirm that the somitic cells do have the ability to invade and colonize the limb bud. This was achieved by first labell ing newly formed somites isolated from the caudal region of 10.5 day e mbryos with Dil and then grafting them into corresponding regions in 8 -11-somite stage hosts. The donor somites were not orientated when the y were implanted into the host. However, this did not disrupt their ab ility to undergo normal somitogenesis. We have detected the presence o f DiI-positive cells in the limb buds of approximately 71% of the 19-3 0-somite stage embryos that have been examined. This is similar to wha t we obtained for the injected embryos. Nevertheless, there is one sli ght difference and that is the stage the somitic cells begin their inv asion of the limb. For the injected embryos, migration began at the 22 -somite stage but in the transplanted embryos, it commenced as early a s the 18-somite stage. We have also investigated the myogenic potentia l of the fore-limb bud at various stages of development to ascertain w hether there is a correlation between the stage the somitic cells firs t appear in the limb bud and the stage the bud acquires the capacity t o form skeletal muscles. This was realized by culturing fore-limb buds excised from 18-30-somite stage embryos conventionally and in the kid ney capsules of adult rats. In both methods, bone and cartilage were p resent in all of the cultures whereas skeletal muscles were only prese nt in cultured explants older than the 21-22-somite stage. The appeara nces of skeletal muscles in the cultures correlated exactly with the s tage somitic cells began their invasion of limb (as seen in the Dil in jected embryos). Based upon the indirect evidence that has been obtain ed, we tentatively propose that the brachial musculature of the rat em bryo is derived from the somites while bone and cartilage are formed b y the somatopleure of the limb. (C) 1993 Wiley-Liss, Inc.