Midline governs axon pathfinding by coordinating expression of two major guidance systems.

Formation of the neural network requires concerted action of multiple axon guidance systems. How neurons orchestrate expression of multiple guidance genes is poorly understood. Here, we show that Drosophila T-box protein Midline controls expression of genes encoding components of two major guidance systems: Frazzled, ROBO, and Slit. In midline mutant, expression of all these molecules are reduced, resulting in severe axon guidance defects, whereas misexpression of Midline induces their expression. Midline is present on the promoter regions of these genes, indicating that Midline controls transcription directly. We propose that Midline controls axon pathfinding through coordinating the two guidance systems.

A conserved developmental program for sensory organ formation in Drosophila melanogaster.

Different sensory organs, such the eye and ear, are widely thought to have separate origins, guided by distinct organ-specific factors that direct all aspects of their development. Previous studies of the D. melanogaster gene eyeless (ey) and its vertebrate homolog Pax6 suggested that this gene acts in such a manner and specifically drives eye development. But diverse sensory organs might instead arise by segment-specific modification of a developmental program that is involved more generally in sensory organ formation. In D. melanogaster, a common proneural gene called atonal (ato) functions in the initial process of development of a number of segment-specific organs, including the compound eye, the auditory organ and the stretch receptor, suggesting that these organs share an evolutionary origin. Here we show that D. melanogaster segment-specific sensory organs form through the integration of decapentaplegic (dpp), wingless (wg) and ecdysone signals into a single cis-regulatory element of ato. The induction of ectopic eyes by ey also depends on these signals for ato expression, and the ey mutant eye imaginal disc allows ato expression if cell death is blocked. These results imply that ey does not induce the entire eye morphogenetic program but rather modifies ato-dependent neuronal development. Our findings strongly suggest that various sensory organs evolved from an ato-dependent protosensory organ through segment specification by ey and Hox genes.

seven-up Controls switching of transcription factors that specify temporal identities of Drosophila neuroblasts.

Drosophila neuronal stem cell neuroblasts (NB) constantly change character upon division, to produce a different type of progeny at the next division. Transcription factors Hunchback (HB), Krüppel (KR), Pdm (PDM), etc. are expressed sequentially in each NB and act as determinants of birth-order identity. How a NB switches its expression profile from one transcription factor to the next is poorly understood. We show that the HB-to-KR switch is directed by the nuclear receptor Seven-up (SVP). SVP expression is confined to a temporally restricted subsection within the NB's lineage. Loss of SVP function causes an increase in the number of HB-positive cells within several NB lineages, whereas misexpression of svp leads to the loss of these early-born neurons. Lineage analysis provides evidence that svp is required to switch off HB at the proper time. Thus, svp modifies the self-renewal stem cell program to allow chronological change of cell fates, thereby generating neuronal diversity.

ROBO directs axon crossing of segmental boundaries by suppressing responsiveness to relocalized Netrin.

Networks in the CNS consist of neural modules that are connected in a repetitive array. Whereas individual modules contain guidance information along which axons track within the unit, these guidance cues hinder axon extension across module boundaries. We investigated how axons solve this 'boundary problem' by analyzing the longitudinal connections of neuromeres in Drosophila melanogaster. The initial trajectory of the longitudinal axons is guided by Netrin, which is localized on commissural axons by its receptor, Frazzled. The Netrin cue on the commissure of the next segment can act as a barrier to longitudinal axons, inhibiting their extension and misguiding them contralaterally along the commissure. We show that, before reaching the segmental boundary, the longitudinal axons' responsiveness to Netrin presented on the commissure is suppressed by Roundabout (ROBO), through counteracting Gq signaling. The absence of suppression causes the robo phenotype: longitudinal axons project toward the midline, as if running around a roundabout (rotary).

Local caspase activity directs engulfment of dendrites during pruning.

Pruning is important for sculpting neural circuits, as it removes excessive or inaccurate projections. Here we show that the removal of sensory neuron dendrites during pruning in Drosophila melanogaster is directed by local caspase activity. Suppressing caspase activity prevented dendrite removal, whereas a global activation of caspases within a neuron caused cell death. A new genetically encoded caspase probe revealed that caspase activity is confined to the degenerating dendrites of pruning neurons.

