In vivo expansion of functionally integrated GABAergic interneurons by targeted increase in neural progenitors.

A central hypothesis for brain evolution is that it might occur via expansion of progenitor cells and subsequent lineage-dependent formation of neural circuits. Here, we report in vivo amplification and functional integration of lineage-specific circuitry in Drosophila Levels of the cell fate determinant Prospero were attenuated in specific brain lineages within a range that expanded not only progenitors but also neuronal progeny, without tumor formation. Resulting supernumerary neural stem cells underwent normal functional transitions, progressed through the temporal patterning cascade, and generated progeny with molecular signatures matching source lineages. Fully differentiated supernumerary gamma-amino butyric acid (GABA)-ergic interneurons formed functional connections in the central complex of the adult brain, as revealed by in vivo calcium imaging and open-field behavioral analysis. Our results show that quantitative control of a single transcription factor is sufficient to tune neuron numbers and clonal circuitry, and provide molecular insight into a likely mechanism of brain evolution.

The mTOR pathway component Unkempt regulates neural stem cell and neural progenitor cell cycle in the Drosophila central nervous system.

The formation of a complex nervous system requires the coordinated action of progenitor cell proliferation, differentiation and maturation. The Drosophila postembryonic central nervous system provides a powerful model for dissecting the cellular and molecular mechanisms underpinning neurogenesis. We previously identified the conserved zinc finger/RING protein Unkempt (Unk) as a key temporal regulator of neuronal differentiation in the Drosophila developing eye and showed that Unk acts downstream of the mechanistic target of rapamycin (mTOR) pathway together with its binding partner Headcase (Hdc). Here we investigate the role of Unk in Drosophila postembryonic thoracic neurogenesis. The Drosophila central nervous system contains neural stem cells, called neuroblasts, and neural progenitors, known as ganglion mother cells (GMCs). Unk is highly expressed in the central brain and ventral nerve cord but is not required to maintain neuroblast numbers or for the regulation of temporal series factor expression in neuroblasts. However, loss of Unk increases the number of neuroblasts and GMCs in S-phase of the cell cycle, resulting in the overproduction of neurons. We also show that Unk interacts with Hdc through its zinc finger domain. The zinc finger domain is required for the synergistic activity of Unk with Hdc during eye development but is not necessary for the activity of Unk in thoracic neurogenesis. Overall, this study shows that Unk and Hdc are novel negative regulators of neurogenesis in Drosophila and indicates a conserved role of mTOR signalling in nervous system development.

Fat cells reactivate quiescent neuroblasts via TOR and glial insulin relays in Drosophila.

Many stem, progenitor and cancer cells undergo periods of mitotic quiescence from which they can be reactivated. The signals triggering entry into and exit from this reversible dormant state are not well understood. In the developing Drosophila central nervous system, multipotent self-renewing progenitors called neuroblasts undergo quiescence in a stereotypical spatiotemporal pattern. Entry into quiescence is regulated by Hox proteins and an internal neuroblast timer. Exit from quiescence (reactivation) is subject to a nutritional checkpoint requiring dietary amino acids. Organ co-cultures also implicate an unidentified signal from an adipose/hepatic-like tissue called the fat body. Here we provide in vivo evidence that Slimfast amino-acid sensing and Target of rapamycin (TOR) signalling activate a fat-body-derived signal (FDS) required for neuroblast reactivation. Downstream of this signal, Insulin-like receptor signalling and the Phosphatidylinositol 3-kinase (PI3K)/TOR network are required in neuroblasts for exit from quiescence. We demonstrate that nutritionally regulated glial cells provide the source of Insulin-like peptides (ILPs) relevant for timely neuroblast reactivation but not for overall larval growth. Conversely, ILPs secreted into the haemolymph by median neurosecretory cells systemically control organismal size but do not reactivate neuroblasts. Drosophila thus contains two segregated ILP pools, one regulating proliferation within the central nervous system and the other controlling tissue growth systemically. Our findings support a model in which amino acids trigger the cell cycle re-entry of neural progenitors via a fat-body-glia-neuroblasts relay. This mechanism indicates that dietary nutrients and remote organs, as well as local niches, are key regulators of transitions in stem-cell behaviour.

