Supplemental Experimental Procedures Neuronal Subtype Labeling, Dissociation, and Purification

For embryonic injections, the day of vaginal plug was designated E0. E17 pregnant mice were deeply anesthetized with Avertin, and each embryo was injected through the uterine wall into the pons, using a VisualSonics Vevo 660 ultrasound-guided microinjection system (VisualSonics, Toronto, Canada) to precisely control the position of the injection site (Figures 1A–1C). Two injections per embryo were performed, with a total of 60–80 nl of green fluorescent microspheres per injection site. The pregnant dam was deeply anesthetized 1 day after surgery, embryos were removed, and sensorimotor cortex was microdissected from both cerebral hemispheres using a fluorescence dissecting microscope to precisely visualize the labeled region (Figure 1D). Similarly, for postnatal injections, P1, P4, and P11 mice were anesthetized by hypothermia (P1, P4) or Avertin (P11) and injected in the pons (P1) or the cervical spinal cord (P4 and P11). Four injections per pup were performed, with a total of 60–80 nl of green fluorescent microspheres per injection site. Pups were returned to the care of their mother and deeply anesthetized at P3, P6, and P14, respectively. Sensorimotor cortex was microdissected as described above. Retrogradely labeled cortex was enzymatically digested in dissociation medium (glucose [2