The distribution of leucokinin (LK) neurons in the central nervous system (CNS) of Drosophila melanogaster was described by immunolabelling many years ago. However, no detailed underlying information of the input or output connections of their neurites was then available. Here, we provide a more accurate morphological description by employing a novel LK-specific GAL4 line that recapitulates LK expression. In order to analyse the possible afferent and efferent neural candidates of LK neurons, we used this lk-GAL4 line together with other CNS-Gal4 lines, combined with antisera against various neuropeptides or neurotransmitters. We found four kinds of LK neurons in the brain. (1) The lateral horn neurons connect the antennal glomerula to the mushroom bodies. (2) The suboesophageal neurons connect the gustatory receptors to the suboesophageal ganglia and ventral nerve cord. (3) The anterior neurons innervate the corpus cardiacum of the ring gland but LK expression is surprisingly not detectable from the third instar onwards in these neurons. (4) A set of abdominal ganglion neurons connect to the dorsal median tract in larvae and send their axons to a segmental muscle 8. Thus, the methods employed in our study can be used to identify individual neuropeptidergic neurons and thereby characterize functional cues or developmental transformations in their differentiation.

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One of the most widely studied phenomena in the establishment of neuronal identity is the determination of neurosecretory phenotype, in which cell-type-specific combinatorial codes direct distinct neurotransmitter or neuropeptide selection. However, neuronal types from divergent lineages may adopt the same neurosecretory phenotype, and it is unclear whether different classes of neurons use different or similar components to regulate shared features of neuronal identity. We have addressed this question by analyzing how differentiation of the Drosophila larval leucokinergic system, which is comprised of only four types of neurons, is regulated by factors known to affect expression of the FMRFamide neuropeptide. We show that all leucokinergic cells express the transcription factor Squeeze (Sqz). However, based on the effect on LK expression of loss- and gain-of-function mutations, we can describe three types of Lk regulation. In the brain LHLK cells, both Sqz and Apterous (Ap) are required for LK expression, but surprisingly, high levels of either Sqz or Ap alone are sufficient to restore LK expression in these neurons. In the suboesophageal SELK cells, Sqz, but not Ap, is required for LK expression. In the abdominal ABLK neurons, inhibition of retrograde axonal transport reduces LK expression, and although sqz is dispensable for LK expression in these cells, it can induce ectopic leucokinergic ABLK-like cells when over-expressed. Thus, Sqz appears to be a regulatory factor for neuropeptidergic identity common to all leucokinergic cells, whose function in different cell types is regulated by cell-specific factors.