ABSTRACT. A partial cDNA encoding zebrafish prothrombin has been cloned and used as a probe to study the temporal expression of prothrombin mRNA during early embryonic development. The results revealed accumulation of prothrombin mRNA in diverse tissues such as the eyes and myotomes in early embryogenesis. We have also examined the enzymatic activity of thrombin in converting fibrinogen to fibrin in individual embryos at different stages of development. We found that the fibrin-forming activity does not temporally correlate with the first presence of thrombin mRNA in the early stages of embryogenesis, but does correlate with the initiation of blood formation. Our ability to observe the fibrin- forming activity in single individual embryo will facilitate studies on identifying recessive mutations affecting blood coagulation, such as the regulatory gene mutations controlling the clotting factor genes. Furthermore, the observation of thrombin activity will also facilitate studies on the blood coagulation pathways in the early embryogenesis in this zebrafish model.

Keywords: Thrombin, cDNA sequence, zebrafish, hemostasis, embryogenesis.

Reprint requests to: Pudur Jagadeeswaran, Ph.D., Department of Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio TX 78284-7762, phone: (210) 567-3848, fax: (210) 567-3803, e-mail: jagadeeswar@uthscsa.edu.

ABSTRACT. Repopulation by donor cells of a bone marrow ablated by irradiation is now recognized to proceed in two phases: initial repopulation that may be temporary followed by permanent engraftment of long-term repopulating cells (LTRC). While a single LTRC has been shown to be capable of restoring the entire lymph-hemopoietic system of an irradiated animal, the identity of the temporary repopulating cells has not been established unequivocally. We used the results of transplantation of subpopulations successively enriched for LTRC and containing varying numbers of CFU-S-12 (colony-forming units in the spleen at day 12 post transplantation) and progenitors to determine the likely cell type and number of cells needed for initial survival after radiation. Subpopulations from untreated and 5- fluorouracil-treated mice were discriminated on the basis of antibody reactivity, Hoechst 33342 and rhodamine 123 fluorescence intensity and light scattering properties. The minimum rescue inocula varied greatly in CFU-GEMM, BFU-E and CFU-GM content. One to two CFU-S-12 were uniformly present in all isolated suspensions that rescued 50% of lethally irradiated animals. In view of the known average seeding efficiency of CFU-S, our studies suggest that transfusion of 10-20 CFU-S day 12/13 is responsible for radioprotection. Evidence that multiple CFU-S day 12/13 are needed for initial repopulation is also supported by quantitative estimates of the number of mature cells that can be produced by CFU-S. Transfusion of a single CFU-S day 12/13 can be shown to be grossly inadequate to provide the number of peripheral blood cells needed to ameliorate the severe pancytopenia following lethal irradiation by day 12-14. Our data also indicate that 5-fluorouracil-treated marrow subpopulations appear inferior to untreated subpopulations in their ability to contribute to initial repopulation when transfused at low cell doses into lethally irradiated recipients.

Keywords: Stem cells, CFU-S, progenitor cells, radioprotection, 5-fluorouracil, flow cytometry.

Reprint requests to: Maria Pallavicini, Ph.D., Cancer Center, Box 0808, University of California, San Francisco, CA 94143, fax: (415) 476-8218, phone: (415) 476-3440, e-mail: pallavicini@cc.ucsf.edu.