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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.

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.

SummaryHippocampal 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.

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).

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.

SummaryDrosophila 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.

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.

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 development1,2. 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 receptor3,4, 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.

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.

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). Development128, 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.

Extracellular factors such as FGF and EGF control various aspects of morphogenesis, patterning and cellular proliferation in both invertebrates and vertebrates. In most systems, it is primarily the distribution of these factors that controls the differential behavior of the responding cells. Here we describe the role of Sprouty in eye development. Sprouty is an extracellular protein that has been shown to antagonize FGF signaling during tracheal branching in Drosophila. It is a novel type of protein with a highly conserved cysteine-rich region. In addition to the embryonic tracheal system, sprouty is also expressed in other tissues including the developing eye imaginal disc, embryonic chordotonal organ precursors and the midline glia. In each of these tissues, EGF receptor signaling is known to participate in the control of the correct number of neurons or glia. We show that, in all three tissues, the loss of sprouty results in supernumerary neurons or glia, respectively. Furthermore, overexpression of sprouty in wing veins and ovarian follicle cells, two other tissues where EGF signaling is required for patterning, results in phenotypes that resemble the loss-of-function phenotypes of Egf receptor. These results suggest that Sprouty acts as an antagonist of EGF as well as FGF signaling pathways. These receptor tyrosine kinase-mediated pathways may share not only intracellular signaling components but also extracellular factors that modulate the strength of the signal.

Antagonists of several growth factor signaling pathways play important roles in developmental patterning by limiting the range of the cognate inducer. Here, we describe an antagonist of FGF signaling that patterns apical branching of the Drosophila airways. In wild-type embryos, the Branchless FGF induces secondary branching by activating the Breathless FGF receptor near the tips of growing primary branches. In sprouty mutants, the FGF pathway is overactive and ectopic branches are induced on the stalks of primary branches. We show that FGF signaling induces sprouty expression in the nearby tip cells, and sprouty acts nonautonomously and in a competitive fashion to block signaling to the more distant stalk cells. sprouty encodes a novel cysteine-rich protein that defines a new family of putative signaling molecules that may similarly function as FGF antagonists in vertebrate development.

The mushroom body (MB) is an important centre for higher order sensory integration and learning in insects. To analyse the development and organisation of the MB neuropile in Drosophila, we performed cell lineage analysis in the adult brain with a new technique that combines the Flippase (flp)/FRT system and the GAL4/UAS system. We showed that the four mushroom body neuroblasts (MBNbs) give birth exclusively to the neurones and glial cells of the MB, and that each of the four MBNb clones contributes to the entire MB structure. The expression patterns of 19 GAL4 enhancer-trap strains that mark various subsets of MB cells revealed overlapping cell types in all four of the MBNb lineages. Partial ablation of MBNbs using hydroxyurea showed that each of the four neuroblasts autonomously generates the entire repertoire of the known MB substructures.

Genetic studies have uncovered many genes that are involved in the first steps of neuronal development in Drosophila. Less is known about the intermediate steps during which individual precursor cells follow either the neuronal pathway or the glial pathway. We report the identification of a novel bHLH gene, biparous, expressed in neuronal and glial precursors in Drosophila. Unlike most bHLH genes, biparous expression continues to the final stages of neurogenesis in the embryo. Expression of biparous is not observed in end stage postmitotic neurons and precedes the expression of repo, a gene activated in later stages of glial differentiation. The bHLH domain is sufficiently different from previously described bHLH domains to imply a novel function.

