(A) Schematic drawing depicts the step-wise development of the nervous system in Drosophila, starting during embryogenesis, progressing through three larval stages and metamorphosing during pupal stage into the adult nervous system. The first connections in the neuromuscular system are formed between MNs and muscles in late Drosophila embryos (stage 16). MNs at this stage expressing UAS-RFP under the control of the motoneuronal driver OK371-GAL4 (OK371>RFP) are color-coded along the ventral nerve cord according to different patterns of Hox gene expression. OK371-RFP positive MNs were FACS sorted and single cells were sequenced by targeted Smart-Seq2 to enrich for Hox gene representation (HoxSeq) as spatial markers (see also Materials and Methods). 