| It has been suggested that during the zebrafish (Danio rerio ) Blastula Period, Ca2+ signals might play an important role during early embryonic development: perhaps mediating cell-to-cell adhesion in the enveloping layer (EVL; Reinhard et al., 1995) or contributing to patterning the dorsal-ventral axis (Westfall et al., 2003). In this study, I extend the investigation of Blastula Period Ca2+ signals using the luminescent Ca2+ reporter aequorin. First, I confirmed that aperiodic Ca2+ signals are generated by EVL cells in small localized domains throughout the Blastula Period. An automated analysis algorithm and a quadrant analysis protocol were developed to analyze the generation of these signals. Applying these tools, I am the first to report that although initially the Blastula Period Ca2+{09}signals are homogeneously distributed throughout the EVL, from around 3.0--3.75 hpf, a 45 minute signaling window is established where a greater number of Ca 2+ signaling events occurred in the future dorsal quadrant of embryos. I have named this period the "dorsal-bias Ca2+-signaling window" (DCW). Three properties of the DCW Ca2+ signals were characterized (duration, fold Ca2+ increase, and area), in both intact and dechorionated embryos, as well as in each of the four blastoderm quadrants (dorsal, ventral, left and right). I also demonstrated that there are two types of DCW Ca2+ signals: (1) Nonpropagating Ca 2+ signals generated within single cells and (2) Propagating intercellular Ca2+ waves that traverse small groups of adjacent cells with a velocity of ~8.0 mum/sec. My new aequorin-based data thus provide new as well as confirmatory information regarding the spatial, temporal and physical properties of the Blastula Period, and in particular, the DCW Ca 2+ signals. Furthermore, using two-photon confocal microscopy, in conjunction with a fluorescent Ca2+ reporter, I confirmed that the DCW Ca2+ signals are generated almost exclusively in the EVL.; In addition, both pharmacological and immunohistochemical studies suggest that IP3Rs are responsible for generating the DCW Ca2+ signals via the release of Ca2+ from internal stores. I also demonstrated that in the early Blastula Period, the IP3R (Type I) protein has a low and homogeneous expression level throughout the blastoderm. However, just prior to the onset of the DCW, the expression level of the protein increases significantly in the outermost cells forming the EVL.; With regards to the possible developmental function of the Blastula Period Ca2+ signals, blocking the release of Ca2+ from the ER during the DCW using an IP3R antagonist, results in a dramatic reduction in the number of Ca2+ transients as well as a loss of EVL integrity, where embryos disintegrate through the compromised EVL. Furthermore, inhibition of the phosphoinositide (PI) pathway and FGF signaling, but not inhibition of G-protein signaling or PDGF signaling, during the DCW also leads to a significant reduction in the number of DCW Ca2+ signals. In addition, FGF signaling and PI pathway inhibition during the DCW results in embryos with a reduced overall body length, a decrease in the number of posterior somites, and a reduction in the size of the otic vesicle, when examined at 24 hpf. Thus, while the links between Blastula Period Ca 2+ signals and various aspects of early embryonic development are highly complex, my data suggest that FGF signaling might play a role in triggering the DCW Ca2+ signals via the PI pathway. |
