Aono H, Ide H
Biological Institute, Tohoku University, Sendai, Japan.
Dev Biol. 1988 Jul;128(1):136-41. doi: 10.1016/0012-1606(88)90275-8.
Limb bud mesoderm of stage 22-23 embryos was dissected into four pieces along the anteroposterior axis and dissociated cells of each region were separately cultured under various conditions. When the cells were cultured in medium containing 0.1% fetal calf serum (serum-poor medium) only a slight increase in cell number occurred in the cultures of all four regions. However, when the cells were cultured in medium containing 10% FCS, only cells of two central regions proliferated rapidly, and no growth promotion was observed in cells in the most anterior and posterior regions. Using the serum-poor medium, we examined the growth-promoting effects of cocultured limb bud fragments and of some growth factors on the cells of four regions. Anterior, distal, and proximal fragments promoted cell proliferation and their promotive effect on the cells of each region was equal. On the other hand, posterior fragments (containing ZPA) showed stronger promotive effects on preaxial cells than on postaxial cells. For comparison with the growth-promotive effect of the posterior fragment, fibroblast growth factor (FGF), epidermal growth factor (EGF), insulin, and retinoic acid were tested in cell culture. FGF showed position-dependent growth promotion, while EGF and insulin promoted growth in the cells of all four regions to a similar degree. Retinoic acid showed no effect on cell growth at low concentrations, and was rather toxic at high concentrations. These results suggest that the cells of the posterior region secrete an FGF-like growth factor(s), which controls normal limb development and experimental duplicate formation.
将22 - 23期胚胎的肢芽中胚层沿前后轴切成四块，并将每个区域解离的细胞在不同条件下分别培养。当细胞在仅含0.1%胎牛血清的培养基（血清含量低的培养基）中培养时，所有四个区域的培养物中细胞数量仅有轻微增加。然而，当细胞在含10%胎牛血清的培养基中培养时，只有两个中央区域的细胞迅速增殖，而最前端和最后端区域的细胞未观察到生长促进现象。使用血清含量低的培养基，我们检测了共培养的肢芽片段和一些生长因子对四个区域细胞的生长促进作用。前端、远端和近端片段促进细胞增殖，并且它们对每个区域细胞的促进作用相同。另一方面，后端片段（含极化活性区）对轴前细胞的促进作用比对轴后细胞更强。为了与后端片段的生长促进作用作比较，在细胞培养中测试了成纤维细胞生长因子（FGF）、表皮生长因子（EGF）、胰岛素和视黄酸。FGF显示出位置依赖性生长促进作用，而EGF和胰岛素对所有四个区域的细胞生长促进程度相似。视黄酸在低浓度时对细胞生长无影响，在高浓度时则有相当大的毒性。这些结果表明，后端区域的细胞分泌一种FGF样生长因子，它控制正常肢体发育和实验性多指（趾）畸形的形成。
