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In the Drosophila larval brain, type I and type II neuroblasts (NBs) undergo a series of asymmetric divisions which give rise to distinct progeny lineages. The intermediate neural progenitors (INPs) exist only in type II NB lineages. In this study, we reveal a novel function of Inscuteable (Insc) that acts to maintain type I NB lineage identity. In insc type I NB clones of mosaic analyses with a repressible cell marker (MARCM), the formation of extra Deadpan (Dpn)+ NB-like and GMC-like cells is observed. The lack of Insc leads to the defective localization and segregation of Numb during asymmetric cell division. By the end of cytokinesis, this results in insufficient Numb in ganglion mother cells (GMCs). The formation of extra Deadpan (Dpn)+ cells in insc clones is prevented by the attenuation of Notch activity. This suggests that Insc functions through the Numb/Notch signaling pathway. We also show that in the absence of Insc in type I NB lineages, the cellular identity of GMCs is altered where they adopt an INP-like cell fate as indicated by the initiation of Dpn expression accompanied by a transient presence of Earmuff (Erm). These INP-like cells have the capacity to divide multiple times. We conclude that Insc is necessary for the maintenance of type I NB lineage identity. Genetic manipulations to eliminate most type I NBs with overproliferating type II NBs in the larval brain lead to altered circadian rhythms and defective phototaxis in adult flies. This indicates that the homeogenesis of NB lineages is important for the adult's brain function.

The central brain of Drosophila consists of the supraesophageal ganglion (SPG) and the subesophageal ganglion (SEG), both of which are generated by neural stem cell-like neuroblasts during embryonic and postembryonic development. Considerable information has been obtained on postembryonic development of the neuroblasts and their lineages in the SPG. In contrast, very little is known about neuroblasts, neural lineages, or any other aspect of the postembryonic development in the SEG. Here we characterize the neuroanatomy of the larval SEG in terms of tracts, commissures, and other landmark features as compared to a thoracic ganglion. We then use clonal MARCM labeling to identify all adult-specific neuroblast lineages in the late larval SEG and find a surprisingly small number of neuroblast lineages, 13 paired and one unpaired. The Hox genes Dfd, Scr, and Antp are expressed in a lineage-specific manner in these lineages during postembryonic development. Hox gene loss-of-function causes lineage-specific defects in axonal targeting and reduction in neural cell numbers. Moreover, it results in the formation of novel ectopic neuroblast lineages. Apoptosis block also results in ectopic lineages suggesting that Hox genes are required for lineage-specific termination of proliferation through programmed cell death. Taken together, our findings show that postembryonic development in the SEG is mediated by a surprisingly small set of identified lineages and requires lineage-specific Hox gene action to ensure the correct formation of adult-specific neurons in the Drosophila brain.

Fragile X syndrome (FXS), caused by loss of FMR1 gene function, is the most common heritable cause of intellectual disability and autism spectrum disorders. The FMR1 protein (FMRP) translational regulator mediates activity-dependent control of synapses. In addition to the metabotropic glutamate receptor (mGluR) hyperexcitation FXS theory, the GABA theory postulates that hypoinhibition is causative for disease state symptoms. Here, we use the Drosophila FXS model to assay central brain GABAergic circuitry, especially within the Mushroom Body (MB) learning center. All 3 GABAA receptor (GABAAR) subunits are reportedly downregulated in dfmr1 null brains. We demonstrate parallel downregulation of glutamic acid decarboxylase (GAD), the rate-limiting GABA synthesis enzyme, although GABAergic cell numbers appear unaffected. Mosaic analysis with a repressible cell marker (MARCM) single-cell clonal studies show that dfmr1 null GABAergic neurons innervating the MB calyx display altered architectural development, with early underdevelopment followed by later overelaboration. In addition, a new class of extra-calyx terminating GABAergic neurons is shown to include MB intrinsic α/β Kenyon Cells (KCs), revealing a novel level of MB inhibitory regulation. Functionally, dfmr1 null GABAergic neurons exhibit elevated calcium signaling and altered kinetics in response to acute depolarization. To test the role of these GABAergic changes, we attempted to pharmacologically restore GABAergic signaling and assay effects on the compromised MB-dependent olfactory learning in dfmr1 mutants, but found no improvement. Our results show that GABAergic circuit structure and function are impaired in the FXS disease state, but that correction of hypoinhibition alone is not sufficient to rescue a behavioral learning impairment.

