Abstract
BackgroundSpatial 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.MethodsWe 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.ResultsLineage 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.ConclusionsWe 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.
Highlights
Neural diversity from flies to mice arises from two major developmental mechanisms
We show that overexpression of Kr/Pdm together, or Nkx6 alone, generates ectopic ventral oblique (VO) motor neurons based on molecular marker expression
The neuroblast 7–1 (NB7–1) lineage has a Kr+ Pdm+ temporal identity window that generates an Nkx6+ motor neuron The existence of a Kr+ Pdm+ temporal identity window had been predicted by computational methods [20], so we sought to confirm this in vivo using a previouslycharacterized highly-specific NB7–1 split gal4 line (NB7–1-gal4 [21]) to express UAS-myr:GFP in NB7–1 and its progeny (Fig. S1, Fig. 1a-d)
Summary
Neural diversity from flies to mice arises from two major developmental mechanisms. First, neural progenitors acquire a unique and heritable spatial identity based on their position along the rostrocaudal or dorsoventral body axes [1, 2]. Temporal patterning is best characterized in Drosophila; neural progenitors (neuroblasts) located in the ventral nerve cord, central brain, and optic lobes all undergo temporal patterning, in which the neuroblast sequentially expresses a cascade of TTFs that specify distinct neuronal identities [3,4,5,6]. 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. 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. We use gain- and loss-of-function strategies to test the role of Kr+ Pdm+ temporal identity and the Nkx transcription factor in specifying VO neuron identity
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