Notch-mediated repression of bantam miRNA contributes to boundary formation in the Drosophila wing

Subdivision of proliferating tissues into adjacent compartments that do not mix plays a key role in animal development. The Actin cytoskeleton has recently been shown to mediate cell sorting at compartment boundaries, and reduced cell proliferation in boundary cells has been proposed as a way of stabilizing compartment boundaries. Cell interactions mediated by the receptor Notch have been implicated in the specification of compartment boundaries in vertebrates and in Drosophila, but the molecular effectors remain largely unidentified. Here, we present evidence that Notch mediates boundary formation in the Drosophila wing in part through repression of bantam miRNA. bantam induces cell proliferation and we have identified the Actin regulator Enabled as a new target of bantam. Increased levels of Enabled and reduced proliferation rates contribute to the maintenance of the dorsal-ventral affinity boundary. The activity of Notch also defines, through the homeobox-containing gene cut, a distinct population of boundary cells at the dorsal-ventral (DV) interface that helps to segregate boundary from non-boundary cells and contributes to the maintenance of the DV affinity boundary.     

Roles for scalloped and vestigial in regulating cell affinity and interactions between the wing blade and the wing hinge

The scalloped and vestigial genes are both required for the formation of the Drosophila wing, and recent studies have indicated that they can function as a heterodimeric complex to regulate the expression of downstream target genes. We have analyzed the consequences of complete loss of scalloped function, ectopic expression of scalloped, and ectopic expression of vestigial on the development of the Drosophila wing imaginal disc. Clones of cells mutant for a strong allele of scalloped fail to proliferate within the wing pouch, but grow normally in the wing hinge and notum. Cells overexpressing scalloped fail to proliferate in both notal and wing-blade regions of the disc, and this overexpression induces apoptotic cell death. Clones of cells overexpressing vestigial grow smaller or larger than control clones, depending upon their distance from the dorsal-ventral compartment boundary. These studies highlight the importance of correct scalloped and vestigial expression levels to normal wing development. Our studies of vestigial-overexpressing clones also reveal two further aspects of wing development. First, in the hinge region vestigial exerts both a local inhibition and a long-range induction of wingless expression. These and other observations imply that vestigial-expressing cells in the wing blade organize the development of surrounding wing-hinge cells. Second, clones of cells overexpressing vestigial exhibit altered cell affinities. Our analysis of these clones, together with studies of scalloped mutant clones, implies that scalloped- and vestigial-dependent cell adhesion contributes to separation of the wing blade from the wing hinge and to a gradient of cell affinities along the dorsal-ventral axis of the wing.     

Osa modulates the expression of Apterous target genes in the Drosophila wing

The establishment of the dorsal-ventral axis of the Drosophila wing depends on the activity of the LIM-homeodomain protein Apterous. Apterous activity depends on the formation of a higher order complex with its cofactor Chip to induce the expression of its target genes. Apterous activity levels are modulated during development by dLMO. Expression of dLMO in the Drosophila wing is regulated by two distinct Chip dependent mechanisms. Early in development, Chip bridges two molecules of Apterous to induce expression of dLMO in the dorsal compartment. Later in development, Chip, independently of Apterous, is required for expression of dLMO in the wing pouch. We have conducted a modular P-element based EP (enhancer/promoter) misexpression screen to look for genes involved in Apterous activity. We have found Osa, a member of the Brahma chromatin-remodeling complex, as a positive modulator of Apterous activity in the Drosophila wing. Osa mediates activation of some Apterous target genes and repression of others, including dLMO. Osa has been shown to bind Chip. We propose that Chip recruits Osa to the Apterous target genes, thus mediating activation or repression of their expression.     

