Delta-dependent Notch activation closes the early neuroblast temporal program to promote lineage progression and neurogenesis termination in Drosophila.

Neuroblasts in Drosophila divide asymmetrically, sequentially expressing a series of intrinsic factors to generate a diversity of neuron types. These intrinsic factors known as temporal factors dictate timing of neuroblast transitions in response to steroid hormone signaling and specify early versus late temporal fates in neuroblast neuron progeny. After completing their temporal programs, neuroblasts differentiate or die, finalizing both neuron number and type within each neuroblast lineage. From a screen aimed at identifying genes required to terminate neuroblast divisions, we identified Notch and Notch pathway components. When Notch is knocked down, neuroblasts maintain early temporal factor expression longer, delay late temporal factor expression, and continue dividing into adulthood. We find that Delta, expressed in cortex glia, neuroblasts, and after division, their GMC progeny, regulates neuroblast Notch activity. We also find that Delta in neuroblasts is expressed high early, low late, and is controlled by the intrinsic temporal program: early factor Imp promotes Delta, late factors Syp/E93 reduce Delta. Thus, in addition to systemic steroid hormone cues, forward lineage progression is controlled by local cell-cell signaling between neuroblasts and their cortex glia/GMC neighbors: Delta transactivates Notch in neuroblasts bringing the early temporal program and early temporal factor expression to a close.

Delta-dependent Notch activation closes the early neuroblast temporal program to promote lineage progression and neurogenesis termination in Drosophila.

Neuroblasts in Drosophila divide asymmetrically, sequentially expressing a series of intrinsic factors to generate a diversity of neuron types. These intrinsic factors known as temporal factors dictate timing of neuroblast transitions in response to steroid hormone signaling and specify early versus late temporal fates in neuroblast neuron progeny. After completing their temporal programs, neuroblasts differentiate or die, finalizing both neuron number and type within each neuroblast lineage. From a screen aimed at identifying genes required to terminate neuroblast divisions, we identified Notch and Notch pathway components. When Notch is knocked down, neuroblasts maintain early temporal factor expression longer, delay late temporal factor expression, and continue dividing into adulthood. We find that Delta, expressed in cortex glia, neuroblasts, and after division, their GMC progeny, regulates neuroblast Notch activity. We also find that Delta in neuroblasts is expressed high early, low late, and is controlled by the intrinsic temporal program: early factor Imp promotes Delta, late factors Syp/E93 reduce Delta. Thus, in addition to systemic steroid hormone cues, forward lineage progression is controlled by local cell-cell signaling between neuroblasts and their cortex glia/GMC neighbors: Delta transactivates Notch in neuroblasts bringing the early temporal program and early temporal factor expression to a close.

Single cell RNA-seq analysis reveals temporally-regulated and quiescence-regulated gene expression in Drosophila larval neuroblasts.

The mechanisms that generate neural diversity during development remains largely unknown. Here, we use scRNA-seq methodology to discover new features of the Drosophila larval CNS across several key developmental timepoints. We identify multiple progenitor subtypes - both stem cell-like neuroblasts and intermediate progenitors - that change gene expression across larval development, and report on new candidate markers for each class of progenitors. We identify a pool of quiescent neuroblasts in newly hatched larvae and show that they are transcriptionally primed to respond to the insulin signaling pathway to exit from quiescence, including relevant pathway components in the adjacent glial signaling cell type. We identify candidate "temporal transcription factors" (TTFs) that are expressed at different times in progenitor lineages. Our work identifies many cell type specific genes that are candidates for functional roles, and generates new insight into the differentiation trajectory of larval neurons.

Neural Stem Cell Reactivation in Cultured Drosophila Brain Explants.

