RNA probes were made by Integrated DNA Technologies. Primary probe solution (2 μl of FLAPY probe + 98 μl of hybridization buffer) was added to samples and incubated in a humidity chamber at 37°C for 5 hours. Embryos were washed three times in smFISH wash buffer (10% formamide in 2× SSC/0.5% Triton X-100) for 5 min each. Embryos were washed for 1 hour at 37°C. Samples were washed twice more in smFISH wash buffer for 5 min each. Eight microliters of 1:3 Vectashield (Thermo Fisher Scientific) DAPI:PBS was added to samples and allowed to incubate for 5 min. A coverslip was placed on slides and sealed with nail polish. smFISH analysis was performed using the FISH-quant program (74 (link)). smFISH foci were localized in 3D space using Gaussian fitting. Quality control for localized spot is determined by its point spread function. Embryos were staged by counting the number of DAPI nuclei.