Drosophila wing development in the absence of dorsal identity

The developing wing disc of Drosophila is divided into distinct lineage-restricted compartments along both the anterior/posterior (A/P) and dorsal/ventral (D/V) axes. At compartment boundaries, morphogenic signals pattern the disc epithelium and direct appropriate outgrowth and differentiation of adult wing structures. The mechanisms by which affinity boundaries are established and maintained, however, are not completely understood. Compartment-specific adhesive differences and inter-compartment signaling have both been implicated in this process. The selector gene apterous (ap) is expressed in dorsal cells of the wing disc and is essential for D/V compartmentalization, wing margin formation, wing outgrowth and dorsal-specific wing structures. To better understand the mechanisms of Ap function and compartment formation, we have rescued aspects of the ap mutant phenotype with genes known to be downstream of Ap. We show that Fringe (Fng), a secreted protein involved in modulation of Notch signaling, is sufficient to rescue D/V compartmentalization, margin formation and wing outgrowth when appropriately expressed in an ap mutant background. When Fng and alphaPS1, a dorsally expressed integrin subunit, are co-expressed, a nearly normal-looking wing is generated. However, these wings are entirely of ventral identity. Our results demonstrate that a number of wing development features, including D/V compartmentalization and wing vein formation, can occur independently of dorsal identity and that inter-compartmental signaling, refined by Fng, plays the crucial role in maintaining the D/V affinity boundary. In addition, it is clear that key functions of the ap selector gene are mediated by only a small number of downstream effectors.     

A novel interaction between hedgehog and Notch promotes proliferation at the anterior-posterior organizer of the Drosophila wing

Notch has multiple roles in the development of the Drosophila melanogaster wing imaginal disc. It helps specify the dorsal-ventral compartment border, and it is needed for the wing margin, veins, and sensory organs. Here we present evidence for a new role: stimulating growth in response to Hedgehog. We show that Notch signaling is activated in the cells of the anterior-posterior organizer that produce the region between wing veins 3 and 4, and we describe strong genetic interactions between the gene that encodes the Hedgehog pathway activator Smoothened and the Notch pathway genes Notch, presenilin, and Suppressor of Hairless and the Enhancer of split complex. This work thus reveals a novel collaboration by the Hedgehog and Notch pathways that regulates proliferation in the 3-4 intervein region independently of Decapentaplegic.     

Development of the Drosophila genital disc requires interactions between its segmental primordia

In both sexes, the Drosophila genital disc comprises three segmental primordia: the female genital primordium derived from segment A8, the male genital primordium derived from segment A9 and the anal primordium derived from segments A10-11. Each segmental primordium has an anterior (A) and a posterior (P) compartment, the P cells of the three segments being contiguous at the lateral edges of the disc. We show that Hedgehog (Hh) expressed in the P compartment differentially signals A cells at the AP compartment border and A cells at the segmental border. As in the wing imaginal disc, cell lineage restriction of the AP compartment border is defined by Hh signalling. There is also a lineage restriction barrier at the segmental borders, even though the P compartment cells of the three segments converge in the lateral areas of the disc. Lineage restriction between segments A9 and A10-11 depends on factors other than the Hh, En and Hox genes. The segmental borders, however, can be permeable to some morphogenetic signals. Furthermore, cell ablation experiments show that the presence of all primordia (either the anal or the genital primordium) during development are required for normal development of genital disc. Collectively, these findings suggest that interaction between segmental primordia is required for the normal development of the genital disc.     

Notch signaling is not sufficient to define the affinity boundary between dorsal and ventral compartments

The developing limbs of Drosophila are subdivided into distinct cells populations known as compartments. Short-range interaction between cells in adjacent compartments induces expression of signaling molecules at the compartment boundaries. In addition to serving as the sources of long-range signals, compartment boundaries prevent mixing of the adjacent cell populations. One model for boundary formation proposes that affinity differences between compartments are defined autonomously as one aspect of compartment-specific cell identity. An alternative is that the affinity boundary depends on signaling between compartments. Here, we present evidence that the dorsal selector gene apterous plays a role in establishing the dorsoventral affinity boundary that is independent of Notch-mediated signaling between dorsal and ventral cells.     

Multiple extracellular activities in Drosophila egg perivitelline fluid are required for establishment of embryonic dorsal-ventral polarity.

Twelve maternal effect genes (the dorsal group and cactus) are required for the establishment of the embryonic dorsal-ventral axis in the Drosophila embryo. Embryonic dorsal-ventral polarity is defined within the perivitelline compartment surrounding the embryo by the ventral formation of a ligand for the Toll receptor. Here, by transplantation of perivitelline fluid we demonstrate the presence of three separate activities present in the perivitelline fluid that can restore dorsal-ventral polarity to mutant easter, snake, and sp?tzle embryos, respectively. These activities are not capable of defining the polarity of the dorsal-ventral axis; instead they restore structures according to the intrinsic dorsal-ventral polarity of the mutant embryos. They appear to be involved in the ventral formation of a ligand for the Toll protein. This process requires serine proteolytic activity; the injection of serine protease inhibitors into the perivitelline space of wild-type embryos results in the formation of dorsalized embryos.     

