Isolation of hArl13b-mCherry-GECO1.2tg and hArl13b-mCherry-GECO1.2tg:GCaMP6ftg:E2aCretg embryos

GCaMP6f (B6;129S-Gt(ROSA)26Sor^{tm95.1(CAG-GCaMP6f)Hze}/J) and E2a-Cre (Tg(EIIa-cre)C5379Lmgd) transgenic animals were obtained from Jackson Laboratories. Embryo isolation was performed as described previously³³. Timed pregnancies resulting from mating wild type C57BL6/6J, Arl13b-mCherry-GECO1.2^tg/− or Arl13b-mCherry-GECO1.2^tg/tg females with Arl13b-mCherry-GECO1.2^tg/tg or GCaMP6f^tg/tg:E2a-Cre^tg/tg males yielded embryos that were then selected for the appropriate developmental stages³⁴. Embryos expressing motile cilia in the embryonic node at stages critical for asymmetric gene expression ³⁵ (starting at developmental stages “early allantoic bud” (EB) up to 2-somite stage) were used for experiments. Embryos were mounted with the embryonic node facing up in a custom-designed embryo mounting plate (Extended Data Fig. 10 b–d). Laser cut holes (diameters 0.5–1.2 mm) in 0.8 mm Delrin ensured a good fit of the embryo into the holding well (Extended Fig. 10 b–c). All embryonic node imaging was performed in DMEM/F12 with 10% fetal calf serum (Invitrogen).

Similar mating strategy was used to obtain Arl13b-mCherry-GECO1.2^tg:GCaMP6f^tg:E2a-Cre^tg E14 embryos. Mouse embryonic fibroblasts were isolated from E14 embryos as described previously¹⁷. Where indicated, MEF cells were serum starved in DMEM containing 0.2% BSA for up to 48 h. To visualize cytoplasmic Ca²⁺ oscillations, Arl13b-mCherry-GECO1.2^tg: GCaMP6f^tg:E2a-Cre^tg embryos from LB to LHF stage were used. In brief, embryos were mounted in the upright position as described above and imaged for 4–6 min at a frame rate of 0.5 Hz on an upright FV1000 confocal system (Olympus, 60x/1.1 N.A. water dipping lens) at either 36°C or 22°C (RT). Cytoplasmic Ca²⁺ oscillations were quantified using ImageJ as described previously³³ with slight modifications: in brief, fluorescence of all frames was averaged and individual frames were divided by average intensity to generate ΔF/F. Images were thresholded to exclude cells with ΔF/F less than 30%. Furthermore, only regions with area >90 pixels² and circularities greater than 0.6 were used to define cells with cytoplasmic Ca²⁺ oscillations. All Ca²⁺ oscillations within 0.01 mm² surrounding the embryonic node were analyzed for occurrence on the left vs. the right side of the node.