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.     

Temporal regulation of apterous activity during development of the Drosophila wing

Dorsoventral axis formation in the Drosophila wing depends on the activity of the selector gene apterous. Although selector genes are usually thought of as binary developmental switches, we find that Apterous activity is negatively regulated during wing development by its target gene dLMO. Apterous-dependent expression of Serrate and fringe in dorsal cells leads to the restricted activation of Notch along the dorsoventral compartment boundary. We present evidence that the ability of cells to participate in this Apterous-dependent cell-interaction is under spatial and temporal control. Apterous-dependent expression of dLMO causes downregulation of Serrate and fringe and allows expression of delta in dorsal cells. This limits the time window during which dorsoventral cell interactions can lead to localized activation of Notch and induction of the dorsoventral organizer. Overactivation of Apterous in the absence of dLMO leads to overexpression of Serrate, reduced expression of delta and concomitant defects in differentiation and cell survival in the wing primordium. Thus, downregulation of Apterous activity is needed to allow normal wing development.     

The relative expression amounts of apterous and its co-factor dLdb/Chip are critical for dorso-ventral compartmentalization in the Drosophila wing.

Dorso-ventral axis formation in the Drosophila wing requires the localized accumulation of the Apterous LIM/homeodomain protein (Ap) in dorsal cells. Here we report that dLdb/Chip encodes a LIM-binding cofactor that controls Ap activity. Both lack and excess of dLdb/Chip function cause the same phenotype as apterous (ap) lack of function; i.e. dorsal to ventral transformations, generation of new wing margins, and wing outgrowths. These results indicate that the normal function of Ap in dorso-ventral compartmentalization requires the correct amount of the DLDB/CHIP co-factor, and suggest that the Ap and DLDB/CHIP proteins form a multimeric functional complex. In support of this model, we show that the dLdb/Chip excess-of-function phenotypes can be rescued by ap overexpression.     

Interactions between chip and the achaete/scute-daughterless heterodimers are required for pannier-driven proneural patterning

The GATA factor Pannier activates the achaete-scute (ASC) proneural complex through enhancer binding and provides positional information for sensory bristle patterning in Drosophila. Chip was previously identified as a cofactor of the dorsal selector Apterous, and we show here that both Apterous and Chip also regulate ASC expression. Chip cooperates with Pannier in bridging the GATA factor with the HLH Ac/Sc and Daughterless proteins to allow enhancer-promoter interactions, leading to activation of the proneural genes, whereas Apterous antagonizes Pannier function. Within the Pannier domain of expression, Pannier and Apterous may compete for binding to their common Chip cofactor, and the accurate stoichiometry between these three proteins is essential for both proneural prepattern and compartmentalization of the thorax.     

A re-evaluation of the contributions of Apterous and Notch to the dorsoventral lineage restriction boundary in the Drosophila wing

The Drosophila limb primordia are subdivided into compartments: cell populations that do not mix during development. The wing is subdivided into dorsal (D) and ventral (V) compartments by the activity of the selector gene apterous in D cells. Apterous causes segregation of D and V cell populations by at least two distinct mechanisms. The LRR transmembrane proteins Capricious and Tartan are transiently expressed in D cells and contribute to initial segregation of D and V cells. Signaling between D and V cells mediated by Notch and Fringe contributes to the maintenance of the DV affinity boundary. Given that Notch is activated symmetrically, in D and V cells adjacent to the boundary, its role in boundary formation remains somewhat unclear. We re-examine the roles of Apterous and Fringe activities in DV boundary formation and present evidence that Fringe cannot, by itself, generate an affinity difference between D and V cells. Although not sufficient, Fringe is required via Notch activation for expression of an Apterous-dependent affinity difference. We propose that Apterous controls expression of surface proteins that confer an affinity difference in conjunction with activated Notch. Thus, we view Apterous as instructive and Notch activity as essential, but permissive.     

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.     

Genes Regulating the Remote Wing Margin Enhancer in the Drosophila Cut Locus

The mechanisms that allow enhancers to activate promoters from thousands of base pairs away are disrupted by the suppressor of Hairy-wing protein (SUHW) of Drosophila. SUHW binds a DNA sequence in the gypsy retrotransposon and prevents enhancers promoter-distal to a gypsy insertion in a gene from activating without affecting promoter-proximal enhancers. Several observations indicate that SUHW does not affect enhancer-binding activators. Instead, SUHW may interfere with factors that structurally facilitate interactions between an enhancer and promoter. To identify putative enhancer facilitators, a screen for mutations that reduce activity of the remote wing margin enhancer in the cut gene was performed. Mutations in scalloped, mastermind, and a previously unknown gene, Chip, were isolated. A TEA DNA-binding domain in the Scalloped protein binds the wing margin enhancer. Interactions between scalloped, mastermind and Chip mutations indicate that mastermind and Chip act synergistically with scalloped to regulate the wing margin enhancer. Chip is essential and also affects expression of a gypsy insertion in Ultrabithorax. Relative to mutations in scalloped or mastermind, a Chip mutation hypersensitizes the wing margin enhancer in cut to gypsy insertions. Therefore, Chip might encode a target of SUHW enhancer-blocking activity.     