DRONC coordinates cell death and compensatory proliferation.

Accidental cell death often leads to compensatory proliferation. In Drosophila imaginal discs, for example, gamma-irradiation induces extensive cell death, which is rapidly compensated by elevated proliferation. Excessive compensatory proliferation can be artificially induced by "undead cells" that are kept alive by inhibition of effector caspases in the presence of apoptotic stimuli. This suggests that compensatory proliferation is induced by dying cells as part of the apoptosis program. Here, we provide genetic evidence that the Drosophila initiator caspase DRONC governs both apoptosis execution and subsequent compensatory proliferation. We examined mutants of five Drosophila caspases and identified the initiator caspase DRONC and the effector caspase DRICE as crucial executioners of apoptosis. Artificial compensatory proliferation induced by coexpression of Reaper and p35 was completely suppressed in dronc mutants. Moreover, compensatory proliferation after gamma-irradiation was enhanced in drice mutants, in which DRONC is activated but the cells remain alive. These results show that the apoptotic pathway bifurcates at DRONC and that DRONC coordinates the execution of cell death and compensatory proliferation.

gone early, a novel germline factor, ensures the proper size of the stem cell precursor pool in the Drosophila ovary.

In order to sustain lifelong production of gametes, many animals have evolved a stem cell-based gametogenic program. In the Drosophila ovary, germline stem cells (GSCs) arise from a pool of primordial germ cells (PGCs) that remain undifferentiated even after gametogenesis has initiated. The decision of PGCs to differentiate or remain undifferentiated is regulated by somatic stromal cells: specifically, epidermal growth factor receptor (EGFR) signaling activated in the stromal cells determines the fraction of germ cells that remain undifferentiated by shaping a Decapentaplegic (Dpp) gradient that represses PGC differentiation. However, little is known about the contribution of germ cells to this process. Here we show that a novel germline factor, Gone early (Goe), limits the fraction of PGCs that initiate gametogenesis. goe encodes a non-peptidase homologue of the Neprilysin family metalloendopeptidases. At the onset of gametogenesis, Goe was localized on the germ cell membrane in the ovary, suggesting that it functions in a peptidase-independent manner in cell-cell communication at the cell surface. Overexpression of Goe in the germline decreased the number of PGCs that enter the gametogenic pathway, thereby increasing the proportion of undifferentiated PGCs. Inversely, depletion of Goe increased the number of PGCs initiating differentiation. Excess PGC differentiation in the goe mutant was augmented by halving the dose of argos, a somatically expressed inhibitor of EGFR signaling. This increase in PGC differentiation resulted in a massive decrease in the number of undifferentiated PGCs, and ultimately led to insufficient formation of GSCs. Thus, acting cooperatively with a somatic regulator of EGFR signaling, the germline factor goe plays a critical role in securing the proper size of the GSC precursor pool. Because goe can suppress EGFR signaling activity and is expressed in EGF-producing cells in various tissues, goe may function by attenuating EGFR signaling, and thereby affecting the stromal environment.

Egfr signaling controls the size of the stem cell precursor pool in the Drosophila ovary.

In many animals, germline progenitors are kept undifferentiated to give rise to germline stem cells (GSCs), enabling continuous production of gametes throughout animal life. In the Drosophila ovary, GSCs arise from a subset of primordial germ cells (PGCs) that stay undifferentiated even after gametogenesis has started. How a certain population of PGCs is protected against differentiation, and the significance of its regulatory mechanisms on GSC establishment remain elusive. Here we show that epidermal growth factor receptor (Egfr) signaling in somatic stromal intermingled cells (ICs), activated by its ligand produced in germ cells, controls the size of the PGC pool at the onset of gametogenesis. Egfr signaling in ICs limits the number of cells that express the heparan sulfate proteoglycan Dally, which is required for the movement and stability of the locally-produced stromal morphogen, Decapentaplegic (Dpp, a BMP2/4 homologue). Dpp is received by PGCs and maintains them in an undifferentiated state. Altering Egfr signaling levels changes the size of the PGC pool and affects the number of GSCs established during development. While excess GSC formation is compensated by the adult stage, insufficient GSC formation can lead to adult ovarioles that completely lack GSCs, suggesting that ensuring an absolute size of the PGC pool is crucial for the GSC system.

