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...[ Read more ]

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⁺ 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 Ca²⁺ 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) Non-propagating Ca²⁺ signals generated within single cells and (2) Propagating intercellular Ca²⁺ waves that traverse small groups of adjacent cells with a velocity of ~8.0 μm/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 Ca²⁺ 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 Ca²⁺ 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.