Multiple signaling pathways and a selector protein sequentially regulate Drosophila wing development

Drosophila wing development is a useful model to study organogenesis, which requires the input of selector genes that specify the identity of various morphogenetic fields (Weatherbee, S. D. and Carroll, S. B. (1999) Cell 97, 283-286) and cell signaling molecules. In order to understand how the integration of multiple signaling pathways and selector proteins can be achieved during wing development, we studied the regulatory network that controls the expression of Serrate (Ser), a ligand for the Notch (N) signaling pathway, which is essential for the development of the Drosophila wing, as well as vertebrate limbs. Here, we show that a 794 bp cis-regulatory element located in the 3' region of the Ser gene can recapitulate the dynamic patterns of endogenous Ser expression during wing development. Using this enhancer element, we demonstrate that Apterous (Ap, a selector protein), and the Notch and Wingless (Wg) signaling pathways, can sequentially control wing development through direct regulation of Ser expression in early, mid and late third instar stages, respectively. In addition, we show that later Ser expression in the presumptive vein cells is controlled by the Egfr pathway. Thus, a cis-regulatory element is sequentially regulated by multiple signaling pathways and a selector protein during Drosophila wing development. Such a mechanism is possibly conserved in the appendage outgrowth of other arthropods and vertebrates.     

The level of DLDB/CHIP controls the activity of the LIM homeodomain protein apterous: evidence for a functional tetramer complex in vivo

The LIM homeodomain (LIM-HD) protein Apterous (Ap) and its cofactor DLDB/CHIP control dorso- ventral (D/V) patterning and growth of Drosophila wing. To investigate the molecular mechanisms of Ap/CHIP function we altered their relative levels of expression and generated mutants in the LIM1, LIM2 and HD domains of Ap, as well as in the LIM-interacting and self-association domains of CHIP. Using in vitro and in vivo assays we found that: (i) the levels of CHIP relative to Ap control D/V patterning; (ii) the LIM1 and LIM2 domains differ in their contributions to Ap function; (iii) Ap HD mutations cause weak dominant negative effects; (iv) overexpression of ChipDeltaSAD mutants mimics Ap lack-of-function, and this dominant negative phenotype is caused by titration of Ap because it can be rescued by adding extra Ap; and (v) overexpression of ChipDeltaLID mutants also causes an Ap lack-of-function phenotype, but it cannot be rescued by extra Ap. These results support the model that the Ap-CHIP active complex in vivo is a tetramer.     

WAVE forms hetero- and homo-oligomeric complexes at integrin junctions in Drosophila visualized by bimolecular fluorescence complementation

Dynamic actin polymerization drives a variety of morphogenetic events during metazoan development. Members of the WASP/WAVE protein family are central nucleation-promoting factors. They are embedded within regulatory networks of macromolecular complexes controlling Arp2/3-mediated actin nucleation in time and space. WAVE (Wiskott-Aldrich syndrome protein family verprolin-homologous protein) proteins are found in a conserved pentameric heterocomplex that contains Abi, Kette/Nap1, Sra-1/CYFIP, and HSPC300. Formation of the WAVE complex contributes to the localization, activity, and stability of the various WAVE proteins. Here, we established the Bimolecular Fluorescence Complementation (BiFC) technique in Drosophila to determine the subcellular localization of the WAVE complex in living flies. Using different split-YFP combinations, we are able to visualize the formation of the WAVE-Abi complex in vivo. We found that WAVE also forms dimers that are capable of forming higher order clusters with endogenous WAVE complex components. The N-terminal WAVE homology domain (WHD) of the WAVE protein mediates both WAVE-Abi and WAVE-WAVE interactions. Detailed localization analyses show that formation of WAVE complexes specifically takes place at basal cell compartments promoting actin polymerization. In the wing epithelium, hetero- and homooligomeric WAVE complexes co-localize with Integrin and Talin suggesting a role in integrin-mediated cell adhesion. RNAi mediated suppression of single components of the WAVE and the Arp2/3 complex in the wing further suggests that WAVE-dependent Arp2/3-mediated actin nucleation is important for the maintenance of stable integrin junctions.