Neural stem cells (NSCs) have the ability to proliferate, differentiate, undergo apoptosis, and even enter and exit quiescence. Many of these processes are controlled by the complex interplay between NSC intrinsic genetic programs with NSC extrinsic factors, local and systemic. In the genetic model organism, Drosophila melanogaster, NSCs, known as neuroblasts (NBs), switch from quiescence to proliferation during the embryonic to larval transition. During this time, larvae emerge from their eggshells and begin crawling, seeking out dietary nutrients. In response to animal feeding, the fat body, an endocrine organ with lipid storage capacity, produces a signal, which is released systemically into the circulating hemolymph. In response to the fat body-derived signal (FBDS), Drosophila insulin-like peptides (Dilps) are produced and released from brain neurosecretory neurons and glia, leading to downstream activation of PI3-kinase growth signaling in NBs and their glial and tracheal niche. Although this is the current model for how NBs switch from quiescence to proliferation, the nature of the FBDS extrinsic cue remains elusive. To better understand how NB extrinsic systemic cues regulate exit from quiescence, a method was developed to culture early larval brains in vitro before animal feeding. With this method, exogenous factors can be supplied to the culture media and NB exit from quiescence assayed. We found that exogenous insulin is sufficient to reactivate NBs from quiescence in whole-brain explants. Because this method is well-suited for large-scale screens, we aim to identify additional extrinsic cues that regulate NB quiescence versus proliferation decisions. Because the genes and pathways that regulate NSC proliferation decisions are evolutionarily conserved, results from this assay could provide insight into improving regenerative therapies in the clinic.

Notch signaling regulates neural stem cell quiescence entry and exit in Drosophila.

Stem cells enter and exit quiescence as part of normal developmental programs and to maintain tissue homeostasis in adulthood. Although it is clear that stem cell intrinsic and extrinsic cues, local and systemic, regulate quiescence, it remains unclear whether intrinsic and extrinsic cues coordinate to control quiescence and how cue coordination is achieved. Here, we report that Notch signaling coordinates neuroblast intrinsic temporal programs with extrinsic nutrient cues to regulate quiescence in Drosophila. When Notch activity is reduced, quiescence is delayed or altogether bypassed, with some neuroblasts dividing continuously during the embryonic-to-larval transition. During embryogenesis before quiescence, neuroblasts express Notch and the Notch ligand Delta. After division, Delta is partitioned to adjacent GMC daughters where it transactivates Notch in neuroblasts. Over time, in response to intrinsic temporal cues and increasing numbers of Delta-expressing daughters, neuroblast Notch activity increases, leading to cell cycle exit and consequently, attenuation of Notch pathway activity. Quiescent neuroblasts have low to no active Notch, which is required for exit from quiescence in response to nutrient cues. Thus, Notch signaling coordinates proliferation versus quiescence decisions.

Correction: Dilp-2-mediated PI3-kinase activation coordinates reactivation of quiescent neuroblasts with growth of their glial stem cell niche.

[This corrects the article DOI: 10.1371/journal.pbio.3000721.].

Steroid hormones, dietary nutrients, and temporal progression of neurogenesis.

Temporal patterning of neural progenitors, in which different factors are sequentially expressed, is an evolutionarily conserved strategy for generating neuronal diversity during development. In the Drosophila embryo, mechanisms that mediate temporal patterning of neural stem cells (neuroblasts) are largely cell-intrinsic. However, after embryogenesis, neuroblast temporal patterning relies on extrinsic cues as well, as freshly hatched larvae seek out nutrients and other key resources in varying natural environments. We recap current understanding of neuroblast-intrinsic temporal programs and discuss how neuroblast extrinsic cues integrate and coordinate with neuroblast intrinsic programs to control numbers and types of neurons produced. One key emerging extrinsic factor that impacts temporal patterning of neuroblasts and their daughters as well as termination of neurogenesis is the steroid hormone, ecdysone, a known regulator of large-scale developmental transitions in insects and arthropods. Lastly, we consider evolutionary conservation and discuss recent work on thyroid hormone signaling in early vertebrate brain development.

Dilp-2-mediated PI3-kinase activation coordinates reactivation of quiescent neuroblasts with growth of their glial stem cell niche.

Dietary nutrients provide macromolecules necessary for organism growth and development. In response to animal feeding, evolutionarily conserved growth signaling pathways are activated, leading to increased rates of cell proliferation and tissue growth. It remains unclear how different cell types within developing tissues coordinate growth in response to dietary nutrients and whether coordinated growth of different cell types is necessary for proper tissue function. Using the early Drosophila larval brain, we asked whether nutrient-dependent growth of neural stem cells (neuroblasts), glia, and trachea is coordinated and whether coordinated growth among these major brain cell types is required for neural development. It is known that in response to dietary nutrients and PI3-kinase activation, brain and ventral nerve cord neuroblasts reactivate from quiescence and ventral nerve cord glia expand their membranes. Here, we assay growth in a cell-type specific manner at short time intervals in the brain and determine that growth is coordinated among different cell types and that coordinated growth is mediated in part through activation of PI3-kinase signaling. Of the 7 Drosophila insulin-like peptides (Dilps), we find that Dilp-2 is required for PI3-kinase activation and growth coordination between neuroblasts and glia in the brain. Dilp-2 induces brain cortex glia to initiate membrane growth and make first contact with quiescent neuroblasts. Once reactivated, neuroblasts promote cortex glia growth to ultimately form a selective membrane barrier. Our results highlight the importance of bidirectional growth signaling between neural stem cells and surrounding cell types in the brain in response to nutrition and demonstrate how coordinated growth among different cell types drives tissue morphogenesis and function.

