Persistent engrailed expression is required to determine sensory axon trajectory, branching, and target choice

The transcription factor Engrailed (En) directs, in the cockroach cercal system, the shape of the axonal arborization and the choice of postsynaptic partners of an identified sensory neuron (6m). Knock-out of En using double-stranded RNA interference transforms 6m so that it resembles a neighboring neuron that normally does not express the en gene, has a different arbor anatomy, and makes different connections. We characterized the development of 6m and perturbed en expression at different stages. Our results show that En is not required before birth for 6m to become a neuron, but that it is required in the postmitotic neuron to control axonal arborization and synaptic specificity. Knock-out of En after 6m has entered the CNS does not change the axonal trajectory and has minor effects on axonal branches but causes the formation of synaptic connections typical of an En-negative cell. This suggests that En controls target recognition molecules independently from those guiding the axon. In contrast, double-stranded RNA injection 1 d later does not have any effects on the phenotype of 6m, suggesting that the period of synapse formation is over by the time En levels have fallen or, if synapse turnover occurs, that En is not required to maintain the specificity of synaptic connections. We conclude that persistent en expression is required to determine successive stages in the differentiation of the neuron, suggesting that it is not far upstream from those genes encoding axon guidance and synaptic recognition molecules.