Wessel G M, McClay D R
Dev Biol. 1987 May;121(1):149-65. doi: 10.1016/0012-1606(87)90148-5.
This study demonstrates that a collagenous extracellular matrix (ECM) is necessary for gastrulation in the sea urchin embryo. The approach taken was to disrupt collagen processing with two types of agents (a lathyritic agent, beta-aminopropionitrile (BAPN), and three types of proline analogs: dehydroproline, cis-OH-proline, and azetidine carboxylic acid) and to assess the effect on embryogenesis by morphological, immunological, and biochemical criteria. Embryos chronically exposed to either of the agents following fertilization displayed no detectable developmental abnormalities before the mesenchyme blastula stage. These embryos, however, did not gastrulate nor differentiate any further and remained at the mesenchyme blastula stage for at least 36 hr. Upon removal of the agents, the embryos resumed a normal developmental schedule and formed pluteus larvae that were indistinguishable from control embryos. By immunofluorescence studies with monospecific antibodies to type I and type IV collagens it is seen that the lathyritic agent BAPN reduces the accumulation of collagens within the ECM. This effect is confirmed and quantitated by use of an ELISA and by a biochemical determination of OH-proline. When the agents are removed from the inhibited embryos, collagen deposition returns to normal, coincident with gastrulation. Western-blot analysis, using monospecific antibodies to collagen, demonstrates that the effect of the lathyritic agent is to reduce the stability of the extracellular collagen by inhibiting the intra- and intermolecular crosslinking of collagen molecules. BAPN exhibits a dose-dependent effect on morphogenesis, but has no effect on respiration nor on protein synthesis of the embryos throughout development. Although the lathyritic agent affects collagen deposition, it is shown to not affect the expression of other molecules of the ECM, nor that of several cell surface molecules. However, a cell surface molecule that is expressed specifically in the endoderm, termed Endo 1, is not expressed in the inhibited embryos. Endo 1 is expressed after removal of the lathyritic agent and its appearance is coincident with gastrulation in the recovered embryos. These results suggest that a collagenous ECM is important for gastrulation and subsequent differentiation in the sea urchin, but not for earlier developmental processes. In addition, the dependence of Endo 1 expression on the collagenous ECM raises the possibility that this cell surface molecule is in some way regulated by interactions of the presumptive endodermal cells with the ECM.
本研究表明，胶原细胞外基质（ECM）对海胆胚胎的原肠胚形成是必需的。所采用的方法是用两种试剂（一种致麻痹剂，β-氨基丙腈（BAPN），以及三种脯氨酸类似物：脱氢脯氨酸、顺式羟基脯氨酸和氮杂环丁烷羧酸）破坏胶原加工过程，并通过形态学、免疫学和生化标准评估对胚胎发生的影响。受精后长期暴露于任何一种试剂的胚胎，在间充质囊胚阶段之前均未显示出可检测到的发育异常。然而，这些胚胎并未进行原肠胚形成，也未进一步分化，而是在间充质囊胚阶段停留至少36小时。去除试剂后，胚胎恢复正常发育进程，形成与对照胚胎无异的长腕幼虫。通过用针对I型和IV型胶原的单特异性抗体进行免疫荧光研究发现，致麻痹剂BAPN减少了ECM内胶原的积累。使用ELISA以及对羟基脯氨酸进行生化测定证实并定量了这种效应。当从受抑制的胚胎中去除试剂时，胶原沉积恢复正常，与原肠胚形成同时发生。使用针对胶原的单特异性抗体进行的蛋白质印迹分析表明，致麻痹剂的作用是通过抑制胶原分子的分子内和分子间交联来降低细胞外胶原的稳定性。BAPN对形态发生表现出剂量依赖性效应，但在整个发育过程中对胚胎的呼吸或蛋白质合成均无影响。虽然致麻痹剂会影响胶原沉积，但研究表明它不会影响ECM其他分子的表达，也不会影响几种细胞表面分子的表达。然而，一种在内胚层中特异性表达的细胞表面分子，称为Endo 1，在受抑制的胚胎中不表达。去除致麻痹剂后Endo 1开始表达，其出现与恢复的胚胎中的原肠胚形成同时发生。这些结果表明，胶原ECM对海胆的原肠胚形成和随后的分化很重要，但对早期发育过程不重要。此外，Endo 1表达对胶原ECM的依赖性增加了这种细胞表面分子在某种程度上受假定内胚层细胞与ECM相互作用调节的可能性。
