Fig. 2

Micromass in vitro differentiation of mesenchymal limb bud cells. Micromass cultures of mesenchymal cells isolated from the limb buds of embryonic day 11.5 (E11.5) wild-type (WT) and Tfap2e ^−/− embryos were maintained for 4 days in vitro. a To compare the differentiation behavior of WT and Tfap2e ^−/− cells, each day, a culture was treated with Alcian Blue solution to stain for secreted glycosaminoglycans. The nodules that are characteristic for this assay became visible at the second day of the culture period. Their number, size and color intensity increased in both genotypes by approximately the same amount. b Directly after initiation of the cultures (d0) and on each following day, mRNA expression of the cartilage differentiation markers aggrecan, collagen, type II, alpha 1 (Col10a1) and collagen, type X, alpha 1 (Col10a1) and matrix metalloproteinase (Mmp13) was analyzed by quantitative RT-PCR. A definite increase in the expression level of all four genes during the differentiation process was detectable in cells of both genotypes. The expression of Col10a1 was enhanced, and expression of Mmp13 tended to be enhanced, at day 4 in Tfap2e ^−/− cells compared with WT cells. The data are given as the means ± standard error of the mean. ns, Not significant; Tfap2e ^−/−, Deficient for activating enhancer binding protein 2, epsilon. *P < 0.05. The assay was carried out four times with cells of four individual WT litters and three times with cells of three individual Tfap2e ^−/− litters