|Budget Amount *help
¥2,300,000 (Direct Cost : ¥2,300,000)
Fiscal Year 1996 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1995 : ¥1,500,000 (Direct Cost : ¥1,500,000)
To understand molecular mechanisms of cell fate determination of the sea urchin embryo, we searched for key genes that encode the micromere-determinant and the archenteron-inducing ligand released from the micromere, and that are activated by lithium ion.
We differentially screened genes whose expression is restricted in the micromeres or the descendants, and is dependent of lithium ion in the mesomere-descendent cells. By the differential display, we obtained approximately 120 DNA fragments that seemed to be the micromere and/or the descendant-specific. Then, we performed the Southern blot analysis to check the specificity. We prepared poly (A^+) RNAs from the micromere and the mesomere as well as their descendants, and used the cDNAs as probes. As the result, we obtained 10 DNA fragments that showed stronger signals to the micromere-probe. By the subtraction PCR,we also detected 1 micromere-specific, 2 micromere descendant-specific, and 1 lithium ion-dependent DNA fraqments. Now, we are under way to examine their expression in the embryo by in situ hybridization
We also tried the expression cloning to isolate the key genes. We prepared poly (A^+) RNA from the ovary, the fertilized egg, as well as the 16-cell embryo. By injecting the RNAs into one of the mesomeres of the 16-cell embryo, we examined the activity that turns the injected cell into the micromere-phenotype, and that induces the adjacent cells to form the archenteron. We however, detected no activity with any RNAs.