Eyeless collaborates with Hedgehog and Decapentaplegic signaling in Drosophila eye induction

eyeless (ey) is a key regulator of the eye development pathway in Drosophila. Ectopic expression of ey can induce the expression of several eye-specification genes (eya, so, and dac) and induce eye formation in multiple locations on the body. However, ey does not induce eye formation everywhere where it is ectopically expressed, suggesting that EY needs to collaborate with additional factors for eye induction. We examined ectopic eye induction by EY in the wing disc and found that eye induction was spatially restricted to the posterior compartment and the anterior-posterior (A/P) compartmental border, suggesting a requirement for both HH and DPP signaling. Although EY in the anterior compartment induced dpp and dac, these were not sufficient for eye induction. Coexpression experiments show that EY needs to collaborate with high level of HH and DPP to induce ectopic eye formation. Ectopic eye formation also requires the activation of an eye-specific enhancer of the endogenous hh gene.     

Gamma neurons mediate dopaminergic input during aversive olfactory memory formation in Drosophila

Mushroom body (MB)-dependent olfactory learning in Drosophila provides a powerful model to investigate memory mechanisms. MBs integrate olfactory conditioned stimulus (CS) inputs with neuromodulatory reinforcement (unconditioned stimuli, US), which for aversive learning is thought to rely on dopaminergic (DA) signaling to DopR, a D1-like dopamine receptor expressed in MBs. A wealth of evidence suggests the conclusion that parallel and independent signaling occurs downstream of DopR within two MB neuron cell types, with each supporting half of memory performance. For instance, expression of the Rutabaga (Rut) adenylyl cyclase in γ neurons is sufficient to restore normal learning to rut mutants, whereas expression of Neurofibromatosis 1 (NF1) in α/β neurons is sufficient to rescue NF1 mutants. DopR mutations are the only case where memory performance is fully eliminated, consistent with the hypothesis that DopR receives the US inputs for both γ and α/β lobe traces. We demonstrate, however, that DopR expression in γ neurons is sufficient to fully support short- and long-term memory. We argue that DA-mediated CS-US association is formed in γ neurons followed by communication between γ and α/β neurons to drive consolidation.     

Antagonistic relationship between Dpp and EGFR signaling in Drosophila head patterning

The Drosophila eye field that gives rise to the visual system and dorsal head epidermis forms an unpaired anlage located in the dorsal head ectoderm. The eye field expresses and requires both Dpp and EGFR signaling for its development. As shown in previous studies, EGFR is required for cell maintenance in the developing visual system. Dpp initially switches on the early eye genes so and eya in the eye field. Consecutively, high levels of Dpp in the dorsal midline inhibit these genes and promote development of head epidermis. We show that Dpp negatively regulates EGFR signaling, thereby increasing the amount of cell death in the dorsal midline. By this mechanism, Dpp controls the formation of a bilateral visual system and indirectly modulates cell death, which is essential for normal head morphogenesis. Loss of either Dpp or its downstream target, Zen, abolishes head epidermis fate and leads to the misexpression of dp-ERK in the dorsal midline. The resulting morphological phenotype consists of cyclopia, reduction of cell death, and failure of head involution. Ectopic expression of activated EGFR inhibits the Dpp target race and thereby causes cyclopia and defective head involution. We discuss possible mechanisms of Dpp and EGFR interaction in the embryo.     

Cooperation of JAK/STAT and Notch signaling in the Drosophila foregut

Temporal and spatial regulation of morphogenesis is pivotal to the formation of organs from simple epithelial tubes. In a genetic screen for novel genes controlling cell movement during posterior foregut development, we have identified and molecularly characterized two alleles of the domeless gene which encodes the Drosophila Janus kinase (JAK)/STAT receptor. We demonstrate that mutants for domeless or any other known component of the canonical JAK/STAT signaling pathway display a failure of coordinated cell movement during the development of the proventriculus, a multiply folded organ which is formed by stereotyped cell rearrangements in the posterior foregut. Whereas the JAK/STAT receptor is expressed in all proventricular precursor cells, expression of upd encoding its ligand and of STAT92E, the signal transducer of the pathway, is locally restricted to cells that invaginate during proventriculus development. We demonstrate by analyzing gene expression mediated by a model Notch response element and by studying the expression of the Notch target gene short stop, which encodes a cytoskeletal crosslinker protein, that JAK/STAT signaling is required for the activation of Notch-dependent gene expression in the foregut. Our results provide strong evidence that JAK/STAT and Notch signaling cooperate in the regulation of target genes that control epithelial morphogenesis in the foregut.     