E93 Integrates Neuroblast Intrinsic State with Developmental Time to Terminate MB Neurogenesis via Autophagy.

Most neurogenesis occurs during development, driven by the cell divisions of neural stem cells (NSCs). We use Drosophila to understand how neurogenesis terminates once development is complete, a process critical for neural circuit formation. We identified E93, a steroid-hormone-induced transcription factor that downregulates phosphatidylinositol 3-kinase (PI3K) levels to activate autophagy for elimination of mushroom body (MB) neuroblasts. MB neuroblasts are a subset of Drosophila NSCs that generate neurons important for memory and learning. MB neurogenesis extends into adulthood when E93 is reduced and terminates prematurely when E93 is overexpressed. E93 is expressed in MB neuroblasts during later stages of pupal development only, which includes the time when MB neuroblasts normally terminate their divisions. Cell intrinsic Imp and Syp temporal factors regulate timing of E93 expression in MB neuroblasts, and extrinsic steroid hormone receptor (EcR) activation boosts E93 levels high for termination. Imp inhibits premature expression of E93 in a Syp-dependent manner, and Syp positively regulates E93 to promote neurogenesis termination. Imp and Syp together with E93 form a temporal cassette, which consequently links early developmental neurogenesis with termination. Altogether, E93 functions as a late-acting temporal factor integrating extrinsic hormonal cues linked to developmental timing with neuroblast intrinsic temporal cues to precisely time neurogenesis ending during development.

Eyeless uncouples mushroom body neuroblast proliferation from dietary amino acids in Drosophila.

Cell proliferation is coupled with nutrient availability. If nutrients become limited, proliferation ceases, because growth factor and/or PI3-kinase activity levels become attenuated. Here, we report an exception to this generality within a subpopulation of Drosophila neural stem cells (neuroblasts). We find that most neuroblasts enter and exit cell cycle in a nutrient-dependent manner that is reversible and regulated by PI3-kinase. However, a small subset, the mushroom body neuroblasts, which generate neurons important for memory and learning, divide independent of dietary nutrient conditions and PI3-kinase activity. This nutrient-independent proliferation is regulated by Eyeless, a Pax-6 orthologue, expressed in mushroom body neuroblasts. When Eyeless is knocked down, mushroom body neuroblasts exit cell cycle when nutrients are withdrawn. Conversely, when Eyeless is ectopically expressed, some non-mushroom body neuroblasts divide independent of dietary nutrient conditions. Therefore, Eyeless uncouples MB neuroblast proliferation from nutrient availability, allowing preferential neurogenesis in brain subregions during nutrient poor conditions.

An Hdac1/Rpd3-Poised Circuit Balances Continual Self-Renewal and Rapid Restriction of Developmental Potential during Asymmetric Stem Cell Division.

How the developmental potential of differentiating stem cell progeny becomes rapidly and stably restricted following asymmetric stem cell division is unclear. In the fly larval brain, earmuff (erm) uniquely functions to restrict the developmental potential of intermediate neural progenitors (INPs) generated by asymmetrically dividing neural stem cells (neuroblasts). Here we demonstrate that the histone deacetylase Hdac1/Rpd3 functions together with self-renewal transcriptional repressors to maintain the erm immature INP enhancer in an inactive but poised state in neuroblasts. Within 2 hr of immature INP birth, downregulation of repressor activities alleviates Rpd3-mediated repression on the erm enhancer, enabling acetylation of multiple histone proteins and activating Erm expression. Erm restricts the developmental potential in immature INPs by repressing genes encoding neuroblast transcriptional activators. We propose that poising the fast-activating enhancers of master regulators of differentiation through continual histone deacetylation in stem cells enables self-renewal and rapid restriction of developmental potential following asymmetric division.