Compartmentalization within neurites: its mechanisms and implications.

Neurons are morphologically characterized by long processes extending from a cell body. These processes, the dendrites and axon, are major sub-cellular compartments defined by morphological, molecular, and functional differences. However, evidence from vertebrates and invertebrates suggests that, based on molecular distribution, individual axons and dendrites are further divided into distinct compartments; many membrane molecules involved in axon guidance and synapse formation are localized to specific segments of axons or dendrites that share a boundary of localization. In this review, we describe recent progress in understanding the mechanisms of intra-neurite patterning, and discuss its potential roles in the development and function of the nervous system. Each protein employs different ways to achieve compartment-specific localization; some membrane molecules localize via cell-autonomous ability of neurons, while others require extrinsic signals for localization. The underlying regulatory mechanisms include transcriptional regulation, local translation, diffusion barrier, endocytosis, and selective membrane targeting. We propose that intra-neurite compartmentalization could provide platforms for structural and functional diversification of individual neurons.

Intra-axonal patterning: intrinsic compartmentalization of the axonal membrane in Drosophila neurons.

In the developing nervous system, distribution of membrane molecules, particularly axon guidance receptors, is often restricted to specific segments of axons. Such localization of membrane molecules can be important for the formation and function of neural networks; however, how this patterning within axons is achieved remains elusive. Here we show that Drosophila neurons in culture establish intra-axonal patterns in a cell-autonomous manner; several membrane molecules localize to either proximal or distal axon segments without cell-cell contacts. This distinct patterning of membrane proteins is not explained by a simple temporal control of expression, and likely involves spatially controlled vesicular targeting or retrieval. Mobility of transmembrane molecules is restricted at the boundary of intra-axonal segments, indicating that the axonal membrane is compartmentalized by a barrier mechanism. We propose that this intra-axonal compartmentalization is an intrinsic property of Drosophila neurons that provides a basis for the structural and functional development of the nervous system.

TFIIH controls developmentally-regulated cell cycle progression as a holocomplex.

Basal transcription factor, TFIIH, is a multifunctional complex that carries out not only transcription but also DNA repair and cell cycle control. TFIIH is composed of two sub-complexes: core TFIIH and Cdk-activating kinase (CAK). In vitro studies suggest that CAK is sufficient for cell cycle regulation, whereas core TFIIH is required for DNA repair. However, the TFIIH complexes that perform these functions in vivo have yet to be identified. Here, we perform an in vivo dissection of TFIIH activity by characterizing mutations in a core subunit p52 in Drosophila. p52 mutants are hypersensitive to UV, suggesting a defect in DNA repair. Nonetheless, mutant cells are able to divide and express a variety of differentiation markers. Although p52 is not essential for cell cycle progression itself, p52 mutant cells in the eye imaginal disc are unable to synchronize their cell cycles and remain arrested at G1. Similar cell cycle phenotypes are observed in mutations in another core subunit XPB and a CAK-component CDK7, suggesting that defects in core TFIIH affect the G1/S transition through modification of CAK activity. We propose that during development the function of TFIIH as a cell cycle regulator is carried out by holo-TFIIH.

Interactions between plexin-A2, plexin-A4, and semaphorin 6A control lamina-restricted projection of hippocampal mossy fibers.