Drosophila seven-up is an orphan receptor of the steroid receptor family that is required to specify photoreceptor neuron subtypes in the developing compound eye. Expression of seven-up is confined to four of the eight photoreceptor precursors, R3/R4/R1/R6. We show that misexpression of seven-up in any of the other cell types within the developing ommatidium interferes with their differentiation. Each cell type responds differently to seven-up misexpression. For example, ectopic expression in the non-neuronal cone cells using the sevenless promoter/enhancer (sev-svp) causes the cone cells to take on a neuronal identity. Ectopic expression of seven-up in R2/R5 using the rough enhancer (ro-svp) causes these neurons to lose aspects of their photoreceptor subtype identity while remaining neuronal. Each cell type appears to have a different developmental time window that is sensitive to misexpressed seven-up. The temporal order of responsiveness of each cell type to misexpressed seven-up is similar but not identical to the order of neuronal differentiation. This suggests that there are processes of specification that are distinct from the specification to become a photoreceptor neuron. We have identified members of the ras signaling pathway as suppressors of the cone cell to R7 neuron transformation caused by sev-svp. Suppression of the sev-svp phenotype can be achieved by decreasing the gene-dosage of any of the members of the ras-pathway. This suggests that the function of seven-up in the cone cells requires ras signaling. However, a decrease in ras signaling results in enhancement of the phenotype caused by the rosvp transgene. We discuss the relationship between decisions controlled by seven-up and those controlled by ras signaling.

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.

During Drosophila ommatidial development, a single cell is selected within the ommatidial cluster to become the R7 photoreceptor neuron. The seven-up gene has been shown to play a role in this process by preventing four other photoreceptor precursors, R3/R4/R1/R6, from adopting the R7 cell fate. The seven-up gene encodes a steroid receptor-like molecule that is expressed only in those four cells that require seven-up function in the developing Drosophila ommatidium. We have examined the functional significance of the spatially restricted expression of seven-up by misexpressing seven-up isoforms. As expected from the function that seven-up performs in R3/R4/R1/R6, ubiquitous expression of seven-up causes transformation of the R7 cell to an R1-R6 cell fate. In addition, depending on the timing and spatial pattern of expression, various other phenotypes are produced including the loss of the R7 cell and the formation of extra R7 cells. Ubiquitous expression of seven-up close to the morphogenetic furrow interferes with R8 differentiation resulting in failure to express the boss protein, the ligand for the sevenless receptor tyrosine kinase, and the R7 cell is lost consequently. Extra R7 cells are formed by recruiting non-neuronal cone cells as photoreceptor neurons in a sevenless and bride of sevenless independent way. Thus, the spatiotemporal pattern of seven-up expression plays an essential role in controlling the number and cellular origin of the R7 neuron in the ommatidium. Our results also suggest that seven-up controls decisions not only between photoreceptor subtypes, but also between neuronal and non-neuronal fates.

Studies on the development of the R7 photoreceptor in the Drosophila eye thus far have identified three genes that specifically affect this cell: sevenless, boss and sina. In each of these mutants the R7 precursor develops instead as the equatorial cone cell (EQC). We have isolated an enhancer trap line, H214, in which beta-galactosidase is primarily expressed in the R7 cell throughout its development. In mutations of sevenless, boss and sina, expression in H214 is initially reduced although still present in the R7 precursor and persists in the EQC into which it develops. The EQC in wild type never expresses lacZ in H214. This result is in contrast to that seen with other enhancer trap lines that display expression in R7, and indicates that some aspect of R7 differentiation is independent of the genetic pathway(s) involving sevenless, boss and sina.

The dorsoventral pattern of the Drosophila embryo is mediated by a gradient of nuclear localization of the dorsal protein which acts as a morphogen. Establishment of the nuclear concentration gradient of dorsal protein requires the activities of the 10 maternal ‘dorsal group’ genes whose function results in the positive regulation of the nuclear uptake of the dorsal protein. Here we show that in contrast to the dorsal group genes, the maternal gene cactus acts as a negative regulator of the nuclear localization of the dorsal protein. While loss of function mutations of any of the dorsal group genes lead to dorsalized embryos, loss of cactus function results in a ventralization of the body pattern. Progressive loss of maternal cactus activity causes progressive loss of dorsal pattern elements accompanied by the expansion of ventrolateral and ventral anlagen. However, embryos still retain dorsoventral polarity, even if derived from germline clones using the strongest available, zygotic lethal cactus alleles. In contrast to the loss-of-function alleles, gain-of-function alleles of cactus cause a dorsalization of the embryonic pattern. Genetic studies indicate that they are not overproducers of normal activity, but rather synthesize products with altered function. Epistatic relationships of cactus with dorsal group genes were investigated by double mutant analysis. The dorsalized phenotype of the dorsal mutation is unchanged upon loss of cactus activity. This result implies that cactus acts via dorsal and has no independent morphogen function. In all other dorsal group mutant backgrounds, reduction of cactus function leads to embryos that express ventrolateral pattern elements and have increased nuclear uptake of the dorsal protein at all positions along the dorsoventral axis. Thus, the cactus gene product can prevent nuclear transport of dorsal protein in the absence of function of the dorsal group genes. Genetic and cytoplasmic transplantation studies suggest that the cactus product is evenly distributed along the dorsoventral axis. Thus the inhibitory function that cactus product exerts on the nuclear transport of the dorsal protein appears to be antagonized on the ventral side. We discuss models of how the action of the dorsal group genes might counteract the cactus function ventrally.