The neurogenic locus brainiac cooperates with the Drosophila EGF receptor to establish the ovarian follicle and to determine its dorsal-ventral polarity.

We have characterized the function of a new neurogenic locus, brainiac (brn), during oogenesis. Homozygous brn females lay eggs with fused dorsal appendages, a phenotype associated with torpedo (top) alleles of the Drosophila EGF receptor (DER) locus. By constructing double mutant females for both brn and top, we have found that brn is required for determining the dorsal-ventral polarity of the ovarian follicle. However, embryos from mature brn eggs develop a neurogenic phenotype which can be zygotically rescued if a wild-type sperm fertilizes the egg. This is the first instance of a Drosophila gene required for determination of dorsal-ventral follicle cell fates that is not required for determination of embryonic dorsal-ventral cell fates. The temperature-sensitive period for brn dorsal-ventral patterning begins at the inception of vitellogenesis. The interaction between brn and DER is also required for at least two earlier follicle cell activities which are necessary to establish the ovarian follicle. Prefollicular cells fail to migrate between each oocyte/nurse cell complex, resulting in follicles with multiple sets of oocytes and nurse cells. brn and DER function is also required for establishing and/or maintaining a continuous follicular epithelium around each oocyte/nurse cell complex. These brn functions as well as the brn requirement for determination of dorsal-ventral polarity appear to be genetically separable functions of the brn locus. Genetic mosaic experiments show that brn is required in the germline during these processes whereas the DER is required in the follicle cells. We propose that brn may be part of a germline signaling pathway differentially regulating successive DER-dependent follicle cell activities of migration, division and/or adhesion and determination during oogenesis. These experiments indicate that brn is required in both tyrosine kinase and neurogenic intercellular signaling pathways. Moreover, the functions of brn in oogenesis are distinct from those of Notch and Delta, two other neurogenic loci that are known to be required for follicular development.     

Developmental biology: a growing role for computer simulations

Keeping cells separated in well-defined domains is essential for development. A new computational-experimental study elucidates the physical mechanisms that establish and maintain the dorsal-ventral compartment boundary in the Drosophila wing disc and demonstrates the increasing value of computer simulations in developmental biology.     

msh specifies dorsal cell fate in the Drosophila wing

Drosophila limbs develop from imaginal discs that are subdivided into compartments. Dorsal-ventral subdivision of the wing imaginal disc depends on apterous activity in dorsal cells. Apterous protein is expressed in dorsal cells and is responsible for (1) induction of a signaling center along the dorsal-ventral compartment boundary (2) establishment of a lineage restriction boundary between compartments and (3) specification of dorsal cell fate. Here, we report that the homeobox gene msh (muscle segment homeobox) acts downstream of apterous to confer dorsal identity in wing development.     

Short-range cell interactions and cell survival in the Drosophila wing

During development of multicellular organisms, cells are often eliminated by apoptosis if they fail to receive appropriate signals from their surroundings. Here, we report on short-range cell interactions that support cell survival in the Drosophila wing imaginal disc. We present evidence showing that cells incorrectly specified for their position undergo apoptosis because they fail to express specific proteins that are found on surrounding cells, including the LRR transmembrane proteins Capricious and Tartan. Interestingly, only the extracellular domains of Capricious and Tartan are required, suggesting that a bidirectional process of cell communication is involved in triggering apoptosis. We also present evidence showing that activation of the Notch signal transduction pathway is involved in triggering apoptosis of cells misspecified for their dorsal-ventral position.     

The Drosophila segment polarity gene patched interacts with decapentaplegic in wing development.

The decapentaplegic (dpp) gene of Drosophila melanogaster encodes a polypeptide of the transforming growth factor-beta family of secreted factors. It is required for the proper development of both embryonic and adult structures, and may act as a morphogen in the embryo. In wing imaginal discs, dpp is expressed and required in a stripe of cells near the anterior-posterior compartment boundary. Here we show that viable mutations in the segment polarity genes patched (ptc) and costal-2 (cos2) cause specific alterations in dpp expression within the anterior compartment of the wing imaginal disc. The interaction between ptc and dpp is particularly interesting; both genes are expressed with similar patterns at the anterior-posterior compartment boundary of the disc, and mis-expressed in a similar way in segment polarity mutant backgrounds like ptc and cos2. This mis-expression of dpp could be correlated with some of the features of the adult mutant phenotypes. We propose that ptc controls dpp expression in the imaginal discs, and that the restricted expression of dpp near the anterior-posterior compartment boundary is essential to maintain the wild-type morphology of the wing disc.     

