Fig. 5

Decreasing the frequency of Ca²⁺ transients leads to ectopic dorsal gene expression in hecate mutant embryos. (a–d) Both wild-type and hec mutant embryos show an expansion of cho expression in the margin at 50% epiboly (a and c: DMSO-treated control; b and d: Thapsigargin-treated, animal views). (e–j) Ectopic cho expression in the EVL is not dependent on β-catenin function. Thapsigargin (f, h, j)- and DMSO (e, g, i)-treated wild-type embryos. β-catenin MO effectively suppresses endogenous wild-type cho expression but not the ectopic expression in the EVL in wild-type embryos (g and h). Standard control MO does not affect the level of cho expression (data not shown). Injection of ΔNtcf suppresses both ectopic thapsigargin-induced cho expression and its endogenous expression in the dorsal region (i, j) (animal views, sphere stage, arrows denote expression in EVL). (k–r) Thapsigargin-induced ectopic cho expression occurs in cells of the EVL. Double labeling of cho mRNA using fluorescent situ hybridization (green in panels n and r) and the DNA stain DAPI (pseudocolored red for clarity in panels n and r). Under these labeling conditions, cho expression is observed diffusely in the cytoplasm and in two bright spots likely representing new transcription at the endogenous loci. (k–n) Control (DMSO)-treated embryo (k) and higher magnification views of the endogenous dorsal cho expression domain (red box in panel k). cho is expressed only in the dorsal region, where it is detected in deep cells and a small fraction of EVL cells. Arrows indicate EVL cells, distinguished by their external position and larger, flattened nuclei. Asterisks next to the arrows indicate EVL cells with some detectable expression. (o–r) Thapsigargin-treated embryos show ectopic cho expression in cells of the EVL layer. White arrows and asterisks as in (k–n). Panels are animal views of embryos at 30% epiboly (4.7 hpf).