Protein phosphatase 4 mediates localization of the Miranda complex during Drosophila neuroblast asymmetric divisions.

Asymmetric localization of cell fate determinants is a crucial step in neuroblast asymmetric divisions. Whereas several protein kinases have been shown to mediate this process, no protein phosphatase has so far been implicated. In a clonal screen of larval neuroblasts we identified the evolutionarily conserved Protein Phosphatase 4 (PP4) regulatory subunit PP4R3/Falafel (Flfl) as a key mediator specific for the localization of Miranda (Mira) and associated cell fate determinants during both interphase and mitosis. Flfl is predominantly nuclear during interphase/prophase and cytoplasmic after nuclear envelope breakdown. Analyses of nuclear excluded as well as membrane targeted versions of the protein suggest that the asymmetric cortical localization of Mira and its associated proteins during mitosis depends on cytoplasmic/membrane-associated Flfl, whereas nuclear Flfl is required to exclude the cell fate determinant Prospero (Pros), and consequently Mira, from the nucleus during interphase/prophase. Attenuating the function of either the catalytic subunit of PP4 (PP4C; Pp4-19C in Drosophila) or of another regulatory subunit, PP4R2 (PPP4R2r in Drosophila), leads to similar defects in the localization of Mira and associated proteins. Flfl is capable of directly interacting with Mira, and genetic analyses indicate that flfl acts in parallel to or downstream from the tumor suppressor lethal (2) giant larvae (lgl). Our findings suggest that Flfl may target PP4 to the MIra protein complex to facilitate dephosphorylation step(s) crucial for its cortical association/asymmetric localization.

Mechanisms of asymmetric progenitor divisions in the Drosophila central nervous system.

The Drosophila central nervous system develops from polarised asymmetric divisions of precursor cells, called neuroblasts. Decades of research on neuroblasts have resulted in a substantial understanding of the factors and molecular events responsible for fate decisions of neuroblasts and their progeny. Furthermore, the cell-cycle dependent mechanisms responsible for asymmetric cortical protein localisation, resulting in the unequal partitioning between daughters, are beginning to be exposed. Disruption to the appropriate partitioning of proteins between neuroblasts and differentiation-committed daughters can lead to supernumerary neuroblast-like cells and the formation of tumours. Many of the factors responsible for regulating asymmetric division of Drosophila neuroblasts are evolutionarily conserved and, in many cases, have been shown to play a functionally conserved role in mammalian neurogenesis. Recent genome-wide studies coupled with advancements in live-imaging technologies have opened further avenues of research into neuroblast biology. We review our current understanding of the molecular mechanisms regulating neuroblast divisions, a powerful system to model mammalian neurogenesis and tumourigenesis.

An Immobilization Technique for Long-Term Time-Lapse Imaging of Explanted Drosophila Tissues.

Time-lapse imaging is an essential tool to study dynamic biological processes that cannot be discerned from fixed samples alone. However, imaging cell- and tissue-level processes in intact animals poses numerous challenges if the organism is opaque and/or motile. Explant cultures of intact tissues circumvent some of these challenges, but sample drift remains a considerable obstacle. We employed a simple yet effective technique to immobilize tissues in medium-bathed agarose. We applied this technique to study multiple Drosophila tissues from first-instar larvae to adult stages in various orientations and with no evidence of anisotropic pressure or stress damage. Using this method, we were able to image fine features for up to 18 h and make novel observations. Specifically, we report that fibers characteristic of quiescent neuroblasts are inherited by their basal daughters during reactivation; that the lamina in the developing visual system is assembled roughly 2-3 columns at a time; that lamina glia positions are dynamic during development; and that the nuclear envelopes of adult testis cyst stem cells do not break down completely during mitosis. In all, we demonstrate that our protocol is well-suited for tissue immobilization and long-term live imaging, enabling new insights into tissue and cell dynamics in Drosophila.

