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

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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