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.

Accidental cell death often leads to compensatory proliferation. In Drosophila imaginal discs, for example, -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 -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. Developing animal tissues have striking regenerative capacity. If a developing tissue is accidentally injured and a large number of cells undergo cell death, the final shape and size of the tissue are often invariant, owing to additional cell proliferation that compensates for the cell loss. The developing Drosophila larval imaginal disc is a tissue with extremely high regenerative capacity. Even when more than half of the cells are lost, the remaining cells undergo extra cell divisions and compensate for the loss (22). This “compensatory proliferation” is, at least in part, a local event that does not depend on the size control mechanism that governs the whole disc. For example, when apoptosis is locally induced by ectopic toxin expression, elevated cell proliferation is observed only around the site of apoptosis (40), suggesting that cells can perceive apoptosis occurring in the vicinity and undergo extra cell divisions until the original cell number is restored. Currently, little is known about how compensatory proliferation is regulated. One attractive hypothesis is that dying cells actively induce proliferation of the neighboring cells by secreting mitogens before they disappear. This hypothesis assumes that the noncell-autonomous induction of compensatory proliferation is a process that is under the control of the intracellular signaling pathway of apoptosis. According to this model, if cells could be stimulated to undergo apoptosis but artificially kept alive, the intracellular apoptosis signaling pathway would be activated

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.

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.

This special issue of Journal of Neurogenetics was born to honor the contribution of Yoshiki Hotta (b. 1938). Yoshiki is not only a major figure in the field of neurogenetics, but an influential le...

Asymmetric cell division is a fundamental strategy for generating cellular diversity during animal development1. Daughter cells manifest asymmetry in their differential gene expression. Transcriptional regulation of this process has been the focus of many studies, whereas cell-type-specific ‘translational’ regulation has been considered to have a more minor role. During sensory organ development in Drosophila, Notch signalling directs the asymmetry between neuronal and non-neuronal lineages2, and a zinc-finger transcriptional repressor Tramtrack69 (TTK69) acts downstream of Notch as a determinant of non-neuronal identity3,4. Here we show that repression of TTK69 protein expression in the neuronal lineage occurs translationally rather than transcriptionally. This translational repression is achieved by a direct interaction between cis-acting sequences in the 3′ untranslated region of ttk69 messenger RNA and its trans-acting repressor, the RNA-binding protein Musashi (MSI)5. Although msi can act downstream of Notch, Notch signalling does not affect MSI expression. Thus, Notch signalling is likely to regulate MSI activity rather than its expression. Our results define cell-type-specific translational control of ttk69 by MSI as a downstream event of Notch signalling in asymmetric cell division.

