Krug E L, Mjaatvedt C H, Markwald R R
Dev Biol. 1987 Apr;120(2):348-55. doi: 10.1016/0012-1606(87)90237-5.
A critical step in early cardiac morphogenesis can be faithfully duplicated in culture using a hydrated collagen substratum, and thereby serves as a useful model system for studying the molecular mechanisms of cell differentiation. Results from previous work suggested that the myocardium in the atrioventricular canal (AV) region of the developing chick heart secretes extracellular proteins into its associated basement membrane, which may function to promote an epithelial-mesenchymal transition of endothelium to form prevalvular fibroblasts (E. L. Krug, R. B. Runyan, and R. R. Markwald, 1985, Dev. Biol. 112, 414-426; C. H. Mjaatvedt, R. C. Lepera, and R. R. Markwald, 1987, Dev. Biol., in press). In the present study we show that an EDTA-soluble extract of embryonic chick hearts can substitute for the presence of myocardium, the presumptive stimulator tissue, in initiating mesenchyme formation from AV endothelium in culture. Ventricular endothelium was unresponsive to this material in keeping with observed in situ behavior. AV endothelial cells did not survive beyond 4-5 days when cultured in the absence of either the EDTA-soluble heart extract, myocardial conditioned medium, or the myocardium itself. Antibody prepared against a particulate fraction of the EDTA-solubilized heart extract immunohistochemically localized this material to the myocardial basement membrane. In addition, conditioned medium from embryonic myocardial cultures effectively induced mesenchyme formation. Neither a variety of growth factors nor a sarcoma basement membrane preparation were effective in promoting mesenchyme formation indicating a selectivity of the responding embryonic AV endothelial cells to myocardial basement membrane. These observations reflect a truly inductive phenomenon as there was an absolute dependence on the presence of the stimulating substance/tissue and retention, in culture, of both the temporal and regional characteristics observed in situ. This is in contrast to the results of others investigating the cytodifferentiation of committed cells whose phenotypic expression can be either accelerated or diminished but not obligatorily regulated by a specific agent, thus making the interpretation of data difficult, if not irrelevant, to the study of differentiation. The results of this study provide direct experimental support for the hypothesis that extracellular matrix can indeed serve as a direct stimulator or "secondary inducer" of cytodifferentiation.
在早期心脏形态发生过程中的一个关键步骤，可以利用水合胶原基质在培养中如实地重现，因此它成为研究细胞分化分子机制的一个有用的模型系统。先前工作的结果表明，发育中的鸡心脏房室管（AV）区域的心肌向其相关的基底膜分泌细胞外蛋白，这可能起到促进内皮细胞上皮-间充质转化以形成瓣膜前成纤维细胞的作用（E.L.克鲁格、R.B.鲁尼恩和R.R.马克瓦尔德，1985年，《发育生物学》112卷，414 - 426页；C.H.米亚特维特、R.C.莱佩拉和R.R.马克瓦尔德，1987年，《发育生物学》，即将发表）。在本研究中，我们表明胚胎鸡心脏的一种可被乙二胺四乙酸（EDTA）溶解的提取物，能够在培养中替代心肌（假定的刺激组织），从而启动房室内皮细胞形成间充质。心室内皮细胞对这种物质没有反应，这与原位观察到的行为一致。当在没有EDTA可溶心脏提取物、心肌条件培养基或心肌本身的情况下培养时，房室内皮细胞在4 - 5天后就无法存活。针对EDTA可溶心脏提取物的颗粒部分制备的抗体，通过免疫组织化学将这种物质定位到心肌基底膜。此外，胚胎心肌培养物的条件培养基能有效地诱导间充质形成。多种生长因子和肉瘤基底膜制剂都不能有效地促进间充质形成，这表明反应性胚胎房室内皮细胞对心肌基底膜具有选择性。这些观察结果反映了一种真正的诱导现象，因为在培养中，对刺激物质/组织的存在以及原位观察到的时间和区域特征的保留存在绝对依赖性。这与其他研究定向细胞的细胞分化的结果形成对比，那些细胞的表型表达可以被加速或减弱，但不一定受特定因子调控，因此使得数据解释对于分化研究即使不是无关紧要，也是困难的。本研究结果为细胞外基质确实可以作为细胞分化的直接刺激物或“二级诱导物”这一假说提供了直接的实验支持。