Neuroblast niche position is controlled by Phosphoinositide 3-kinase-dependent DE-Cadherin adhesion.

Correct positioning of stem cells within their niche is essential for tissue morphogenesis and homeostasis. How stem cells acquire and maintain niche position remains largely unknown. Here, we show that a subset of brain neuroblasts (NBs) in Drosophila utilize Phosphoinositide 3-kinase (PI3-kinase) and DE-cadherin to build adhesive contact for NB niche positioning. NBs remain within their native microenvironment when levels of PI3-kinase activity and DE-cadherin are elevated in NBs. This occurs through PI3-kinase-dependent regulation of DE-Cadherin-mediated cell adhesion between NBs and neighboring cortex glia, and between NBs and their ganglion mother cell daughters. When levels of PI3-kinase activity and/or DE-Cadherin are reduced in NBs, NBs lose niche position and relocate to a non-native brain region that is rich in neurosecretory neurons, including those that secrete some of the Drosophila insulin-like peptides. Linking levels of PI3-kinase activity to the strength of adhesive attachment could provide cancer stem cells and hematopoietic stem cells with a means to cycle from trophic-poor to trophic-rich microenvironments.

Inactivation of both Foxo and reaper promotes long-term adult neurogenesis in Drosophila.

Adult neurogenesis occurs in specific locations in the brains of many animals, including some insects, and relies on mitotic neural stem cells. In mammals, the regenerative capacity of most of the adult nervous system is extremely limited, possibly because of the absence of neural stem cells. Here we show that the absence of adult neurogenesis in Drosophila results from the elimination of neural stem cells (neuroblasts) during development. Prior to their elimination, their growth and proliferation slows because of decreased insulin/PI3 kinase signaling, resulting in nuclear localization of Foxo. These small neuroblasts are typically eliminated by caspase-dependent cell death, and not exclusively by terminal differentiation as has been proposed. Eliminating Foxo, together with inhibition of reaper family proapoptotic genes, promotes long-term survival of neuroblasts and sustains neurogenesis in the adult mushroom body (mb), the center for learning and memory in Drosophila. Foxo likely activates autophagic cell death, because simultaneous inhibition of ATG1 (autophagy-specific gene 1) and apoptosis also promotes long-term mb neuroblast survival. mb neurons generated in adults incorporate into the existing mb neuropil, suggesting that their identity and neuronal pathfinding cues are both intact. Thus, inhibition of the pathways that normally function to eliminate neural stem cells during development enables adult neurogenesis.

Microtubule-induced cortical cell polarity.

Most cells are polarized. Embryonic and stem cells can use their polarity to generate cell diversity by asymmetric cell division, whereas differentiated cells use their polarity to execute specific functions. For example, fibroblasts form an actin-rich leading edge required for cell migration, neurons form distinctive axonal and dendritic compartments important for directional signaling, and epithelial cells have apical and basolateral cortical domains necessary for maintaining tissue impermeability. It is well established that actin and actin-associated proteins are essential for generating molecular and morphological cell polarity, but only recently has it become accepted that microtubules can induce and/or maintain polarity. One common feature among different cell types is that microtubules can establish the position of cortical polarity, but are not required for cortical polarity per se. In this review, we discuss how different cell types utilize microtubules and microtubule-associated signaling pathways to generate cortical cell polarity, highlight common mechanisms, and discuss open questions for directing future research.

Brat is a Miranda cargo protein that promotes neuronal differentiation and inhibits neuroblast self-renewal.

An important question in stem cell biology is how a cell decides to self-renew or differentiate. Drosophila neuroblasts divide asymmetrically to self-renew and generate differentiating progeny called GMCs. Here, we report that the Brain tumor (Brat) translation repressor is partitioned into GMCs via direct interaction with the Miranda scaffolding protein. In brat mutants, another Miranda cargo protein (Prospero) is not partitioned into GMCs, GMCs fail to downregulate neuroblast gene expression, and there is a massive increase in neuroblast numbers. Single neuroblast clones lacking Prospero have a similar phenotype. We conclude that Brat suppresses neuroblast stem cell self-renewal and promotes neuronal differentiation.

Extrinsic cues orient the cell division axis in Drosophila embryonic neuroblasts.

