Fig. 2

Fig. 2

Reduced calcium release underlies furrow formation defects in zebrafish nebel mutants. (A,B) Fluorescence after injection of Fura-2 during second cleavage furrow formation, with calcium-saturated (340 nm) and calcium-free (380 nm) images converted to a pseudocolor ratio. Intracellular calcium increases are observed prior to and during furrow formation in wild-type embryos (A), whereas nebel mutants show repeated intracellular increases of reduced intensity (B). Animal views, with numbers indicating the lapsed fraction of the cell cycle. Arrows indicate sites of high intracellular calcium, corresponding to SCWs. (C-H) In nebel mutants, furrow initiation appears normal [black arrowheads during the first (D,E versus C) and third (G,H versus F) cell division]. In rare cases, the furrow fails to initiate (white arrowhead in E; insert depicts a clearer focal plane). Black arrow (F) indicates the adhesive septum in wild type, which fails to form in mutants (G,H, white arrows). (I-L) Ratiometric analysis using Oregon Green 488 BAPTA-Dextran and Rhodamine in wild-type (I), nebel mutant (J) and 3-NP-treated wild-type (K) embryos, with representative kymographs (I′-K′, white line in I-K) and average time in seconds between calcium pulses (L, n>6 furrows). Error bars indicate the s.e.m.; two-tailed P-values were assessed using an unpaired t-test. (M-P) Pseudocleavages in unfertilized wild-type eggs (M, arrow) do not form in nebel mutant eggs (N) but are induced by CaCl₂ injection (O), an effect independent of the KCl co-injected to improve egg survival (P).