library_construction_protocol: The hindbrains of embryos were dissected  in ice-cold HEBG media (0.8X B27, 0.25X GlutaMAX in Hibernate E media). For each timepoint, at least 3 embryos were collected for each two independent replicates. Tissues were incubated with Worthington Papain solution at 37°C for 30 min at 800 rpm. At the end of incubation, the samples were transferred to 15 mL falcon tube, followed by gentle trituration with serologic pipette. The cell pellets were collected by centrifuge at 200 rcf for 3 min at 4°C, washed and resuspended in ice-cold sorting buffer (PBS with 0.05% fetal bovine serum). The single-cell resuspension was loaded to 30 µm cell drainer to remove debris, followed by DAPI staining at RT for 5 min. GFP+DAPI-, GFP+TdTom+DAPI-, or TdTom+DAPI- cells were sorted by Sony SH800S. The cells were pelleted by centrifuge at 200 rcf for 5 min at 4°C and resuspended in sorting buffer to make the final concentration of 1,000 cells/µL. The cDNA libraries were constructed by 10x Genomics 3' v3.1 kit following the user guide. Briefly, ~16,500 cells from one sample were mixed with reversed transcription master mix before loaded into Chromium Chip G. GEM containing cells, reversed transcription reagents, and barcoded gel beads were generated by Chromium Controller. The first strand cDNA was amplified, fragmentated, and ligated with sequencing adaptors and sample indices.