Neurons of the Drosophila central brain fall into approximately 100 paired groups, termed lineages. Each lineage is derived from a single asymmetrically-dividing neuroblast. Embryonic neuroblasts produce 1,500 primary neurons (per hemisphere) that make up the larval CNS followed by a second mitotic period in the larva that generates approximately 10,000 secondary, adult-specific neurons. Clonal analyses based on previous works using lineage-specific Gal4 drivers have established that such lineages form highly invariant morphological units. All neurons of a lineage project as one or a few axon tracts (secondary axon tracts, SATs) with characteristic trajectories, thereby representing unique hallmarks. In the neuropil, SATs assemble into larger fiber bundles (fascicles) which interconnect different neuropil compartments. We have analyzed the SATs and fascicles formed by lineages during larval, pupal, and adult stages using antibodies against membrane molecules (Neurotactin/Neuroglian) and synaptic proteins (Bruchpilot/N-Cadherin). The use of these markers allows one to identify fiber bundles of the adult brain and associate them with SATs and fascicles of the larval brain. This work lays the foundation for assigning the lineage identity of GFP-labeled MARCM clones on the basis of their close association with specific SATs and neuropil fascicles, as described in the accompanying paper (Wong et al., 2013. Postembryonic lineages of the Drosophila brain: II. Identification of lineage projection patterns based on MARCM clones. Submitted.).

SummaryWe describe a new repressible binary expression system based on the regulatory genes from the Neurospora qa gene cluster. This “Q system” offers attractive features for transgene expression in Drosophila and mammalian cells: low basal expression in the absence of the transcriptional activator QF, high QF-induced expression, and QF repression by its repressor QS. Additionally, feeding flies quinic acid can relieve QS repression. The Q system offers many applications, including (1) intersectional “logic gates” with the GAL4 system for manipulating transgene expression patterns, (2) GAL4-independent MARCM analysis, and (3) coupled MARCM analysis to independently visualize and genetically manipulate siblings from any cell division. We demonstrate the utility of the Q system in determining cell division patterns of a neuronal lineage and gene function in cell growth and proliferation, and in dissecting neurons responsible for olfactory attraction. The Q system can be expanded to other uses in Drosophila and to any organism conducive to transgenesis.

The Golgi staining method has served neuroscience well for more than a century. In this assay I review recent progresses using genetic methods to recapitulate and extend the Golgi staining method. These methods enable new discoveries on organization and development of neuronal circuits in the fly and mouse brains.

Axonal selection of synaptic partners is generally believed to determine wiring specificity in the nervous system. However, we have recently found evidence for specific dendritic targeting in the olfactory system of Drosophila: second order olfactory neurons (Projection Neurons) from the anterodorsal (adPN) and lateral (lPN) lineages send their dendrites to stereotypical, intercalating but non-overlapping glomeruli. Here we show that POU domain transcription factors, Acj6 and Drifter, are expressed in adPNs and lPNs respectively, and are required for their dendritic targeting. Moreover, misexpression of Acj6 in lPNs, or Drifter in adPNs, results in dendritic targeting to glomeruli normally reserved for the other PN lineage. Thus, Acj6 and Drifter translate PN lineage information into distinct dendritic targeting specificity. Acj6 also controls stereotypical axon terminal arborization of PNs in a central target, suggesting that the connectivity of PN axons and dendrites in different brain centers is coordinately regulated.

