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.

Neural progenitor cells, in both vertebrates and invertebrates, go through temporal competence changes, evidenced by the generation of different classes of neurons and glia at different time points (Okano and Temple, 2009). These programmed changes are likely to be controlled by a combination of both extrinsic and intrinsic cues, and evidence points to the existence of both mechanisms in vertebrates and invertebrates. With respect to intrinsic cues, major progress has been made in the Drosophila melanogaster system, in particular in the embryonic ventral nerve cord (VNC). Here, temporal competence changes have been shown to be under control of an intrinsic temporal cascade of transcription factors, the temporal gene cascade (Brody and Odenwald, 2002;Jacob et al., 2008;Pearson and Doe, 2004). This cascade consist of the sequential expression, and function, of the Hunchback (Hb), Kruppel (Kr), Nubbin and Pdm2 (denoted collectively Pdm herein), Castor (Cas) and Grainy head (Grh) transcription factors, in a HbrKrrPdmrCasrGrh cascade. The precise progression of this cascade is an effect of mutually activating and repressing actions of the factors upon each other. In addition, studies have also identified factors that facilitate this progression, i.e. 'switching factors'. Here, the seven up (svp) and distal antenna/distal antenna related (collectively referred to as dan herein) genes have been shown to play important roles in ensuring the switch from HbrKr, by suppressing Hb (Kanai et al., 2005;Kohwi et al., 2011;Mettler et al., 2006). Both Svp and Dan display a second wave of expression, but their function here is unknown. Finally, our previous studies have also identified the existence of so-called 'sub-temporal' genes, which act downstream of the temporal genes, do not regulate temporal genes, and act to sub-divide larger temporal windows (Baumgardt et al., 2009). However, in spite of the progress in understanding temporal competence changes, it is not clear how neuroblasts switch from one competence window to the next, how window size is controlled and how windows are subdivided. Moreover, recent mathematical modelling of the temporal cascades, indicate the existence of additional players involved in the temporal competence changes observed in vivo (Nakajima et al., 2010).To address these issues, we are using the Drosophila embryonic thoracic neuroblast 5-6 (NB5-6T) as a model. This neuroblast, which can be readily identified by the specific expression of reporter genes under the control of an enhancer fragment from the ladybird early gene [lbe(K)] (De Graeve et al., 2004), is generated in each of the six thoracic VNC hemisegments. Each NB5-6T produces a mixed lineage of 20 cells, and the four last cells to be born are a set of four interneurons expressing the Apterous (Ap) LIM-homeodomain transcription factor: the Ap neurons (Baumgardt et al., 2009). The four Ap neurons can be further subdivided into three different neuronal sub-types: the Ap1/Nplp1 and Ap4/FMRFa neurons, expressing the Nplp1 and FMRFamide neurop...

SUMMARYIdentification of the genetic mechanisms underlying the specification of large numbers of different neuronal cell fates from limited numbers of progenitor cells is at the forefront of developmental neurobiology. In Drosophila, the identities of the different neuronal progenitor cells, the neuroblasts, are specified by a combination of spatial cues. These cues are integrated with temporal competence transitions within each neuroblast to give rise to a specific repertoire of cell types within each lineage. However, the nature of this integration is poorly understood. To begin addressing this issue, we analyze the specification of a small set of peptidergic cells: the abdominal leucokinergic neurons. We identify the progenitors of these neurons, the temporal window in which they are specified and the influence of the Notch signaling pathway on their specification. We also show that the products of the genes klumpfuss, nab and castor play important roles in their specification via a genetic cascade.

Specification of the myriad of unique neuronal subtypes found in the nervous system depends upon spatiotemporal cues and terminal selector gene cascades, often acting in sequential combinatorial codes to determine final cell fate. However, a specific neuronal cell subtype can often be generated in different parts of the nervous system and at different stages, indicating that different spatiotemporal cues can converge on the same terminal selectors to thereby generate a similar cell fate. However, the regulatory mechanisms underlying such convergence are poorly understood. The Nplp1 neuropeptide neurons in the Drosophila ventral nerve cord can be subdivided into the thoracic-ventral Tv1 neurons and the dorsal-medial dAp neurons. The activation of Nplp1 in Tv1 and dAp neurons depends upon the same terminal selector cascade: col>ap/eya>dimm>Nplp1. However, Tv1 and dAp neurons are generated by different neural progenitors (neuroblasts) with different spatiotemporal appearance. Here, we find that the same terminal selector cascade is triggered by Kr/pdm>grn in dAp neurons, but by Antp/hth/exd/lbe/cas in Tv1 neurons. Hence, two different spatiotemporal combinations can funnel into a common downstream terminal selector cascade to determine a highly related cell fate.