The Ets protein Pointed P1 represses Asense expression in type II neuroblasts by activating Tailless.

Intermediate neural progenitors (INPs) boost the number and diversity of neurons generated from neural stem cells (NSCs) by undergoing transient proliferation. In the developing Drosophila brains, INPs are generated from type II neuroblasts (NBs). In order to maintain type II NB identity and their capability to produce INPs, the proneural protein Asense (Ase) needs to be silenced by the Ets transcription factor pointed P1 (PntP1), a master regulator of type II NB development. However, the molecular mechanisms underlying the PntP1-mediated suppression of Ase is still unclear. In this study, we utilized genetic and molecular approaches to determine the transcriptional property of PntP1 and identify the direct downstream effector of PntP1 and the cis-DNA elements that mediate the suppression of ase. Our results demonstrate that PntP1 directly activates the expression of the transcriptional repressor, Tailless (Tll), by binding to seven Ets-binding sites, and Tll in turn suppresses the expression of Ase in type II NBs by binding to two hexameric core half-site motifs. We further show that Tll provides positive feedback to maintain the expression of PntP1 and the identity of type II NBs. Thus, our study identifies a novel direct target of PntP1 and reveals mechanistic details of the specification and maintenance of the type II NB identity by PntP1.

Homeodomain protein Six4 prevents the generation of supernumerary Drosophila type II neuroblasts and premature differentiation of intermediate neural progenitors.

In order to boost the number and diversity of neurons generated from neural stem cells, intermediate neural progenitors (INPs) need to maintain their homeostasis by avoiding both dedifferentiation and premature differentiation. Elucidating how INPs maintain homeostasis is critical for understanding the generation of brain complexity and various neurological diseases resulting from defects in INP development. Here we report that Six4 expressed in Drosophila type II neuroblast (NB) lineages prevents the generation of supernumerary type II NBs and premature differentiation of INPs. We show that loss of Six4 leads to supernumerary type II NBs likely due to dedifferentiation of immature INPs (imINPs). We provide data to further demonstrate that Six4 inhibits the expression and activity of PntP1 in imINPs in part by forming a trimeric complex with Earmuff and PntP1. Furthermore, knockdown of Six4 exacerbates the loss of INPs resulting from the loss of PntP1 by enhancing ectopic Prospero expression in imINPs, suggesting that Six4 is also required for preventing premature differentiation of INPs. Taken together, our work identified a novel transcription factor that likely plays important roles in maintaining INP homeostasis.

JNK signaling coordinates with ecdysone signaling to promote pruning of Drosophila sensory neuron dendrites.

Developmental pruning of axons and dendrites is crucial for the formation of precise neuronal connections, but the mechanisms underlying developmental pruning are not fully understood. Here, we have investigated the function of JNK signaling in dendrite pruning using Drosophila class IV dendritic arborization (c4da) neurons as a model. We find that loss of JNK or its canonical downstream effectors Jun or Fos led to dendrite-pruning defects in c4da neurons. Interestingly, our data show that JNK activity in c4da neurons remains constant from larval to pupal stages but the expression of Fos is specifically activated by ecdysone receptor B1 (EcRB1) at early pupal stages, suggesting that ecdysone signaling provides temporal control of the regulation of dendrite pruning by JNK signaling. Thus, our work not only identifies a novel pathway involved in dendrite pruning and a new downstream target of EcRB1 in c4da neurons, but also reveals that JNK and Ecdysone signaling coordinate to promote dendrite pruning.

Notch maintains Drosophila type II neuroblasts by suppressing expression of the Fez transcription factor Earmuff.

Notch signaling is crucial for maintaining neural stem cell (NSC) self-renewal and heterogeneity; however, the underlying mechanism is not well understood. In Drosophila, loss of Notch prematurely terminates the self-renewal of larval type II neuroblasts (NBs, the Drosophila NSCs) and transforms type II NBs into type I NBs. Here, we demonstrate that Notch maintains type II NBs by suppressing the activation of earmuff (erm) by Pointed P1 (PntP1). We show that loss of Notch or components of its canonical pathway leads to PntP1-dependent ectopic Erm expression in type II NBs. Knockdown of Erm significantly rescues the loss-of-Notch phenotypes, and misexpression of Erm phenocopies the loss of Notch. Ectopically expressed Erm promotes the transformation of type II NBs into type I NBs by inhibiting PntP1 function and expression in type II NBs. Our work not only elucidates a key mechanism of Notch-mediated maintenance of type II NB self-renewal and identity, but also reveals a novel function of Erm.