DNase II deficiency impairs innate immune function in Drosophila

DNase II enzymes are highly conserved proteins that are required for the degradation of DNA within phagolysosomes. Engulfment of apoptotic cells and/or bacteria by phagocytic cells requires the function of DNase II to completely destroy ingested DNA. Mutation of the dnase II gene results in an increase of undegraded apoptotic DNA within phagocytic cells in mice and nematodes. Additionally, reduction of DNase II enzymatic activity in Drosophila melanogaster has been shown to lead to increased accumulation of DNA in the ovaries. Due to the importance of DNA clearance during infection, we hypothesized that a severe reduction of DNase II activity would result in diminished immune function and viability. To test this hypothesis, we knocked down DNase II activity in flies using RNAi. As expected, expression of a dnase II-RNAi construct in flies resulted in a dramatic reduction of DNase II activity and a significant decrease in total hemocyte numbers. Furthermore, infection of dnase II-RNAi flies with Gram negative or positive bacteria resulted in a severe reduction in fly viability. These results confirm that DNase II and the ability to clear macromolecular DNA is essential for maintaining proper immune function in Drosophila.     

Functional and evolutionary trade-offs co-occur between two consolidated memory phases in Drosophila melanogaster

Memory is a complex and dynamic process that is composed of different phases. Its evolution under natural selection probably depends on a balance between fitness benefits and costs. In Drosophila, two separate forms of consolidated memory phases can be generated experimentally: anaesthesia-resistant memory (ARM) and long-term memory (LTM). In recent years, several studies have focused on the differences between these long-lasting memory types and have found that, at the functional level, ARM and LTM are antagonistic. How this functional relationship will affect their evolutionary dynamics remains unknown. We selected for flies with either improved ARM or improved LTM over several generations, and found that flies selected specifically for improvement of one consolidated memory phase show reduced performance in the other memory phase. We also found that improved LTM was linked to decreased longevity in male flies but not in females. Conversely, males with improved ARM had increased longevity. We found no correlation between either improved ARM or LTM and other phenotypic traits. This is, to our knowledge, the first evidence of a symmetrical evolutionary trade-off between two memory phases for the same learning task. Such trade-offs may have an important impact on the evolution of cognitive capacities. On a neural level, these results support the hypothesis that mechanisms underlying these forms of consolidated memory are, to some degree, antagonistic.     

Six1 is essential for early neurogenesis in the development of olfactory epithelium

The olfactory epithelium (OE) is derived from the olfactory placode (OP) during mouse development. At embryonic day (E) 10.0-E10.5, “early neurogenesis” occurs in the OE, which includes production of pioneer neurons that emigrate out of the OE and other early-differentiated neurons. Around E12.5, the OE becomes organized into mature pseudostratified epithelium and shows “established neurogenesis,” in which olfactory receptor neurons (ORNs) are differentiated from basal progenitors. Little is known about the molecular pathway of early neurogenesis. The homeodomain protein Six1 is expressed in all OP cells and neurogenic precursors in the OE. Here we show that early neurogenesis is severely disturbed despite the unaltered expression of Mash1 at E10.5 in the Six1-deficient mice (Six1^−/−). Expression levels of neurogenin1 (Ngn1) and NeuroD are reduced and those of Hes1 and Hes5 are augmented in the OE of Six1^−/− at E10.5. Pioneer neurons and cellular aggregates, which are derived from the OP/OE and situated in the mesenchyme between the OE and forebrain, are completely absent in Six1^−/−. Moreover, ORN axons and the gonadotropin-releasing hormone-positive neurons fail to extend and migrate to the forebrain, respectively. Our study indicates that Six1 plays critical roles in early neurogenesis by regulating Ngn1, NeuroD, Hes1, and Hes5.     

Combinatorial expression of Prospero,Seven-up,and Elav identifies progenitor cell types during sense-organ differentiation in the Drosophila antenna

The Drosophila antenna has a diversity of chemosensory organs within a single epidermal field. We have some idea from recent studies of how the three broad categories of sense-organs are specified at the level of progenitor choice. However, little is known about how cell fates within single sense-organs are specified. Selection of individual primary olfactory progenitors is followed by organization of groups of secondary progenitors, which divide in a specific order to form a differentiated sensillum. The combinatorial expression of Prospero Elav, and Seven-up allows us to distinguish three secondary progenitor fates. The lineages of these cells have been established by clonal analysis and marker distribution following mitosis. High Notch signaling and the exclusion of these markers identifies PIIa; this cell gives rise to the shaft and socket. The sheath/neuron lineage progenitor PIIb, expresses all three markers; upon division, Prospero asymmetrically segregates to the sheath cell. In the coeloconica, PIIb undergoes an additional division to produce glia. PIIc is present in multiinnervated sense-organs and divides to form neurons. An understanding of the lineage and development of olfactory sense-organs provides a handle for the analysis of how olfactory neurons acquire distinct terminal fates.     

