Fig. 3

Reduced Ca²⁺ wave causes egg activation defects in brom bones that is rescued by either Ca²⁺ or IP₃. (A) Representative profiles of luminescence from aequorin-loaded zebrafish eggs (red: strongly affected brom bones mutant egg; blue: moderately affected mutant egg; green: mildly affected mutant egg). The profiles illustrate the total luminescent output for the entire activation process with a temporal resolution of 1 second. Arrow indicates the addition of 0.5% fructose in egg water to the egg-injection chamber to activate the eggs. Asterisks indicate the time window when the photon counting was suspended and a bright-field image of the egg was taken. Bright-field images show the egg morphology after activation, with the color of the box outline corresponding to the luminescence profile. Arrowheads indicate the degree of chorion elevation after activation. Scale bar: 100 μm. (B) Calcium injection rescues the egg activation defects in brom bones mutants. The degree of egg activation was examined after injection of KCl (control) or CaCl₂ into brom bones eggs. brb1, brb2 and brb3 were three brom bones mutant females that produced eggs in this experiment. Eggs from brb2 and brb3 showed a similar range of phenotype and rescue, and were therefore combined for simplicity. The egg activation phenotype was scored as a percentage of the total number of eggs, with the strength of egg activation divided into five classes (shown in the bright-field images, right). (C-E)IP₃ injection rescues the egg activation defects in brom bones mutants. (C,D) Eggs from a single clutch at 60 mpa after injection of buffer (C) or IP₃ (D). (E) Percentages of eggs showing different degrees of egg activation after injection of buffer or IP3. brb1 and brb2 were two mutant females that produced eggs for this experiment and are different from the females in B.