Cell polarity must be integrated with tissue polarity for proper development. The Drosophila embryonic central nervous system (CNS) is a highly polarized tissue; neuroblasts occupy the most apical layer of cells within the CNS, and lie just basal to the neural epithelium. Neuroblasts are the CNS progenitor cells and undergo multiple rounds of asymmetric cell division, ;budding off' smaller daughter cells (GMCs) from the side opposite the epithelium, thereby positioning neuronal/glial progeny towards the embryo interior. It is unknown whether this highly stereotypical orientation of neuroblast divisions is controlled by an intrinsic cue (e.g. cortical mark) or an extrinsic cue (e.g. cell-cell signal). Using live imaging and in vitro culture, we find that neuroblasts in contact with epithelial cells always ;bud off' GMCs in the same direction, opposite from the epithelia-neuroblast contact site, identical to what is observed in vivo. By contrast, isolated neuroblasts 'bud off' GMCs at random positions. Imaging of centrosome/spindle dynamics and cortical polarity shows that in neuroblasts contacting epithelial cells, centrosomes remained anchored and cortical polarity proteins localize at the same epithelia-neuroblast contact site over subsequent cell cycles. In isolated neuroblasts, centrosomes drifted between cell cycles and cortical polarity proteins showed a delay in polarization and random positioning. We conclude that embryonic neuroblasts require an extrinsic signal from the overlying epithelium to anchor the centrosome/centrosome pair at the site of epithelial-neuroblast contact and for proper temporal and spatial localization of cortical Par proteins. This ensures the proper coordination between neuroblast cell polarity and CNS tissue polarity.

Microtubule-induced Pins/Galphai cortical polarity in Drosophila neuroblasts.

Cortical polarity regulates cell division, migration, and differentiation. Microtubules induce cortical polarity in yeast, but few examples are known in metazoans. We show that astral microtubules, kinesin Khc-73, and Discs large (Dlg) induce cortical polarization of Pins/Galphai in Drosophila neuroblasts; this cortical domain is functional for generating spindle asymmetry, daughter-cell-size asymmetry, and distinct sibling fates. Khc-73 localizes to astral microtubule plus ends, and Dlg/Khc-73 and Dlg/Pins coimmunoprecipitate, suggesting that microtubules induce Pins/Galphai cortical polarity through Dlg/Khc-73 interactions. The microtubule/Khc-73/Dlg pathway acts in parallel to the well-characterized Inscuteable/Par pathway, but each provides unique spatial and temporal information: The Inscuteable/Par pathway initiates at prophase to coordinate neuroblast cortical polarity with CNS tissue polarity, whereas the microtubule/Khc-73/Dlg pathway functions at metaphase to coordinate neuroblast cortical polarity with the mitotic spindle axis. These results identify a role for microtubules in polarizing the neuroblast cortex, a fundamental step for generating cell diversity through asymmetric cell division.

Molecular and phylogenetic characterization of Zyx102, a Drosophila orthologue of the zyxin family that interacts with Drosophila Enabled.

Adherens junctions, which are cadherin-mediated junctions between cells, and focal adhesions, which are integrin-mediated junctions between cells and the extracellular matrix, are protein complexes that link the actin cytoskeleton to the plasma membrane and, in turn, to the extracellular environment. Zyxin is a LIM domain protein that is found in vertebrate adherens junctions and focal adhesions. Zyxin's molecular architecture and binding partner repertoire suggest roles in actin assembly and dynamics, cell motility, and nuclear-cytoplasmic communication. In order to study the function of zyxin in development, we have identified a zyxin orthologue in Drosophila melanogaster that we have termed Zyx102. Like its vertebrate counterparts, Zyx102 displays three carboxy-terminal LIM domains, a potential nuclear export signal, and three proline-rich motifs, one of which matches the consensus for mediating an interaction with Ena/VASP (Drosophila Enabled/Vasodilator-stimulated phosphoprotein) proteins. Here we show that Zyx102 and Enabled (Ena), the Drosophila member of the Ena/VASP family, can interact specifically in vitro and that this interaction does not occur when a particular mutant form of Ena, encoded by the lethal ena210 allele, is used. Lastly, we show that the zyx102 gene and Drosophila Ena are co-expressed during oogenesis and early embryogenesis, indicating that the two proteins may be able to interact during the development of the Drosophila egg chamber and early embryo.