Netrin is a secreted protein that can act as a chemotropic axon guidance cue1,2. Two classes of Netrin receptor, DCC3,4,5 and UNC-5 (refs 6,7,8,9), are required for axon guidance3,4,6,7,8,9,10,11 and are thought to mediate Netrin signals in growth cones through their cytoplasmic domains12,13. However, in the guidance of Drosophila photoreceptor axons, the DCC orthologue Frazzled3 is required not in the photoreceptor neurons but instead in their targets, indicating that Frazzled also has a non-cell-autonomous function14. Here we show that Frazzled can capture Netrin and ‘present’ it for recognition by other receptors. Moreover, Frazzled itself is actively localized within the axon through its cytoplasmic domain, and thereby rearranges Netrin protein into a spatial pattern completely different from the pattern of Netrin gene expression. Frazzled-dependent guidance of one pioneer neuron in the central nervous system can be accounted for solely on the basis of this ability of Frazzled to control Netrin distribution, and not by Frazzled signalling. We propose a model of patterning mechanism in which a receptor rearranges secreted ligand molecules, thereby creating positional information for other receptors.

klingon is a member of the Immunoglobulin superfamily and is expressed in a restricted pattern of neurons during embryonic neurogenesis and in the R7 photoreceptor precursor throughout its development. Starting from the H214 enhancer trap line, we identified a transcription unit, klingon, that encodes a putative protein of 528 amino acids and contains three C2-type Immunoglobulin-like domains followed by one fibronectin type III repeat. When Klingon is expressed in S2 tissue culture cells, it is associated with the cell membrane by a glycosyl-phosphatidylinositol linkage and can mediate homophilic adhesion. Genetic analysis has revealed that klingon is an essential gene that participates in the development of the R7 neuron. Ectopic expression of klingon in all neurons in a sevenless background can alter the position of the R8 rhabdomere.

Publisher Summary This chapter describes procedures for an enhancer trap screen, using P-element vectors containing lacZ fusion genes, to isolate genes involved during Drosophila neurogenesis. The ability of the enhancer trap method to reveal the expression pattern of a gene should greatly facilitate the identification of a number of genes operating in any neural developmental process, regardless of their function at earlier stages or in other tissues. This method should complement genetic screens for mutant phenotypes and allow the isolation of genes that would stay undetected in standard genetic saturation screens. The chapter describes a screening method based on the assumption that some of the genes involved in the specification of individual cell fates during neurogenesis would be expressed in a restricted subset of cells. The expression patterns of the svp and sea genes that have been identified in such a screen are very similar to those of the lacZ reporter gene in the respective transformant lines, confirming the utility of this approach to identify genes expressed in spatially restricted patterns during nervous system development.

The Drosophila seven-up (svp) gene was isolated as a lethal insertion in an "enhancer trap" screen. It is expressed and required in photoreceptor cell precursors R1, R3, R4, and R6 during eye development. The absence of svp + function causes a transformation of these cells toward an R7 cell fate, as judged by morphology and expression of an R7-specific marker. This transformation depends in part on the sevenless gene product. Our results show that svp is involved in control of cell fate during the generation of neuronal diversity. Molecular analysis of svp reveals that it is a member of the steroid receptor gene superfamily and is likely to be a Drosophila homolog of the human transcription factor COUP.

INTRODUCTION ...... 147 PHENOTYPIC EFFECTS OF HOMEOTIC MUTATIONS . .. ....... 149 GENETIC AND MOLECULAR ORGANIZATION ..... 154 The Antennapedia Complex . . 154 The Bithorax Complex . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157 THE HOMEOBOX . . .. ...... . .. . ...... . ......... .. ........ . . . . . . 158 EVOLUTIONARY CONSIDERATIONS ... 160 EXPRESSION OF THE HOMEOTIC GENES 161 REGULATION OF HOMEOTIC GENES ..... ... .... 162 Transregulatory Genes . .. . . . . . . . . . .. 163 cis-Acting Control Sequences . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163 Interactions Among Homeotic Genes . . . . . . . ......... . .. . . 164 MECHANISM OF ACTION . . . . . . . . . . . . ..... 165

Segmentation genes control cell identities during early pattern formation in Drosophila. One of these genes, fushi tarazu (ftz), is now shown also to control cell fate during neurogenesis. Early in development, ftz is expressed in a striped pattern at the blastoderm stage. Later, it is transiently expressed in a specific subset of neuronal precursor cells, neurons (such as aCC, pCC, RP1, and RP2), and glia in the developing central nervous system (CNS). The function of ftz in the CNS was determined by creating ftz mutant embryos that express ftz in the blastoderm stripes but not in the CNS. In the absence of ftz CNS expression, some neurons appear normal (for example, the aCC, pCC, and RP1), whereas the RP2 neuron extends its growth cone along an abnormal pathway, mimicking its sibling (RP1), suggesting a transformation in neuronal identity.

