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, oopl asmic segregation and cleavage. To explore new roles of Ca2+ in verteb rate development, we injected the Ca2+ indicator aequorin into zebrafi sh eggs and imaged Ca2+ throughout the first day of development. Durin g early cleavages, a high Ca2+ zone is seen in the cleavage furrows. T he high Ca2+ zone during first cleavage spreads as a slow wave (0.5 mu m/second) and is preceded by three Ca2+ pulses within the animal pole region of the egg. When Ca2+ concentrations are clamped at the restin g level by BAPTA buffer injection into the zygote, all signs of develo pment are blocked. In later development, Ca2+ patterns are associated with cell movements during gastrulation, with neural induction, with b rain regionalization, with formation of the somites and neural keel, w ith otic placode formation, with muscle movements and with formation o f 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+ c hanges are damped by injection of low concentrations of BAPTA, fish fo rm 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 differentiati on and pattern formation.