Cell-cell interaction by mouse limb cells during in vitro chondrogenesis: analysis of the brachypod mutation

Owens, E.M.; Solursh, M.

Developmental Biology 91(2): 376-388


ISSN/ISBN: 0012-1606
PMID: 7095270
DOI: 10.1016/0012-1606(82)90043-4
Accession: 004890910

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Observations and experiments are presented which collectively demonstrate a requirement for cell-cell interactions among limb bud mesenchyme cells during chondrogenic differentiation. Limb bud cells isolated from brachypodismH (bpH) and wild-type mouse embryos between Thieler stage 16-17 and midstage 21 were compared with respect to their abilities to undergo chondrogenic differentiation in high-density micromas cultures. Nodules formed by dissociated day 12 (stage 20) bpH limb bud cells were reported previously to be abnormally reduced in size and number, and delayed in formation. bpH cultures prepared from earlier-stage limb buds (between stages 16-17 and early stage 21) are progressively more like wild-type cultures. Stage 16-17 bpH cultures at 72 h actually contain normal numbers of and size nodules, while stage 18 bpH cultures are intermediate between stages 16-17 and stage 21 in nodule formation. The initial rate of aggregate formation is normal even in bpH cultures prepared from stage 20 cultures in which nodule formation is not normal. Preparation of cultures composed primarily of early stage 21 bpH limb bud cells mixed with small quantities (e.g., 5%) of stage 16-17 wild-type limb bud cells showed significant increases in cartilage nodule formation over control cultures composed only of early stage 21 bpH cells. Greater proportions of wild-type cells obtained from embryos older than stages 16-17 were required for the same degree of normalization, supporting the hypothesis that a specific cell type, whose proportion decreases normally in the limb bud over time, is required to increase in vitro chondrogenesis by bpH cells. Additionally, cultures containing stage 23 chick limb cells and early stage bpH cells at a ratio of 1:20 contained wild-type levels of nodules per mm2 of culture. bpH cells appear to respond to chondrogenic inductive signals from normal limb mesenchyme cells. In order to test for the ability of bpH limb bud mesenchyme to induce chondrogenesis, stage 16-17 bpH and wild-type limb bud cells, which form identical numbers of aggregates and nodules in culture, were each mixed with early stage 21 bpH cells at ratios of 1:20, 1:10 and 1:3. Although low proportion of wild-type stage 17 cells significantly increased the number of aggregates and nodules in these mixed cultures, low proportions of bpH stage 16-17 cells did not. The primary defect of the bpH mutation is likely to reside in the reduced ability of a specific mesenchyme cell subpopulation to provide an inductive stimulus for chondrogenesis.