Further analysis of developmental potentialities expressed by the footplate of 11-day mouse embryo hindlimb buds explanted in organ culture.

Abstract

The developmental potentialities expressed in vitro by hindlimb buds or excised footplates of 11-day mouse embryos have been analyzed using different kinds of organ culture procedures. As compared with unsatisfactory results obtained with explants transferred in submerged culture, others cultured on Nuclepore membranes following the Trowell technique exhibited growth and pattern formation performances which could be correlated with the CR length of their donor embryos. According to the case, their distal segment appeared hypotrophic and strongly necrotic, triangular shaped with 2 or 3 converging digital rays, four toed shaped with 3 or 4 digital rays, and pentadactylous. Insufficient distal morphogenesis observed in younger limb buds could be related to selective cell death in the A.E.R. and subridge mesoderm, as well as to early dilation of the marginal vein. Prospective area of digit 1 proved to be particularly sensitive to culture conditions. However, observations made in explants excised from potentially polydactylous embryos showed that the X-linked polydactyly (Xpl) phenotype can be expressed in vitro. Three technical variants contributed to improve the quality of pattern formation in the distal segment: the use of collagen-coated nuclepore membranes and the addition of bFGF or TGF-beta in the culture medium. The presence of bFGF seems to provide a protection against mesodermal cell death; that of TGF-beta appeared to promote digital outgrowth. The preskeletal pattern achieved in cultured footplates was in good agreement with maps of prospective areas previously established for the mouse hindlimb bud.