Formation of the extracellular matrix during the epimorphic anterior regeneration of Owenia fusiformis: autoradiographical and in situ hybridization studies.

During post-traumatic regeneration of the polychaete annelid Owenia fusiformis, the extracellular matrix (ECM) formation was studied by light and electron microscopy and by histoautoradiography after incorporation of tritiated proline as marker for collagenic proteins. Three days after amputation, a new basement membrane was reformed in the blastema between the ectoderm and the mesoderm. At the same time, the cytoskeleton and the anchoring structures (hemidesmosomes) were differentiated in the basal part of the ectodermal cells. Four days after amputation, collagen fibers appeared in the extracellular matrix newly reformed between the ectodermal and mesodermal layers. The existence of a proximo-distal gradient in the organization of the new extracellular matrix and the accumulation of molecules labeled by 3H-proline was shown. This accumulation started at the level of the injured segment of the stump. Differences in labeling intensity were seen in the regenerate. Within specific organogenetic zones, i.e. the epidermal gland analagen, the branchial buds and the stomodeal invagination, the labeling between the ectodermal and mesodermal layers was less intense than in other parts of the regenerate. In the mesodermal connective septa (dissepiments), located between consecutive segments, the labeling and the accumulation of extracellular material occurred later than the formation of the ectodermal basement membrane. In situ hybridization of a DNA molecular probe corresponding partially to the coding region of the collagen-like gene Ocg8, showed a spatio-temporal expression of this gene. Northern blot analysis showed a single transcript of 6.6 kb. Four days after amputation the accumulation of this transcript was exclusively localized at the level of the ectodermal layer during differentiation of the regenerate. The ectoderm was thus shown to play a dynamic role during the first stages of traumatic regeneration, although it did not seem to be directly involved in the early events of the metameric process.