A novel temporal identity window generates alternating Eve(+)/Nkx6(+) motor neuron subtypes in a single progenitor lineage

BACKGROUND: Spatial patterning specifies neural progenitor identity, with further diversity generated by temporal patterning within individual progenitor lineages. In vertebrates, these mechanisms generate “cardinal classes” of neurons that share a transcription factor identity and common morphology. In Drosophila, two cardinal classes are Even-skipped (Eve)(+) motor neurons projecting to dorsal longitudinal muscles, and Nkx6(+) motor neurons projecting to ventral oblique muscles. Cross-repressive interactions prevent stable double-positive motor neurons. The Drosophila neuroblast 7–1 (NB7–1) lineage uses a temporal transcription factor cascade to generate five distinct Eve(+) motor neurons; the origin and development of Nkx6(+) motor neurons remains unclear. METHODS: We use a neuroblast specific Gal4 line, sparse labelling and molecular markers to identify an Nkx6(+) VO motor neuron produced by the NB7–1 lineage. We use lineage analysis to birth-date the VO motor neuron to the Kr(+) Pdm(+) neuroblast temporal identity window. We use gain- and loss-of-function strategies to test the role of Kr(+) Pdm(+) temporal identity and the Nkx6 transcription factor in specifying VO neuron identity. RESULTS: Lineage analysis identifies an Nkx6(+) neuron born from the Kr(+) Pdm(+) temporal identity window in the NB7–1 lineage, resulting in alternation of cardinal motor neuron subtypes within this lineage (Eve>Nkx6 > Eve). Co-overexpression of Kr/Pdm generates ectopic VO motor neurons within the NB7–1 lineage – the first evidence that this TTF combination specifies neuronal identity. Moreover, the Kr/Pdm combination promotes Nkx6 expression, which itself is necessary and sufficient for motor neuron targeting to ventral oblique muscles, thereby revealing a molecular specification pathway from temporal patterning to cardinal transcription factor expression to motor neuron target selection. CONCLUSIONS: We show that one neuroblast lineage generates interleaved cardinal motor neurons fates; that the Kr/Pdm TTFs form a novel temporal identity window that promotes expression of Nkx6; and that the Kr/Pdm > Nkx6 pathway is necessary and sufficient to promote VO motor neuron targeting to the correct ventral muscle group.