Drosophila dopamine synthesis pathway genes regulate tracheal morphogenesis

While studying the developmental functions of the Drosophila dopamine synthesis pathway genes, we noted interesting and unexpected mutant phenotypes in the developing trachea, a tubule network that has been studied as a model for branching morphogenesis. Specifically, Punch (Pu) and pale (ple) mutants with reduced dopamine synthesis show ectopic/aberrant migration, while Catecholamines up (Catsup) mutants that over-express dopamine show a characteristic loss of migration phenotype. We also demonstrate expression of Punch, Ple, Catsup and dopamine in tracheal cells. The dopamine pathway mutant phenotypes can be reproduced by pharmacological treatments of dopamine and a pathway inhibitor 3-iodotyrosine (3-IT), implicating dopamine as a direct mediator of the regulatory function. Furthermore, we show that these mutants genetically interact with components of the endocytic pathway, namely shibire/dynamin and awd/nm23, that promote endocytosis of the chemotactic signaling receptor Btl/FGFR. Consistent with the genetic results, the surface and total cellular levels of a Btl-GFP fusion protein in the tracheal cells and in cultured S2 cells are reduced upon dopamine treatment, and increased in the presence of 3-IT. Moreover, the transducer of Btl signaling, MAP kinase, is hyper-activated throughout the tracheal tube in the Pu mutant. Finally we show that dopamine regulates endocytosis via controlling the dynamin protein level.     

JNK signaling pathway required for wound healing in regenerating Drosophila wing imaginal discs

We have examined wound healing during regeneration of Drosophila wing imaginal discs fragments by confocal microscopy and assessed the role of components of the JNK pathway in this process. After cutting, columnar and peripodial epithelia cells at the wound edge start to close the wound through formation and contraction of an actin cable. This is followed by a zipping process through filopodial protrusions from both epithelia knitting the wound edges from proximal to distal areas of the disc. Activation of the JNK pathway is involved in such process. puckered (puc) expression is induced in several rows of cells at the edge of the wound, whereas absence of JNK pathway activity brought about by hemipterous, basket, and Dfos mutants impair wound healing. These defects are accompanied by lowered or loss of expression of puc. In support of a role of puc in wound healing, hep mutant phenotypes are rescued by reducing puc function, whereas overexpression of puc inhibits wound healing. Altogether, these results demonstrate a role for the JNK pathway in imaginal disc wound healing, similar to that reported for other healing processes such as embryonic dorsal closure, thoracic closure, and adult epithelial wound healing in Drosophila. Differences with such processes are also highlighted.     

Notch signaling relieves the joint-suppressive activity of Defective proventriculus in the Drosophila leg

Segmentation plays crucial roles during morphogenesis. Drosophila legs are divided into segments along the proximal-distal axis by flexible structures called joints. Notch signaling is necessary and sufficient to promote leg growth and joint formation, and is activated in distal cells of each segment in everting prepupal leg discs. The homeobox gene defective proventriculus (dve) is expressed in regions both proximal and distal to the intersegmental folds at 4 h after puparium formation (APF). Dve-expressing region partly overlaps with the Notch-activated region, and they become a complementary pattern at 6 h APF. Interestingly, dve mutant legs resulted in extra joint formation at the center of each tarsal segment, and the forced expression of dve caused a jointless phenotype. We present evidence that Dve suppresses the potential joint-forming activity, and that Notch signaling represses Dve expression to form joints.     

Therapeutic peptide production in Drosophila

Bioactive peptides are important therapeutic drugs, yet conventional methods of peptide synthesis are challenged to meet increasing demand. We developed a novel and efficient means of metabolic engineering: therapeutic peptide production in Drosophila and as a proof of concept, we demonstrate production of fully matured human insulin. This in vivo system offers an innovative means to produce valuable bioactive peptides for therapies, its inherent flexibility facilitates drug development, and its ease of producing fully processed peptides simplifies metabolic engineering of new peptide products.     

