Soluble tubulin complexes, gamma-tubulin, and their changing distribution in the zebrafish (Danio rerio) ovary, oocyte and embryo

gamma-tubulin is a key centrosomal protein which plays many central roles in zebrafish early development. A novel finding in this dissertation revealed that there were soluble pools of tubulins in zebrafish oocytes that were sequestered and maintained in a temporary "oligomeric" state, which failed to assemble into microtubules (MTs) spontaneously in vivo. Soluble alpha-tubulin was found to be associated with large molecular weight complexes which were reduced both in amount and size by the blastula stage. Smaller complexes then increased after gastrulation. Two different anti-gamma-tubulin antibodies, GTU 88 and TU 30, revealed the existence of soluble gamma-tubulin in both zebrafish oocytes and embryos, which also decreased by the blastula stage and increased in the gastrula stage. A proportion of ovarian soluble alpha-tubulin could be co-immunoprecipitated with gamma-tubulin, and vice versa. Microarray data demonstrated that ovary and eggs showed similar patterns of tubulin gene product expression while differing from day 4 larva. In situ hybridization with gamma-tubulin oligo probes revealed diffuse label in oocytes, with a marked localization to the blastodisc upon maturation. In addition, injection of anti-sense oligos to gamma-tubulin resulted in anterior-posterior axis defects. Using epi-immunofluorescent and confocal microscopy analysis, gamma-tubulin was found to translocate from oocyte cortex to the future blastodisc region during oocyte maturation. In eggs and 1-cell embryos, gamma-tubulin proteins were found to be uniformly distributed in the future blastodisc area. Beginning with the second embryonic division and especially at the eight cell stage, long, linear shaped centrosomes could be detected.;In addition, the orientation of the linear centrosome array and nuclear division alternated by about 90 degrees for each cell cycle. Two antibodies specific for Xenopus gamma-tubulin ring complex proteins revealed the existence of Xgrip109 and Xgrip195 homologues in zebrafish. Two models, "wrap-around" and "gamma-tubulin cycling," are proposed to explain our original hypothesis: maternal, soluble gamma-tubulin was accumulated and maintained in a temporary "oligomeric" form that was composed of large molecular weight complexes during zebrafish oogenesis. In addition, these large tubulin complexes would be subsequently sequestered and reallocated into many other complexes (e.g. centrosomes) and incorporated into embryonic MTs networks in ensuing embryogenesis.