Determination Mechanism of Organization Center for Pattern Formation in Palleal Buds of Ascidians

在这个项目的早期工作中，我发现肠域是多柱目海囊动物(如多年生动物和多雄果)中形成瓣状花蕾的前后模式形成的组织中心。组织中心以全能干细胞、造血细胞的聚集和表面转化为特征，并进入快速细胞周期(T_c=12.5hr)。最近，我们从味佐氏疟原虫中分离纯化了一个钙依赖半乳糖结合14 kDa凝集素(TC-14)，并测定了它的氨基酸序列。FITC标记的凝集素或生物素标记的造血细胞。抗TC-14多克隆抗体与芽发育早期出现在组织中心的细胞外基质(ECM)反应。它阻止造血细胞分化为胃上皮细胞。这些结果有力地表明，凝集素阳性的ECM在血细胞向上皮细胞聚集方面起作用。穿心莲粗提物…蛋白免疫印迹分析进一步的SDS-PAGE分析表明，TC-14与64 kDa糖蛋白的亚基--20 kDa多肽(GP-)有亲和力。这一反应需要钙离子，表明GP-是TC-14的体内靶分子。TC-14在体内和体外对造血细胞均有生长抑制作用，而GP-对其体外增殖有明显的促进作用。这些结果有力地表明TC-14和GP-之间的分子相互作用在组织中心的全能性、细胞生长和上皮转化中起关键作用。我还研究了非肽效应分子，其中前列腺素(PGs)参与了上皮细胞中凝集素颗粒的胞吐。在组织中心附近的血细胞中，血清学检测到PGH合成酶。吲哚美辛是PGs生物合成的特异性抑制剂，可阻断凝集素颗粒的胞吐。吲哚美辛的作用被PGs部分抵消，组织中心重新出现。结果表明，鳞芽组织中心的确定涉及TC-14、GP-等多种功能分子胞吐作用的时空整合。较少

In earlier works of this project, I found that the gut domain is an organization center for antero-posterior pattern formation of a palleal bud in polystyelid ascidians such as Polyzoa and Polyandrocarpa. The organization center was characterized by the aggregation and epithe ial transformation of totipotent stem cells, hemoblasts, and by their entrance into quick cell cycling (Tc=12.5hr). Recently, we have purified a calcium-dependent galactosebinding 14kDa lectin (TC-14) from P. misakiensis, and determined its amino acid sequence. The lectin labeled with FITC or biotin stained hemoblasts. Anti TC-14 polyclonal antibody reacted with the extracellular matrix (ECM) that appeared in the organization center at the earliest stage of bud development. It blocked hemoblasts to differentiate into the gastric epithelium. The results strongly suggested that lectin-positive ECM plays a role in aggregation of hemoblasts toward the epithelium. After western blotting of Polyandrocarpa crude extracts … More on SDS-PAGE, TC-14 showed an affinity for 20kDa polypeptide that was a subunit of 64kDa glycoprotein (GP-64). This reaction required Ca^<2+>, showing that GP-64 is an in vivo target molecule of TC-14. TC-14 had a growthinhibitory effect on hemoblasts in in vivo and in vitro bioassays, whereas GP-64 enhanced their in vivtro proliferation rem arkably. These results strongly suggested that molecular interactions between TC-14 and GP-64 play a key role in totipotency, cell growth and epithelial transformation of hemoblasts at the organization center.I also studied non-peptide effector molecules, of which Prostaglandins (PGs) were found to be involved in exocytosis of lectin granules included in epithelial cells. PGH synthetase was found serologically in blood cells in the vicinity of organization center. Indomethacin, a specific inhibitor of PGs biosynthesis, blocked the exocytosis of lectin granules. The effect of indomethacin was canceled in part by PGs and the organization center reappeared. It is concluded that the determination of organization center in palleal buds involves the spatio-temporal integration of exocytosis of various functional molecules such as TC-14 and GP-64. Less