Ca²⁺ is a highly versatile intra- and intercellular signal that has been reported to regulate a variety of different cellular processes during early development. Preliminary imaging data suggested that localized Ca²⁺ transients generated in the lateral intermediate mesoderm (LIM) of developing zebrafish embryos coincided with the appearance of the bilateral pronephric rods, the first visible components of the embryonic kidney of teleosts. The objectives of this study were, therefore, to further investigate the possible role(s) that Ca²⁺ might play in the differentiation and positioning of the pronephric rods within the LIM....[ Read more ]

Ca²⁺ is a highly versatile intra- and intercellular signal that has been reported to regulate a variety of different cellular processes during early development. Preliminary imaging data suggested that localized Ca²⁺ transients generated in the lateral intermediate mesoderm (LIM) of developing zebrafish embryos coincided with the appearance of the bilateral pronephric rods, the first visible components of the embryonic kidney of teleosts. The objectives of this study were, therefore, to further investigate the possible role(s) that Ca²⁺ might play in the differentiation and positioning of the pronephric rods within the LIM.

From 11.5 hpf (i.e., just before the pronephric rods are morphologically distinguishable in the LIM) to 16 hpf, embryos were treated with a variety of membrane permeable pharmacological reagents known to modulate intracellular Ca²⁺ levels. The effect of these treatments on pronephric rod formation was determined in both live (through staining with BODIPY FL C₅-ceramide) and fixed specimens (via in situ hybridization using a variety of probes specific to the forming pronephros, i.e., cdh17, pax2.1 and sim1). The use of these pharmacological reagents also allowed the determination of the probable source and the Ca²⁺-release mechanism involved. Data recorded from the application of the plasma membrane Ca²⁺ channel antagonists CdCl₂, NiCl₂ or nifedipine, as well as incubation of embryos in Ca²⁺-free conditions, suggested that extracellular Ca²⁺ did not play a role in pronephric rod development or positioning. On the other hand, however, the application of antagonists to the PI signaling pathway (U73122), the IP₃R (2-APB) or the SERCA pump (thapsigargin), but not the ryanodine receptor (dantrolene), indicated that Ca²⁺ released from intracellular stores (most likely the ER) via IP₃Rs, appeared to play a significant role in the positioning and the morphological development of the pronephric rods (i.e., following treatment the two pronephric rods were located further apart and appeared abnormally flatter). The application of these various Ca²⁺ release antagonists had no apparent effect on the fate determination of the LIM cells that form the pronephric rods (i.e., as pronephros markers were always still expressed, albeit ectopically), suggesting that fate specification of this cell population was either Ca²⁺-independent or occurred at some time prior to 11.5 hpf. It is proposed that the effect on pronephric rod location and morphology induced by the application of the intracellular Ca²⁺ release antagonists is via their inhibitory effect on the convergent/extension movements of LIM cells toward the embryonic midline.

In addition to determining the effects of Ca²⁺ release antagonists on the development and positioning of the pronephric rods, an attempt was made via aequorin-based imaging to further characterized the precise location, timing, nature and reproducibility of any Ca²⁺ transients generated in the LIM during the period of pronephric rod formation. During the 5 hr imaging period (i.e., 11 hpf to 16 hpf), no obvious zone of elevated Ca²⁺ or specific pattern of Ca²⁺ transients were seen to be generated in the LIM at the approximate location from which the pronephric rods form. What was revealed, however, were stochastic patterns of localized Ca²⁺ transients generated both within the LIM and the paraxial mesoderm throughout the 5 hr imaging period. Preliminary data suggests that these stochastic transients are also generated by Ca²⁺ release via IP₃Rs. The developmental function and significance of these stochastic Ca²⁺ transients generated during the early Segmentation Period of zebrafish embryos is currently unknown.