The Ets protein Pointed prevents both premature differentiation and dedifferentiation of Drosophila intermediate neural progenitors.

Intermediate neural progenitors (INPs) need to avoid both dedifferentiation and differentiation during neurogenesis, but the underlying mechanisms are not well understood. In Drosophila, the Ets protein Pointed P1 (PntP1) is required to generate INPs from type II neuroblasts. Here, we investigated how PntP1 promotes INP generation. By generating pntP1-specific mutants and using RNAi knockdown, we show that the loss of PntP1 leads to both an increase in type II neuroblast number and the elimination of INPs. The elimination of INPs results from the premature differentiation of INPs due to ectopic Prospero expression in newly generated immature INPs (imINPs), whereas the increase in type II neuroblasts results from the dedifferentiation of imINPs due to loss of Earmuff at later stages of imINP development. Furthermore, reducing Buttonhead enhances the loss of INPs in pntP1 mutants, suggesting that PntP1 and Buttonhead act cooperatively to prevent premature INP differentiation. Our results demonstrate that PntP1 prevents both the premature differentiation and the dedifferentiation of INPs by regulating the expression of distinct target genes at different stages of imINP development.

bHLH-O proteins balance the self-renewal and differentiation of Drosophila neural stem cells by regulating Earmuff expression.

Balancing self-renewal and differentiation of stem cells requires differential expression of self-renewing factors in two daughter cells generated from the asymmetric division of the stem cells. In Drosophila type II neural stem cell (or neuroblast, NB) lineages, the expression of the basic helix-loop-helix-Orange (bHLH-O) family proteins, including Deadpan (Dpn) and E(spl) proteins, is required for maintaining the self-renewal and identity of type II NBs, whereas the absence of these self-renewing factors is essential for the differentiation of intermediate neural progenitors (INPs) generated from type II NBs. Here, we demonstrate that Dpn maintains type II NBs by suppressing the expression of Earmuff (Erm). We provide evidence that Dpn and E(spl) proteins suppress Erm by directly binding to C-sites and N-boxes in the cis-regulatory region of erm. Conversely, the absence of bHLH-O proteins in INPs allows activation of erm and Erm-mediated maturation of INPs. Our results further suggest that Pointed P1 (PntP1) mediates the dedifferentiation of INPs resulting from the loss of Erm or overexpression of Dpn or E(spl) proteins. Taken together, these findings reveal mechanisms underlying the regulation of the maintenance of type II NBs and differentiation of INPs through the differential expression of bHLH-O family proteins.

Drosophila Valosin-Containing Protein is required for dendrite pruning through a regulatory role in mRNA metabolism.

The dendritic arbors of the larval Drosophila peripheral class IV dendritic arborization neurons degenerate during metamorphosis in an ecdysone-dependent manner. This process-also known as dendrite pruning-depends on the ubiquitin-proteasome system (UPS), but the specific processes regulated by the UPS during pruning have been largely elusive. Here, we show that mutation or inhibition of Valosin-Containing Protein (VCP), a ubiquitin-dependent ATPase whose human homolog is linked to neurodegenerative disease, leads to specific defects in mRNA metabolism and that this role of VCP is linked to dendrite pruning. Specifically, we find that VCP inhibition causes an altered splicing pattern of the large pruning gene molecule interacting with CasL and mislocalization of the Drosophila homolog of the human RNA-binding protein TAR-DNA-binding protein of 43 kilo-Dalton (TDP-43). Our data suggest that VCP inactivation might lead to specific gain-of-function of TDP-43 and other RNA-binding proteins. A similar combination of defects is also seen in a mutant in the ubiquitin-conjugating enzyme ubcD1 and a mutant in the 19S regulatory particle of the proteasome, but not in a 20S proteasome mutant. Thus, our results highlight a proteolysis-independent function of the UPS during class IV dendritic arborization neuron dendrite pruning and link the UPS to the control of mRNA metabolism.

Ets transcription factor Pointed promotes the generation of intermediate neural progenitors in Drosophila larval brains.