Spatiotemporally controlled genetic perturbation for efficient large-scale studies of cell non-autonomous effects.

Studies in genetic model organisms have revealed much about the development and pathology of complex tissues. Most have focused on cell-intrinsic gene functions and mechanisms. Much less is known about how transformed, or otherwise functionally disrupted, cells interact with healthy ones toward a favorable or pathological outcome. This is largely due to technical limitations. We developed new genetic tools in Drosophila melanogaster that permit efficient multiplexed gain- and loss-of-function genetic perturbations with separable spatial and temporal control. Importantly, our novel tool-set is independent of the commonly used GAL4/UAS system, freeing the latter for additional, non-autonomous, genetic manipulations; and is built into a single strain, allowing one-generation interrogation of non-autonomous effects. Altogether, our design opens up efficient genome-wide screens on any deleterious phenotype, once plasmid or genome engineering is used to place the desired miRNA(s) or ORF(s) into our genotype. Specifically, we developed tools to study extrinsic effects on neural tumor growth but the strategy presented has endless applications within and beyond neurobiology, and in other model organisms.

A mosaic genetic screen for novel mutations affecting Drosophila neuroblast divisions.

BACKGROUND: The asymmetric segregation of determinants during cell division is a fundamental mechanism for generating cell fate diversity during development. In Drosophila, neural precursors (neuroblasts) divide in a stem cell-like manner generating a larger apical neuroblast and a smaller basal ganglion mother cell. The cell fate determinant Prospero and its adapter protein Miranda are asymmetrically localized to the basal cortex of the dividing neuroblast and segregated into the GMC upon cytokinesis. Previous screens to identify components of the asymmetric division machinery have concentrated on embryonic phenotypes. However, such screens are reaching saturation and are limited in that the maternal contribution of many genes can mask the effects of zygotic loss of function, and other approaches will be necessary to identify further genes involved in neuroblast asymmetric division. RESULTS: We have performed a genetic screen in the third instar larval brain using the basal localization of Miranda as a marker for neuroblast asymmetry. In addition to the examination of pupal lethal mutations, we have employed the MARCM (Mosaic Analysis with a Repressible Cell Marker) system to generate postembryonic clones of mutations with an early lethal phase. We have screened a total of 2,300 mutagenized chromosomes and isolated alleles affecting cell fate, the localization of basal determinants or the orientation of the mitotic spindle. We have also identified a number of complementation groups exhibiting defects in cell cycle progression and cytokinesis, including both novel genes and new alleles of known components of these processes. CONCLUSION: We have identified four mutations which affect the process of neuroblast asymmetric division. One of these, mapping to the imaginal discs arrested locus, suggests a novel role for the anaphase promoting complex/cyclosome (APC/C) in the targeting of determinants to the basal cortex. The identification and analysis of the remaining mutations will further advance our understanding of the process of asymmetric cell division. We have also isolated a number of mutations affecting cell division which will complement the functional genomics approaches to this process being employed by other laboratories. Taken together, these results demonstrate the value of mosaic screens in the identification of genes involved in neuroblast division.

Phosphorylation and dephosphorylation events allow for rapid segregation of fate determinants during Drosophila neuroblast asymmetric divisions.