Hippocampal mossy fibers project preferentially to the stratum lucidum, the proximal-most lamina of the suprapyramidal region of CA3. The molecular mechanisms that govern this lamina-restricted projection are still unknown. We examined the projection pattern of mossy fibers in mutant mice for semaphorin receptors plexin-A2 and plexin-A4, and their ligand, the transmembrane semaphorin Sema6A. We found that plexin-A2 deficiency causes a shift of mossy fibers from the suprapyramidal region to the infra- and intrapyramidal regions, while plexin-A4 deficiency induces inappropriate spreading of mossy fibers within CA3. We also report that the plexin-A2 loss-of-function phenotype is genetically suppressed by Sema6A loss of function. Based on these results, we propose a model for the lamina-restricted projection of mossy fibers: the expression of plexin-A4 on mossy fibers prevents them from entering the Sema6A-expressing suprapyramidal region of CA3 and restricts them to the proximal-most part, where Sema6A repulsive activity is attenuated by plexin-A2.

Cell-type specific utilization of multiple negative feedback loops generates developmental constancy.

Signaling pathways generally contain multiple negative regulators that are induced by the signal they repress, constructing negative feedback loops. Although such negative regulators are often expressed in a tissue- or cell-type specific manner during development, little is known about the significance of their differential expression patterns and possible interactions. We show the role and interplay of two cell-type specific negative feedback loops during specification of photoreceptor neurons in the Drosophila compound eye, a process that occurs via epidermal growth factor (EGF)-mediated sequential induction through the activation of the Ras/MAPK signaling pathway. Inducing cells secreting EGF express a negative regulator Sprouty (SPRY) that lowers Ras/MAPK signaling activity, and as a consequence reduces the signal-dependent expression of a secreted EGF inhibitor, Argos (AOS). Induced cells in turn express an orphan nuclear receptor Seven-up (SVP), which represses SPRY expression thereby allowing expression and secretion of AOS, preventing further induction. When this intricate system fails, as in spry mutants, sequential induction is no longer constant and the number of photoreceptor neurons becomes variable. Thus, cell-type specific utilization of multiple negative feedback loops not only confers developmental robustness through functional redundancy, but is a key component in generating consistent patterning.

EDL/MAE regulates EGF-mediated induction by antagonizing Ets transcription factor Pointed.

Inductive patterning mechanisms often use negative regulators to coordinate the effects and efficiency of induction. During Spitz EGF-mediated neuronal induction in the Drosophila compound eye and chordotonal organs, Spitz causes activation of Ras signaling in the induced cells, resulting in the activation of Ets transcription factor Pointed P2. We describe developmental roles of a novel negative regulator of Ras signaling, EDL/MAE, a protein with an Ets-specific Pointed domain but not an ETS DNA-binding domain. The loss of EDL/MAE function results in reduced number of photoreceptor neurons and chordotonal organs, suggesting a positive role in the induction by Spitz EGF. However, EDL/MAE functions as an antagonist of Pointed P2, by binding to its Pointed domain and abolishing its transcriptional activation function. Furthermore, edl/mae appears to be specifically expressed in cells with inducing ability. This suggests that inducing cells, which can respond to Spitz they themselves produce, must somehow prevent activation of Pointed P2. Indeed hyperactivation of Pointed P2 in inducing cells interferes with their inducing ability, resulting in the reduction in inducing ability. We propose that EDL/MAE blocks autocrine activation of Pointed P2 so that inducing cells remain induction-competent. Inhibition of inducing ability by Pointed probably represents a novel negative feedback system that can prevent uncontrolled spread of induction of similar cell fates.

Context-dependent utilization of Notch activity in Drosophila glial determination.

During Drosophila neurogenesis, glial differentiation depends on the expression of glial cells missing (gcm). Understanding how glial fate is achieved thus requires knowledge of the temporal and spatial control mechanisms directing gcm expression. A recent report showed that in the adult bristle lineage, gcm expression is negatively regulated by Notch signaling ( Van De Bor, V. and Giangrande, A. (2001). Development 128, 1381-1390). Here we show that the effect of Notch activation on gliogenesis is context-dependent. In the dorsal bipolar dendritic (dbd) sensory lineage in the embryonic peripheral nervous system (PNS), asymmetric cell division of the dbd precursor produces a neuron and a glial cell, where gcm expression is activated in the glial daughter. Within the dbd lineage, Notch is specifically activated in one of the daughter cells and is required for gcm expression and a glial fate. Thus Notch activity has opposite consequences on gcm expression in two PNS lineages. Ectopic Notch activation can direct gliogenesis in a subset of embryonic PNS lineages, suggesting that Notch-dependent gliogenesis is supported in certain developmental contexts. We present evidence that POU-domain protein Nubbin/PDM-1 is one of the factors that provide such context.

