(A) In singleton embryos, injection of the caged molecule (BHQ-O-5HT) at the 1-cell stage, paired with uncaging at the 32-cell stage, induces significant levels of heterotaxia. Photoactivation of 5-HT at the 8-cell stage did not significantly increase rates of LR patterning defects. (B) BHQ-O-5HT was injected at the 1-cell stage and conjoined twins were induced at the 8- or 16-cell stage. When 5-HT was uncaged at the 32-cell stage, prior to the induction of conjoined twins, LR patterning in the induced twin was randomized. When 5-HT was uncaged at stage 8, as XSiamois begins to induce the development of a conjoined twin, LR patterning defects were still observed. (C) BHQ-O-5HT and XSiamois were co-injected in the ventral vegetal left blastomere at the 8- or 16-cell stage, and 5-HT was uncaged at either 32-cell, stage 8, or stage 10. Regardless of when the 5-HT was released, heterotaxia in the induced twin was observed. In all panels, the numbers on the graphs indicate the sample size, *p<0.05 compared to embryos injected with BHQ-O-5HT but not uncaged, Chi Square test. Red bars indicate inverted hearts, blue indicates inverted stomach and/or gall bladder, and purple indicates inverted heart plus stomach and/or gall bladder.