Drosophila neuroblasts display remarkable asymmetry throughout mitosis. The most prominent asymmetry is the size difference between daughter cells at cytokinesis. The larger cell retains stem cell identity, i.e., remains a neuroblast while the smaller cell, called a ganglion mother cell (GMC), will generate differentiated neural and glial progeny. Preceding this size difference, several protein complexes localize to opposite sides of the neuroblast cortex (apical and basal in the embryo and, by analogy, referred to as such in larval neuroblasts although their asymmetry no longer correlates with such axis). The plane of division is coordinated with this molecular asymmetry such that apical and basal complexes are unequally partitioned between the two daughter cells: apical complexes are inherited by the self-renewing neuroblast while basal complexes are inherited by the GMC. This unequal segregation has been extensively shown to be functionally significant. Apical complexes contain factors required for cellular selfrenewal and basal complexes contain factors required for the differentiation of the GMC progeny. Curiously, however, some "basal" neuroblast proteins such as the scaffold protein Miranda (Mira) and its associated fate determinant Prospero (Pros), are initially apically localized prior to translocating to the opposite side of the cell cortex by the onset of mitosis. This is because mira mRNA is apically enriched, where it remains throughout the cell cycle, suggesting that Mira protein is translated within the apical environment.1,2 The transition from apical to basal enrichment of Mira and Pros takes place within minutes.2 Here, we summarize the known phosphorylation events and roles during neuroblast asymmetric divisions, as well as very recent work, including our own, identifying the first protein phosphatases implicated in this process. We then discuss models previously proposed, as well as a new model, for apical-to-basal transition of the Mira complex in light of our new results.

Regulating neural proliferation in the Drosophila CNS.

Neural stem and progenitor cells generate the central nervous system (CNS) in organisms as diverse as insects and mammals. In Drosophila, multipotent asymmetrically dividing progenitors called neuroblasts produce neurons and glia throughout the developing CNS. Nevertheless, the time-windows of mitotic activity, the division modes, the termination mechanisms and the lineage sizes of individual neuroblasts all vary considerably from region-to-region. Recent studies shed light on some of the mechanisms underlying this neuroblast diversity and, in particular, how proliferation is boosted in two brain regions. In the central brain, some specialised neuroblasts generate intermediate neural progenitors that can each divide multiple times, thus increasing overall lineage size. In the optic lobe, an alternative expansion strategy involves symmetrically dividing neuroepithelial cells generating large numbers of asymmetrically dividing neuroblasts. Evidence is also emerging for a cell-intrinsic timer that alters the properties of each neuroblast with increasing developmental age. The core mechanism corresponds to a series of transcription factors that coordinates temporal changes in neuronal/glial identity with transitions in neuroblast cell-cycle speed, entry into quiescence and, ultimately, with termination.

Interplay between the transcription factor Zif and aPKC regulates neuroblast polarity and self-renewal.

How a cell decides to self-renew or differentiate is a critical issue in stem cell and cancer biology. Atypical protein kinase C (aPKC) promotes self-renewal of Drosophila larval brain neural stem cells, neuroblasts. However, it is unclear how aPKC cortical polarity and protein levels are regulated. Here, we have identified a zinc-finger protein, Zif, which is required for the expression and asymmetric localization of aPKC. aPKC displays ectopic cortical localization with upregulated protein levels in dividing zif mutant neuroblasts, leading to neuroblast overproliferation. We show that Zif is a transcription factor that directly represses aPKC transcription. We further show that Zif is phosphorylated by aPKC both in vitro and in vivo. Phosphorylation of Zif by aPKC excludes it from the nucleus, leading to Zif inactivation in neuroblasts. Thus, reciprocal repression between Zif and aPKC act as a critical regulatory mechanism for establishing cell polarity and controlling neuroblast self-renewal.

Characterizing embryonic gene expression patterns in the mouse using nonredundant sequence-based selection.

This article investigates the expression patterns of 160 genes that are expressed during early mouse development. The cDNAs were isolated from 7.5 d postcoitum (dpc) endoderm, a region that comprises visceral endoderm (VE), definitive endoderm, and the node-tissues that are required for the initial steps of axial specification and tissue patterning in the mouse. To avoid examining the same gene more than once, and to exclude potentially ubiquitously expressed housekeeping genes, cDNA sequence was derived from 1978 clones of the Endoderm library. These yielded 1440 distinct cDNAs, of which 123 proved to be novel in the mouse. In situ hybridization analysis was carried out on 160 of the cDNAs, and of these, 29 (18%) proved to have restricted expression patterns.