Coactivator MBF1 preserves the redox-dependent AP-1 activity during oxidative stress in Drosophila.

Basic leucine zipper proteins Jun and Fos form the dimeric transcription factor AP-1, essential for cell differentiation and immune and antioxidant defenses. AP-1 activity is controlled, in part, by the redox state of critical cysteine residues within the basic regions of Jun and Fos. Mutation of these cysteines contributes to oncogenic potential of Jun and Fos. How cells maintain the redox-dependent AP-1 activity at favorable levels is not known. We show that the conserved coactivator MBF1 is a positive modulator of AP-1. Via a direct interaction with the basic region of Drosophila Jun (D-Jun), MBF1 prevents an oxidative modification (S-cystenyl cystenylation) of the critical cysteine and stimulates AP-1 binding to DNA. Cytoplasmic MBF1 translocates to the nucleus together with a transfected D-Jun protein, suggesting that MBF1 protects nascent D-Jun also in Drosophila cells. mbf1-null mutants live shorter than mbf1+ controls in the presence of hydrogen peroxide (H2O2). An AP-1-dependent epithelial closure becomes sensitive to H2O2 in flies lacking MBF1. We conclude that by preserving the redox-sensitive AP-1 activity, MBF1 provides an advantage during oxidative stress.

Drosophila MBF1 is a co-activator for Tracheae Defective and contributes to the formation of tracheal and nervous systems.

During gene activation, the effect of binding of transcription factors to cis-acting DNA sequences is transmitted to RNA polymerase by means of co-activators. Although co-activators contribute to the efficiency of transcription, their developmental roles are poorly understood. We used Drosophila to conduct molecular and genetic dissection of an evolutionarily conserved but unique co-activator, Multiprotein Bridging Factor 1 (MBF1), in a multicellular organism. Through immunoprecipitation, MBF1 was found to form a ternary complex including MBF1, TATA-binding protein (TBP) and the bZIP protein Tracheae Defective (TDF)/Apontic. We have isolated a Drosophila mutant that lacks the mbf1 gene in which no stable association between TBP and TDF is detectable, and transcription of a TDF-dependent reporter gene is reduced by 80%. Although the null mutants of mbf1 are viable, tdf becomes haploinsufficient in mbf1-deficient background, causing severe lesions in tracheae and the central nervous system, similar to those resulting from a complete loss of tdf function. These data demonstrate a crucial role of MBF1 in the development of tracheae and central nervous system.

Drosophila homeodomain protein REPO controls glial differentiation by cooperating with ETS and BTB transcription factors.

In Drosophila, cell-fate determination of all neuroectoderm-derived glial cells depends on the transcription factor Glial cells missing (GCM), which serves as a binary switch between the neuronal and glial cell fates. Because the expression of GCM is restricted to the early phase of glial development, other factors must be responsible for the terminal differentiation of glial cells. Expression of three transcription factors, Reversed Polarity (REPO), Tramtrack p69 (TTK69) and PointedP1 (PNTP1), is induced by GCM in glial cells. REPO is a paired-like homeodomain protein, expressed exclusively in glial cells, and is required for the migration and differentiation of embryonic glial cells. To understand how REPO functions in glial terminal differentiation, we have analyzed the mechanism of gene regulation by REPO. We show that REPO can act as a transcriptional activator through the CAATTA motif in glial cells, and define three genes whose expression in vivo depends on REPO function. In different types of glial cells, REPO can act alone, or cooperate with either TTK69 or PNTP1 to regulate different target genes. Coordination of target gene expression by these three transcription factors may contribute to the diversity of glial cell types. In addition to promoting glial differentiation, we found that REPO is also necessary to suppress neuronal development, cooperating with TTK69. We propose that REPO plays a key role in both glial development and diversification.