D-JNK signaling in visceral muscle cells controls the laterality of the Drosophila gut

Although bilateral animals appear to have left-right (LR) symmetry from the outside, their internal organs often show directional and stereotypical LR asymmetry. The mechanisms by which the LR axis is established in vertebrates have been extensively studied. However, how each organ develops its LR asymmetric morphology with respect to the LR axis is still unclear. Here, we showed that Drosophila Jun N-terminal kinase (D-JNK) signaling is involved in the LR asymmetric looping of the anterior-midgut (AMG) in Drosophila. Mutant embryos of puckered (puc), which encodes a D-JNK phosphatase, showed random laterality of the AMG. Directional LR looping of the AMG required D-JNK signaling to be down-regulated by puc in the trunk visceral mesoderm. Not only the down-regulation, but also the activation of D-JNK signaling was required for the LR asymmetric looping. We also found that the LR asymmetric cell rearrangement in the circular visceral muscle (CVM) was regulated by D-JNK signaling and required for the LR asymmetric looping of the AMG. Rac1, a Rho family small GTPase, augmented D-JNK signaling in this process. Our results also suggest that a basic mechanism for eliciting LR asymmetric gut looping may be conserved between vertebrates and invertebrates.     

Ap-let neurons--a peptidergic circuit potentially controlling ecdysial behavior in Drosophila

Here we describe a novel set of peptidergic neurons conserved throughout all developmental stages in the Drosophila central nervous system (CNS). We show that a small complement of 28 apterous-expressing cells (Ap-let neurons) in the ventral nerve cord (VNC) of Drosophila larvae co-express numerous gene products. The products include the neuroendocrine-specific bHLH regulator called Dimmed (Dimm), four neuropeptide biosynthetic enzymes (PC2, Fur1, PAL2, and PHM), and a specific dopamine receptor subtype (dDA1). For the PC2, Fur1, and PAL2 enzymes, and for the dDA1 receptor, this neuronal pattern represents the vast majority of their total expression in the VNC. In addition, while Dimm and PHM are present in the peritracheal Inka cells in larvae, pupae, and adults, Ap, PC2, Fur1, PAL2, and dDA1 are not. PC2, PAL2, and DA1 receptor expression were all controlled by both dimm and ap. Previous genetic analysis of animals deficient in PC2 revealed an abnormal larval ecdysis phenotype. Together, these data support the hypothesis that the small cohort of Ap-let interneurons regulates larval ecdysis behavior by secretion of an unidentified amidated peptide(s). This hypothesis further predicts that the production of the Ap-let neuropeptide(s) is dependent on each of four specific enzymes, and that a certain aspect(s) of its production and/or release is regulated by dopamine input.     

amnesiac regulates sleep onset and maintenance in Drosophila melanogaster

The adenylate cyclase/cAMP signaling pathway and adult mushroom bodies (MBs) have been shown to play an important role in sleep regulation in Drosophila. The amnesiac (amn) gene, encodes a neuropeptide that is homologous with vertebrate pituitary adenylate cyclase-activating peptide (PACAP), is expressed in dorsal paired medial (DPM) neurons and is required for the middle-term memory (MTM) in flies. However, the role of amn on regulation of sleep is as yet unknown. Here we provide evidence that amn plays a major role on sleep maintenance and onset in Drosophila. Flies with the amnesiac allele, loss-of-function amn^X8 mutation, showed a fragmented sleep pattern and short sleep latency. Moreover, homeostatic regulation was disrupted in amn^X8 mutants after sleep deprivation. Sleep maintenance was also influenced by disruption of neurotransmission in DPM neurons with increased sleep bout number and decreased sleep bout length. Furthermore, age-related sleep fragmentation and initiation were inhibited in amn^X8 mutant flies. These data suggest that amn is required in initiation and maintenance of sleep.     

klumpfuss regulates cell death in the Drosophila retina

Programmed cell death (PCD) plays a central role in the sculpting and maturation of developing epithelia. In adult tissue, PCD plays a further role in the prevention of malignancy though removal of damaged cells. Here, we report that mutations in klumpfuss result in an excess of support cells during maturation of the developing Drosophila pupal retina. These ectopic cells are the result of a partial and specific failure of apoptotic death during normal cell fate selection. klumpfuss is required and differentially expressed in the cells that choose the life or death cell fate. We also provide genetic and biochemical evidence that klumpfuss regulates this process through down-regulation of the Epidermal Growth Factor Receptor/dRas1 signaling pathway. Based on its sequence Klumpfuss is an EGR-class nuclear factor, and our results suggest a mechanism by which mutations in EGR-class factors such as Wilms' Tumor Suppressor-1 may result in oncogenic events such as pediatric kidney tumors.