Intermediate neural progenitor (INP) cells are transient amplifying neurogenic precursor cells generated from neural stem cells. Amplification of INPs significantly increases the number of neurons and glia produced from neural stem cells. In Drosophila larval brains, INPs are produced from type II neuroblasts (NBs, Drosophila neural stem cells), which lack the proneural protein Asense (Ase) but not from Ase-expressing type I NBs. To date, little is known about how Ase is suppressed in type II NBs and how the generation of INPs is controlled. Here we show that one isoform of the Ets transcription factor Pointed (Pnt), PntP1, is specifically expressed in type II NBs, immature INPs, and newly mature INPs in type II NB lineages. Partial loss of PntP1 in genetic mosaic clones or ectopic expression of the Pnt antagonist Yan, an Ets family transcriptional repressor, results in a reduction or elimination of INPs and ectopic expression of Ase in type II NBs. Conversely, ectopic expression of PntP1 in type I NBs suppresses Ase expression the NB and induces ectopic INP-like cells in a process that depends on the activity of the tumor suppressor Brain tumor. Our findings suggest that PntP1 is both necessary and sufficient for the suppression of Ase in type II NBs and the generation of INPs in Drosophila larval brains.

Kin17 regulates proper cortical localization of Miranda in Drosophila neuroblasts by regulating Flfl expression.

During asymmetric division of Drosophila larval neuroblasts, the fate determinant Prospero (Pros) and its adaptor Miranda (Mira) are segregated to the basal cortex through atypical protein kinase C (aPKC) phosphorylation of Mira and displacement from the apical cortex, but Mira localization after aPKC phosphorylation is not well understood. We identify Kin17, a DNA replication and repair protein, as a regulator of Mira localization during asymmetric cell division. Loss of Kin17 leads to aberrant localization of Mira and Pros to the centrosome, cytoplasm, and nucleus. We provide evidence to show that the mislocalization of Mira and Pros is likely due to reduced expression of Falafel (Flfl), a component of protein phosphatase 4 (PP4), and defects in dephosphorylation of serine-96 of Mira. Our work reveals that Mira is likely dephosphorylated by PP4 at the centrosome to ensure proper basal localization of Mira after aPKC phosphorylation and that Kin17 regulates PP4 activity by regulating Flfl expression.

Drosophila sensory neurons require Dscam for dendritic self-avoidance and proper dendritic field organization.

A neuron's dendrites typically do not cross one another. This intrinsic self-avoidance mechanism ensures unambiguous processing of sensory or synaptic inputs. Moreover, some neurons respect the territory of others of the same type, a phenomenon known as tiling. Different types of neurons, however, often have overlapping dendritic fields. We found that Down's syndrome Cell Adhesion Molecule (Dscam) is required for dendritic self-avoidance of all four classes of Drosophila dendritic arborization (da) neurons. However, neighboring mutant class IV da neurons still exhibited tiling, suggesting that self-avoidance and tiling differ in their recognition and repulsion mechanisms. Introducing 1 of the 38,016 Dscam isoforms to da neurons in Dscam mutants was sufficient to significantly restore self-avoidance. Remarkably, expression of a common Dscam isoform in da neurons of different classes prevented their dendrites from sharing the same territory, suggesting that coexistence of dendritic fields of different neuronal classes requires divergent expression of Dscam isoforms.

Identification of E2/E3 ubiquitinating enzymes and caspase activity regulating Drosophila sensory neuron dendrite pruning.

Ubiquitin-proteasome system (UPS) is a multistep protein degradation machinery implicated in many diseases. In the nervous system, UPS regulates remodeling and degradation of neuronal processes and is linked to Wallerian axonal degeneration, though the ubiquitin ligases that confer substrate specificity remain unknown. Having shown previously that class IV dendritic arborization (C4da) sensory neurons in Drosophila undergo UPS-mediated dendritic pruning during metamorphosis, we conducted an E2/E3 ubiquitinating enzyme mutant screen, revealing that mutation in ubcD1, an E2 ubiquitin-conjugating enzyme, resulted in retention of C4da neuron dendrites during metamorphosis. Further, we found that UPS activation likely leads to UbcD1-mediated degradation of DIAP1, a caspase-antagonizing E3 ligase. This allows for local activation of the Dronc caspase, thereby preserving C4da neurons while severing their dendrites. Thus, in addition to uncovering E2/E3 ubiquitinating enzymes for dendrite pruning, this study provides a mechanistic link between UPS and the apoptotic machinery in regulating neuronal process remodeling.

