Using the bioluminescent Ca²⁺ reporter, f-aequorin, in conjunction with luminescence microscopy, I have identified both short-range fast (i.e., travelling at ~49 μm/sec over a distance of ~80 μm) and long-range slow (i.e., travelling at ~4 μm/sec over a distance of ~235 μm) propagating Ca²⁺ waves that are generated in the external yolk syncytial layer (E-YSL) of developing zebrafish embryos. Embryos were microinjected with f-aequorin into the top of the yolk cell at the 128-cell stage in order to preferentially load the Ca²⁺ reporter into the E-YSL as it formed and developed. These two classes of Ca²⁺ waves begin during the late blastula period, between the dome stage and 30% epiboly, and continue for ~90 minutes (i.e., until the germ ring/shield stage). The majority of both cla...[ Read more ]

Using the bioluminescent Ca²⁺ reporter, f-aequorin, in conjunction with luminescence microscopy, I have identified both short-range fast (i.e., travelling at ~49 μm/sec over a distance of ~80 μm) and long-range slow (i.e., travelling at ~4 μm/sec over a distance of ~235 μm) propagating Ca²⁺ waves that are generated in the external yolk syncytial layer (E-YSL) of developing zebrafish embryos. Embryos were microinjected with f-aequorin into the top of the yolk cell at the 128-cell stage in order to preferentially load the Ca²⁺ reporter into the E-YSL as it formed and developed. These two classes of Ca²⁺ waves begin during the late blastula period, between the dome stage and 30% epiboly, and continue for ~90 minutes (i.e., until the germ ring/shield stage). The majority of both classes of waves are generated in the dorsal quadrant of the embryo and move in both directions around the E-YSL. The E-YSL was identified as the site of Ca²⁺ wave generation by: (i) combined luminescence/fluorescence imaging of embryos injected with f-aequorin and SYTOX Green, the latter being used to specifically label the yolk syncytial nuclei (YSN); and (ii) multi-photon imaging of embryos loaded with the fluorescent Ca²⁺ reporter, Calcium Green-1 dextran. I also investigated the localisation of the YSN and endoplasmic reticulum (ER) in the E-YSL during the early stages of epiboly when the E-YSL Ca²⁺ waves are known to be actively propagating, and subsequently proposed a hypothesis that involves YSN clustering, and their associated peri-nuclear ER, as well as a requirement for a short inter-nuclear distance for effective E-YSL Ca²⁺ wave generation and propagation. In addition, treating embryos with a Ca²⁺ chelator (5,5’-dibromo BAPTA), or an IP₃R antagonist (2-APB) resulted in the inhibition of the E-YSL Ca²⁺ signals and a subsequent delay in late epiboly. On the other hand, treatment with antagonists of RyRs (ryanodine and dantrolene) had no significant effect on the Ca²⁺ signals or on development. Morpholino knockdown of regulator of G-protein signalling 3 (rgs3) reproducibly and reliably suppressed the E-YSL Ca²⁺ waves and led to a delay in late epiboly, as well as dorsalisation of embryos. The possible roles of Rgs3 in the regulation of Ca²⁺ signalling in the E-YSL, and in zebrafish development are discussed.