We have modified an FLP/FRT-based genetic mosaic system to label either neurons derived from a common progenitor or isolated single neurons, in the Drosophila CNS. These uniquely labeled neurons can also be made homozygous for a mutation of interest within an otherwise phenotypically wild-type brain. Using this new mosaic system, not only can normal brain development be described with unprecedented single cell resolution, but also the underlying molecular mechanisms can be investigated by identifying genes that are required for these developmental processes.

castor (cas) encodes a zink finger protein expressed in a subset of Drosophila embryonic neuroglioblasts where it controls neuronal differentiation. We show here that cas is expressed at larval and pupal stages in brain cell clusters where it participates in the elaboration of the adult structures. In particular using the MARCM system (mosaic analysis with a repressible cell marker), we show that cas is required postembryonically for correct axon pathfinding of the central complex (CX) and mushroom body (MB) neurons. The linotte (lio) gene encodes a transmembrane protein expressed at larval/pupal stage in a glial structure, the TIFR, and interacts with the no-bridge (nob) gene. We show that cas interacts genetically with lio and nob. These interactions do not involve direct transcription regulation but probably cellular communication processes.

We describe a genetic mosaic system in Drosophila, in which a dominant repressor of a cell marker is placed in trans to a mutant gene of interest. Mitotic recombination events between homologous chromosomes generate homozygous mutant cells, which are exclusively labeled due to loss of the repressor. Using this system, we are able to visualize axonal projections and dendritic elaboration in large neuroblast clones and single neuron clones with a membrane-targeted GFP marker. This new method allows for the study of gene functions in neuroblast proliferation, axon guidance, and dendritic elaboration in the complex central nervous system. As an example, we show that the short stop gene is required in mushroom body neurons for the extension and guidance of their axons.

The spatio-temporal proliferation pattern of postembryonic neuroblasts in the central brain region of the supra-esophageal ganglion of Drosophila melanogaster was studied by labeling DNA replicating cells with 5-bromo-2'-deoxyuridine (BrdU). There are five proliferating neuroblasts per hemisphere in larvae just after hatching: one in the ventro-lateral, and the other four in the postero-dorsal region of the brain. Dividing neuroblasts increase during the late first-late second instar larval stages, reaching a plateau of about 85 neuroblasts per hemisphere. Most neuroblasts cease dividing 20-30 hr after puparium formation (APF), while only four in the postero-dorsal region continue making progenies until 85-90 hr APF. The four distinct neuroblasts proliferating in the early larval and late pupal stages are identical; they lie in the cortex above the calyces of the mushroom bodies (corpora pedunculata), proliferating over a period twice as long as that for the other neuroblasts. Their daughter neurons project into the mushroom body neuropile, and hence are likely to be the Kenyon cells. The cell-cycle period of the four neuroblasts (named mushroom body neuroblasts: MBNbs) is rather constant (1.1-1.5 hr) during the mid larval-early pupal stages and is longer before and after that. The total number of the MBNb progenies made throughout the embryonic and postembryonic development was estimated to be 800-1200 per hemisphere.

Postembryonic neuroepithelial stem cells (NESCs) in the outer proliferation center (OPC) of the Drosophila optic lobe initially divide symmetrically to expand the pool of stem or progenitor cells, and are consequently converted into postembryonic neuroblasts (pNBs). pNBs generate both progenitor cells (pNBs) and differentiating cells (GMCs; ganglion mother cells) by asymmetric division, the latter of which then generate two neurons. Thus far the mechanism underlying the conversion of NESCs into pNBs has been unknown. We have shown that the NESCs dividing symmetrically at the border between NESCs and pNBs generate two NESCs, one of which is then converted into pNB during G1 phase by losing epithelial features including belted adherence junction (AJ) and high level of DE-cadherin expression at the AJ, which is followed by upregulation of pan-neural gene Asense. We found that Notch signaling is activated just before the start of conversion and rapidly downregulated upon completion of the conversion, which is marked by high level of Asense expression. Thus, we examined the role of Notch signaling in the conversion of NESCs by MARCM clonal analysis and found that Notch signaling regulates the timing of conversion through initiating morphological changes and upregulation of Asense in the NESCs.