Differential Requirement for Translation Initiation Factor Pathways during Ecdysone-Dependent Neuronal Remodeling in Drosophila.

Dendrite pruning of Drosophila sensory neurons during metamorphosis is induced by the steroid hormone ecdysone through a transcriptional program. In addition, ecdysone activates the eukaryotic initiation factor 4E-binding protein (4E-BP) to inhibit cap-dependent translation initiation. To uncover how efficient translation of ecdysone targets is achieved under these conditions, we assessed the requirements for translation initiation factors during dendrite pruning. We found that the canonical cap-binding complex eIF4F is dispensable for dendrite pruning, but the eIF3 complex and the helicase eIF4A are required, indicating that differential translation initiation mechanisms are operating during dendrite pruning. eIF4A and eIF3 are stringently required for translation of the ecdysone target Mical, and this depends on the 5' UTR of Mical mRNA. Functional analyses indicate that eIF4A regulates eIF3-mRNA interactions in a helicase-dependent manner. We propose that an eIF3-eIF4A-dependent alternative initiation pathway bypasses 4E-BP to ensure adequate translation of ecdysone-induced genes.

Requirement of Cul3 for axonal arborization and dendritic elaboration in Drosophila mushroom body neurons.

Cul3 belongs to the family of cullin proteins, which function as scaffold proteins of E3 ubiquitin ligase complexes. Here we show cell-autonomous involvement of Cul3 in axonal arborization and dendritic elaboration of Drosophila mushroom body neurons. Cul3 mutant neurons are defective in terminal morphogenesis of neurites. Interestingly, mutant axons often terminate around branching points. In addition, dendritic elaboration is severely affected in Cul3 mutant neurons. However, loss of Cul3 function does not affect extension of the axons that rarely arborize. Function of cullin-type proteins has been shown to require covalent attachment of Nedd8 (neural precursor cell-expressed developmentally downregulated), a ubiquitin-like protein. Consistent with this notion, Cul3 is inactivated by a mutation in its conserved neddylation site, and Nedd8 mutant neurons exhibit similar neuronal morphogenetic defects. Together, Cul3 plays an essential role in both axonal arborization and proper elaboration of dendrites and may require neddylation for its proper function.

EAG2 potassium channel with evolutionarily conserved function as a brain tumor target.

Over 20% of the drugs for treating human diseases target ion channels, but no cancer drug approved by the US Food and Drug Administration (FDA) is intended to target an ion channel. We found that the EAG2 (Ether-a-go-go 2) potassium channel has an evolutionarily conserved function for promoting brain tumor growth and metastasis, delineate downstream pathways, and uncover a mechanism for different potassium channels to functionally cooperate and regulate mitotic cell volume and tumor progression. EAG2 potassium channel was enriched at the trailing edge of migrating medulloblastoma (MB) cells to regulate local cell volume dynamics, thereby facilitating cell motility. We identified the FDA-approved antipsychotic drug thioridazine as an EAG2 channel blocker that reduces xenografted MB growth and metastasis, and present a case report of repurposing thioridazine for treating a human patient. Our findings illustrate the potential of targeting ion channels in cancer treatment.

The Drosophila Sp8 transcription factor Buttonhead prevents premature differentiation of intermediate neural progenitors.

Intermediate neural progenitor cells (INPs) need to avoid differentiation and cell cycle exit while maintaining restricted developmental potential, but mechanisms preventing differentiation and cell cycle exit of INPs are not well understood. In this study, we report that the Drosophila homolog of mammalian Sp8 transcription factor Buttonhead (Btd) prevents premature differentiation and cell cycle exit of INPs in Drosophila larval type II neuroblast (NB) lineages. We show that the loss of Btd leads to elimination of mature INPs due to premature differentiation of INPs into terminally dividing ganglion mother cells. We provide evidence to demonstrate that Btd prevents the premature differentiation by suppressing the expression of the homeodomain protein Prospero in immature INPs. We further show that Btd functions cooperatively with the Ets transcription factor Pointed P1 to promote the generation of INPs. Thus, our work reveals a critical mechanism that prevents premature differentiation and cell cycle exit of Drosophila INPs.

The bHLH repressor Deadpan regulates the self-renewal and specification of Drosophila larval neural stem cells independently of Notch.