Regulation of imaginal disc cell size, cell number and organ size by Drosophila class I(A) phosphoinositide 3-kinase and its adaptor.

BACKGROUND: Class I(A) phosphoinositide 3-kinases (PI 3-kinases) have been implicated in the regulation of several cellular processes including cell division, cell survival and protein synthesis. The size of Drosophila imaginal discs (epithelial structures that give rise to adult organs) is maintained by factors that can compensate for experimentally induced changes in these PI 3-kinase-regulated processes. Overexpression of the gene encoding the Drosophila class I(A) PI 3-kinase, Dp110, in imaginal discs, however, results in enlarged adult organs. These observations have led us to investigate the role of Dp100 and its adaptor, p60, in the control of imaginal disc cell size, cell number and organ size. RESULTS: Null mutations in Dp110 and p60 were generated and used to demonstrate that they are essential genes that are autonomously required for imaginal disc cells to achieve their normal adult size. In addition, modulating Dp110 activity increases or reduces cell size in the developing imaginal disc, and does so throughout the cell cycle. The inhibition of Dp110 activity reduces the rate of increase in cell number in the imaginal discs, suggesting that Dp110 normally promotes cell division and/or cell survival. Unlike direct manipulation of cell-cycle progression, manipulation of Dp110 activity in one compartment of the disc influences the size of that compartment and the size of the disc as a whole. CONCLUSIONS: We conclude that during imaginal disc development, Dp110 and p60 regulate cell size, cell number and organ size. Our results indicate that Dp110 and p60 signalling can affect growth in multiple ways, which has important implications for the function of signalling through class I(A) PI 3-kinases.     

The endocytic pathway and formation of the Wingless morphogen gradient

Controlling the spread of morphogens is crucial for pattern formation during development. In the Drosophila wing disc, Wingless secreted at the dorsal-ventral compartment boundary forms a concentration gradient in receiving tissue, where it activates short- and long-range target genes. The glypican Dally-like promotes Wingless spreading by unknown mechanisms, while Dynamin-dependent endocytosis is thought to restrict Wingless spread. We have utilized short-term expression of dominant negative Rab proteins to examine the polarity of endocytic trafficking of Wingless and its receptors and to determine the relative contributions of endocytosis, degradation and recycling to the establishment of the Wingless gradient. Our results show that Wingless is internalized via two spatially distinct routes: one on the apical, and one on the basal, side of the disc. Both restrict the spread of Wingless, with little contribution from subsequent degradation or recycling. As previously shown for Frizzled receptors, depleting Arrow does not prevent Wingless from entering endosomes. We find that both Frizzled and Arrow are internalized mainly from the apical membrane. Thus, the basal Wingless internalization route must be independent of these proteins. We find that Dally-like is not required for Wingless spread when endocytosis is blocked, and propose that Dally-like promotes the spread of Wingless by directing it to lateral membranes, where its endocytosis is less efficient. Thus, subcellular localization of Wingless along the apical-basal axis of receiving cells may be instrumental in shaping the Wingless gradient.     

Plasma membrane localization of the Toll protein in the syncytial Drosophila embryo: importance of transmembrane signaling for dorsal-ventral pattern formation

Formation of the Drosophila embryo's dorsal-ventral pattern requires the maternal product of the Toll gene. DNA sequence and genetic analyses together suggested that the Toll gene product is a transmembrane protein which communicates information from an extracytoplasmic compartment to the cytoplasm. Using antibodies as probes, we show that the Toll protein is a 135 x 10(3) Mr glycoprotein which is tightly associated with embryonic membranes. During the syncytial stage when dorsal-ventral polarity is established, the maternal Toll protein is associated with the plasma membrane around the entire embryo. During later embryonic stages, the Toll protein is expressed zygotically on many cell surfaces, possibly to promote cell adhesion. The plasma membrane localization of the Toll protein in the syncytial embryo suggests that transmembrane signaling from the extracellular perivitelline space to the cytoplasm is required for establishment of the embryonic dorsal-ventral pattern.