Previous studies described three different classes of glial cells in the developing CNS of the early Drosophila embryo that prefigure and ensheath the major CNS axon tracts. Among these are 6 longitudinal glial cells on each side of each segment that overlie the longitudinal axon tracts. Here we use transformant lines carrying a P element containing a 130 by sequence from the fushi tarazu gene in front of the lacZ reporter gene to direct β-galactosidase expression in the longitudinal glia. Using this molecular lineage marker, we show that 1 of the "neuroblasts" in each hemisegment is actually a glioblast, which divides once symmetrically, in contrast to the typical asymmetric neuroblast divisions, producing 2 glial cells, which migrate medially and divide to generate the 6 longitudinal glial cells. As with neuroblasts, mutations in Notch and other neurogenic genes lead to supernumerary glioblasts. The results indicate that the glioblast is similar to other neuroblasts; however, the positionally specified fate of this blast cell is to generate a specific lineage of glia rather than a specific family of neurons.

We have identified four mutations affecting the actin III isoform in the indirect flight muscles (IFM) of Drosophila. One mutation does not produce any protein product, and three direct the synthesis of electrophoretic variants of actin. Complementation tests and recombination mapping indicate that all mutations are alleles of an actin gene at chromosomal band 88F (act88F gene). The effect of these mutations is restricted to the IFM. We conclude that the act88F gene is expressed only in the IFM to encode actin III, which is its major isoform. In two of the actin mutants, heat shock proteins are constitutively expressed in the IFM. Genetic evidence strongly suggest that this anomaly is primarily caused by the mutations in the act88F structural gene.

Two Drosophila mutants KM75 and HH5, which are mutated in the act88F actin gene specific for the indirect flight muscles (IFM), synthesize heat shock proteins (hsps) constitutively in a tissue-specific manner. We have introduced cloned mutant act88F genes into a strain containing the wild-type act88F allele by P-element-mediated transformation. Flies transformed with a 4.05 kb KM75 act88F gene fragment encoding the p42 actin variant express both p42 and hsps specifically in the IFM. Using normal/mutant chimeric genes, the mutation sites of KM75 and HH5 were mapped within the sequence encoding the last 72 amino acids of actin. An in vitro mutated gene encoding a protein that lacks the 72 carboxy-terminal amino acids also induces constitutive hsp synthesis.

Fushi tarazu (ftz) is one of the Drosophila homeo-box-containing genes required to establish the segmental pattern. Ftz+ transcripts accumulate in cells that form seven equally spaced bands at the blastoderm stage. We have analyzed cis-acting controlling sequences of the ftz gene by germ line transformation. The functional ftz gene includes a transcription unit of 1.9 kb and approximately 6.1 kb of the 5'-flanking sequences. Fusion of the 5'-flanking sequences to the E. coli lacZ gene directs expression of beta-galactosidase in a "zebra" pattern in transformed embryos. The fusion gene is also expressed in certain neuroblasts that contribute to ganglion cells of all segments. Various morphogenetic controlling elements have been identified within the 5'-flanking sequences.

The Drosophila segmentation gene fushi tarazu (ftz) is expressed in a pattern of seven stripes at the blastoderm stage. Two cis-acting control elements are required for this expression: the zebra element, which confers the striped pattern by mediating the effects of a subset of segmentation genes; and the upstream element, an enhancer element requiring ftz+ activity for its action. Fusion of the upstream element to a basal promoter results in activation of the heterologous promoter in a ftz-dependent striped pattern, supporting the idea that ftz regulates itself by acting through its enhancer. The upstream element can also confer expression patterns similar to that of the homeotic gene Antennapedia, suggesting that a similar element may play a role in the activation of Antennapedia.