Neural stem cells (NSCs) are able to self-renew while giving rise to neurons and glia that comprise a functional nervous system. However, how NSC self-renewal is maintained is not well understood. Using the Drosophila larval NSCs called neuroblasts (NBs) as a model, we demonstrate that the Hairy and Enhancer-of-Split (Hes) family protein Deadpan (Dpn) plays important roles in NB self-renewal and specification. The loss of Dpn leads to the premature loss of NBs and truncated NB lineages, a process likely mediated by the homeobox protein Prospero (Pros). Conversely, ectopic/over-expression of Dpn promotes ectopic self-renewing divisions and maintains NB self-renewal into adulthood. In type II NBs, which generate transit amplifying intermediate neural progenitors (INPs) like mammalian NSCs, the loss of Dpn results in ectopic expression of type I NB markers Asense (Ase) and Pros before these type II NBs are lost at early larval stages. Our results also show that knockdown of Notch leads to ectopic Ase expression in type II NBs and the premature loss of type II NBs. Significantly, dpn expression is unchanged in these transformed NBs. Furthermore, the loss of Dpn does not inhibit the over-proliferation of type II NBs and immature INPs caused by over-expression of activated Notch. Our data suggest that Dpn plays important roles in maintaining NB self-renewal and specification of type II NBs in larval brains and that Dpn and Notch function independently in regulating type II NB proliferation and specification.

Integrins regulate repulsion-mediated dendritic patterning of drosophila sensory neurons by restricting dendrites in a 2D space.

Dendrites of the same neuron usually avoid each other. Some neurons also repel similar neurons through dendrite-dendrite interaction to tile the receptive field. Nonoverlapping coverage based on such contact-dependent repulsion requires dendrites to compete for limited space. Here we show that Drosophila class IV dendritic arborization (da) neurons, which tile the larval body wall, grow their dendrites mainly in a 2D space on the extracellular matrix (ECM) secreted by the epidermis. Removing neuronal integrins or blocking epidermal laminin production causes dendrites to grow into the epidermis, suggesting that integrin-laminin interaction attaches dendrites to the ECM. We further show that some of the previously identified tiling mutants fail to confine dendrites in a 2D plane. Expansion of these mutant dendrites in three dimensions results in overlap of dendritic fields. Moreover, overexpression of integrins in these mutant neurons effectively reduces dendritic crossing and restores tiling, revealing an additional mechanism for tiling.

Gradients of the Drosophila Chinmo BTB-zinc finger protein govern neuronal temporal identity.

Many neural progenitors, including Drosophila mushroom body (MB) and projection neuron (PN) neuroblasts, sequentially give rise to different subtypes of neurons throughout development. We identified a novel BTB-zinc finger protein, named Chinmo (Chronologically inappropriate morphogenesis), that governs neuronal temporal identity during postembryonic development of the Drosophila brain. In both MB and PN lineages, loss of Chinmo autonomously causes early-born neurons to adopt the fates of late-born neurons from the same lineages. Interestingly, primarily due to a posttranscriptional control, MB neurons born at early developmental stages contain more abundant Chinmo than their later-born siblings. Further, the temporal identity of MB progeny can be transformed toward earlier or later fates by reducing or increasing Chinmo levels, respectively. Taken together, we suggest that a temporal gradient of Chinmo (Chinmo(high) --> Chinmo(low)) helps specify distinct birth order-dependent cell fates in an extended neuronal lineage.

Development of the Drosophila mushroom bodies: elaboration, remodeling and spatial organization of dendrites in the calyx.

One Drosophila mushroom body (MB) is derived from four indistinguishable cell lineages, development of which involves sequential generation of multiple distinct types of neurons. Differential labeling of distinct MB clones reveals that MB dendrites of different clonal origins are well mixed at the larval stage but become restricted to distinct spaces in adults. Interestingly, a small dendritic domain in the adult MB calyx remains as a fourfold structure that, similar to the entire larval calyx, receives dendritic inputs from all four MB clones. Mosaic analysis of single neurons demonstrates that MB neurons, which are born around pupal formation, acquire unique dendritic branching patterns and consistently project their primary dendrites into the fourfold dendritic domain. Distinct dendrite distribution patterns are also observed for other subtypes of MB neurons. In addition, pruning of larval dendrites during metamorphosis allows for establishment of adult-specific dendrite elaboration/distribution patterns. Taken together, subregional differences exist in the adult Drosophila MB calyx, where processing and integration of distinct types of sensory information begin.