Patterns of free calcium in zebrafish embryos.

Direct knowledge of Ca2+ patterns in vertebrate development is largely restricted to early stages, in which they control fertilization, ooplasmic segregation and cleavage. To explore new roles of Ca2+ in vertebrate development, we injected the Ca2+ indicator aequorin into zebrafish eggs and imaged Ca2+ throughout the first day of development. During early cleavages, a high Ca2+ zone is seen in the cleavage furrows. The high Ca2+ zone during first cleavage spreads as a slow wave (0.5 microm/second) and is preceded by three Ca2+ pulses within the animal pole region of the egg. When Ca2+ concentrations are clamped at the resting level by BAPTA buffer injection into the zygote, all signs of development are blocked. In later development, Ca2+ patterns are associated with cell movements during gastrulation, with neural induction, with brain regionalization, with formation of the somites and neural keel, with otic placode formation, with muscle movements and with formation of the heart. Particularly remarkable is a sharp boundary between high Ca2+ in the presumptive forebrain and midbrain versus low Ca2+ in the presumptive hindbrain starting at 10 hours of development. When Ca2+ changes are damped by injection of low concentrations of BAPTA, fish form with greatly reduced eyes and hearts. The present study provides a first overview of Ca2+ patterns during prolonged periods of vertebrate development and points to new roles of Ca2+ in cellular differentiation and pattern formation.