In vitro chondrogenesis of limb mesoderm from normal and brachypod mouse embryos

This paper describes the cytodifferentiation of hind limb mesodermal cells from 12-day-old normal and brachypod (pbH) mouse embryos grown in vitro at high densities. Over a 3-day culture period normal cells underwent aggregation, nodule formation, and coalescence of nodules into large masses of cartilage. This was associated at the biochemical level with a cessation of cell division, with a concomitant increase in metachromatic matrix, and synthesis of collagen. Under the described culture conditions the collagen synthesized by 48 h cultures was predominantly of cartilage type with an alpha1:alpha2 ratio of 9:1. A change in the collagen synthetic program was observed when the entire medium was replaced after 48 h incubation with fresh, serum-free medium. Under these conditions the alpha1:alpha2 ratio was 4:1. In contrast, brachypod cells plated at the same density appeared large, flattened, and stellate. Upon aggregation, normal nodule morphology was only rarely observed. More often large, irregular clusters formed from suspended cells loosely attaching to the surface aggregates. Concomitant with the marked changes in the morphology of the mutant cells was a linear increase in DNA synthesis and the appearance of many mitotic figures. A biochemical transformation in matrix synthesis was not observed, however. After a 24 h delay, mutant matrix accumulated and stained intensely with toluidine blue. Collagen was synthesized at approximately the normal rate and was of the cartilage type with an alpha1:alpha2 ratio of 9:1. When incubated in fresh, serum-free medium, the response of collagen subunit synthesis was identical to the normal cultures. In view of these results the possible manner in which brachypodism causes developmental anomalies of the limb skeleton is suggested.