Using complementary luminescent- and fluorescent-based Ca(2+) imaging techniques, we have re-examined the Ca(2+) dynamics that occur during the Blastula Period (BP) of zebrafish development. We confirm that aperiodic, localized Ca(2+) transients are generated predominately in the superficial epithelial cells (SECs). At the start of the BP, these Ca(2+) transients are distributed homogeneously throughout the entire superficial epithelium. Following the mid-blastula transition (MBT), however, their distribution becomes asymmetrical, where a significantly greater number are generated in the presumptive dorsal quadrant of the superficial epithelium. This asymmetry in Ca(2+) signaling lasts for around 60 min, after which the total number of transients generated from the entire superficial epithelium falls to less than one per minute until the end of the BP. We have thus called this asymmetry the "dorsal-biased Ca(2+) signaling window". The application of pharmacological agents indicates that the post-MBT SEC Ca(2+) transients are generated via the phosphatidylinositol (PI) signaling pathway. This suggests that the previously reported ventralizing function attributed to the homogeneously distributed PI pathway-generated SEC Ca(2+) transients is most likely to occur earlier in development, prior to the MBT.

Carboxypeptidase A6 (CPA6) is an extracellular protease that cleaves carboxy-terminal hydrophobic amino acids and has been implicated in the defective innervation of the lateral rectus muscle by the VIth cranial nerve in Duane syndrome. In order to investigate the role of CPA6 in development, in particular its potential role in axon guidance, the zebrafish ortholog was identified and cloned. Zebrafish CPA6 was secreted and interacted with the extracellular matrix where it had a neutral pH optimum and specificity for C-terminal hydrophobic amino acids. Transient mRNA expression was found in newly formed somites, pectoral fin buds, the stomodeum and a conspicuous condensation posterior to the eye. Markers showed this tissue was not myogenic in nature. Rather, the CPA6 localization overlapped with a chondrogenic site which subsequently forms the walls of a myodome surrounding the lateral rectus muscle. No other zebrafish CPA gene exhibited a similar expression profile. Morpholino-mediated knockdown of CPA6 combined with retrograde labeling and horizontal eye movement analyses demonstrated that deficiency of CPA6 alone did not affect either VIth nerve development or function in the zebrafish. We suggest that mutations in other genes and/or enhancer elements, together with defective CPA6 expression, may be required for altered VIth nerve pathfinding. If mutations in CPA6 contribute to Duane syndrome, our results also suggest that Duane syndrome can be a chondrogenic rather than a myogenic or neurogenic developmental disorder.

During the early blastula period of zebrafish embryos, the outermost blastomeres begin to undergo a significant thinning in the apical ⁄ basolateral dimension to form the first distinct cellular domain of the embryo, the enveloping layer (EVL). During this shape transformation, only the EVL-precursor cells generate a coincidental series of highly restricted Ca 2+ transients. To investigate the role of these localized Ca 2+ transients in this shape-change process, embryos were treated with a Ca 2+ chelator (5,5¢-difluoro BAPTA AM; DFB), or the Ca 2+ ionophore (A23187), to downregulate and upregulate the transients, respectively, while the shape-change of the forming EVL cells was measured. DFB was shown to significantly slow, and A23187 to significantly facilitate the shape change of the forming EVL cells. In addition, to investigate the possible involvement of the phosphoinositide and Wnt ⁄ Ca 2+ signaling pathways in the Ca 2+ transient generation and ⁄ or shape-change processes, embryos were treated with antagonists (thapsigargin, 2-APB and U73122) or an agonist (Wnt-5A) of these pathways. Wnt-5A upregulated the EVL-restricted Ca 2+ transients and facilitated the change in shape of the EVL cells, while 2-APB downregulated the Ca 2+ transients and significantly slowed the cell shape-change process. Furthermore, thapsigargin and U73122 also both inhibited the EVL cell shape-change. We hypothesize, therefore, that the highly localized and coincidental Ca 2+ transients play a necessary role in initiating the shape-change of the EVL cells.

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