Characterization of Ca²⁺ signaling in the external yolk syncytial layer during the late blastula and early gastrula periods of zebrafish development

Preferential loading of the complementary bioluminescent (f-aequorin) and fluorescent (Calcium Green-1 dextran) Ca²⁺ reporters into the yolk syncytial layer (YSL) of zebrafish embryos, revealed the generation of stochastic patterns of fast, short-range, and slow, long-range Ca²⁺ waves that propagate exclusively through the external YSL (E-YSL). Starting abruptly just after doming (~4.5h post-fertilization: hpf), and ending at the shield stage (~6.0hpf) these distinct classes of waves propagated at mean velocities of ~50 and ~4μm/s, respectively. Although the number and pattern of these waves varied between embryos, their initiation site and arcs of propagation displayed a distinct dorsal bias, suggesting an association with the formation and maintenance of the nascent dorsal-ventral axis. Wave initiation coincided with a characteristic clustering of YSL nuclei (YSN), and their associated perinuclear ER, in the E-YSL. Furthermore, the inter-YSN distance (IND) appeared to be critical such that Ca²⁺ wave propagation occurred only when this was <~8μm; an IND >~8μm was coincidental with wave termination at shield stage. Treatment with the IP₃R antagonist, 2-APB, the Ca²⁺ buffer, 5,5'-dibromo BAPTA, and the SERCA-pump inhibitor, thapsigargin, resulted in a significant disruption of the E-YSL Ca²⁺ waves, whereas exposure to the RyR antagonists, ryanodine and dantrolene, had no significant effect. These findings led us to propose that the E-YSL Ca²⁺ waves are generated mainly via Ca²⁺ release from IP₃Rs located in the perinuclear ER, and that the clustering of the YSN is an essential step in providing a CICR pathway required for wave propagation. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.