The role of brinker in eggshell patterning

Drosophila oogenesis provides a useful system to study signal transduction pathways and their interactions. Through clonal analysis, we found that brinker (brk), a repressor of Dpp signaling, plays an important role in the Drosophila ovary, where its function is essential for dorsal appendage formation. In the absence of brk, operculum fates are specified at the expense of dorsal appendage fates. Brk is expressed by most of the oocyte associated follicle cells, starting from stage 8 of oogenesis. Transforming Growth Factor beta (TGFbeta) signaling represses brk expression in both the early stage egg chambers and in the anterior follicle cells. In brk mutant follicle cell clones at the dorsal anterior region, Broad Complex (BR-C) expression is down-regulated in a larger domain than in wild type. We show that BR-C is required for dorsal appendage development. In large anterior BR-C mutant clones, dorsal appendages are absent, and instead, the eggshell has an enlarged operculum like region at the anterior. In addition, we show that the Epidermal Growth Factor (EGF) receptor signaling represses the TGFbeta signaling in oogenesis by up-regulating brk expression. From our results and previously published data, it appears that anterior follicle cells integrate the levels of EGF receptor activation and TGFbeta receptor activation. Operculum fate results when the sum of the level of activation of both pathways reaches a threshold level, and reduction of activity of one pathway can be compensated to some extent by increase in the other pathway.     

Sequential Notch Signalling at the Boundary of Fringe Expressing and Non-Expressing Cells

Wing development in Drosophila requires the activation of Wingless (Wg) in a small stripe along the boundary of Fringe (Fng) expressing and non-expressing cells (FB), which coincides with the dorso-ventral (D/V) boundary of the wing imaginal disc. The expression of Wg is induced by interactions between dorsal and ventral cells mediated by the Notch signalling pathway. It appears that mutual signalling from dorsal to ventral and ventral to dorsal cells by the Notch ligands Serrate (Ser) and Delta (Dl) respectively establishes a symmetric domain of Wg that straddles the D/V boundary. The directional signalling of these ligands requires the modification of Notch in dorsal cells by the glycosyltransferase Fng and is based on the restricted expression of the ligands with Ser expression to the dorsal and that of Dl to the ventral side of the wing anlage. In order to further investigate the mechanism of Notch signalling at the FB, we analysed the function of Fng, Ser and Dl during wing development at an ectopic FB and at the D/V boundary. We find that Notch signalling is initiated in an asymmetric fashion on only one side of the FB. During this initial asymmetric phase, only one ligand is required, with Ser initiating Notch-signalling at the D/V and Dl at the ectopic FB. Furthermore, our analysis suggests that Fng has also a positive effect on Ser signalling. Because of these additional properties, differential expression of the ligands, which has been a prerequisite to restrict Notch activation to the FB in the current model, is not required to restrict Notch signalling to the FB.     

Cell-fate choice and boundary formation by combined action of Notch and engrailed in the Drosophila hindgut

The Drosophila large intestine is initially subdivided into dorsal and ventral domains with distinct cell types, and a one-cell-wide strand of boundary cells is induced between them. Here we show that cell identity and localization of the boundary cells are determined by the combined action of Delta, Notch, and engrailed genes. The prospective dorsal domain of the hindgut primordium expresses engrailed. Engrailed represses Delta, which is ubiquitously expressed throughout the prospective hindgut region in early blastodermal stages, in the dorsal domain, and thus generates a Delta-positive/negative prepattern. Expression of Engrailed protein determines the dorsal domain, while an Engrailed-negative (Delta-positive) region is differentiated into the ventral domain. Delta-positive ventral cells activate a Notch cascade in abutting dorsal cells, and thus induce their differentiation into boundary cells. Mis-expression of a constitutively active Notch intracellular domain causes the entire large intestine to develop as boundary cells. It was also found that the transducing activity of a transmembrane form of activated Notch, which requires further proteolytic processing to generate intracellular fragments, is suppressed in the Delta-positive domain. Delta acts in two distinct ways: it activates the Notch signaling pathway in adjacent Delta-negative cells, and, at the same time, autonomously blocks Notch signaling in Delta-positive cells by affecting Notch processing.     

EGFR-dependent network interactions that pattern Drosophila eggshell appendages

Similar to other organisms, Drosophila uses its Epidermal Growth Factor Receptor (EGFR) multiple times throughout development. One crucial EGFR-dependent event is patterning of the follicular epithelium during oogenesis. In addition to providing inductive cues necessary for body axes specification, patterning of the follicle cells initiates the formation of two respiratory eggshell appendages. Each appendage is derived from a primordium comprising a patch of cells expressing broad (br) and an adjacent stripe of cells expressing rhomboid (rho). Several mechanisms of eggshell patterning have been proposed in the past, but none of them can explain the highly coordinated expression of br and rho. To address some of the outstanding issues in this system, we synthesized the existing information into a revised mathematical model of follicle cell patterning. Based on the computational model analysis, we propose that dorsal appendage primordia are established by sequential action of feed-forward loops and juxtacrine signals activated by the gradient of EGFR signaling. The model describes pattern formation in a large number of mutants and points to several unanswered questions related to the dynamic interaction of the EGFR and Notch pathways.     

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.     

Dpp and Notch specify the fusion cell fate in the dorsal branches of the Drosophila trachea.

Decapentaplegic (Dpp) signaling determines the number of cells that migrate dorsally to form the dorsal primary branch during tracheal development. We report that Dpp signaling is also required for the differentiation of one of three different cell types in the dorsal branches, the fusion cell. In Mad mutant embryos or in embryos expressing dominant negative constructs of the two type I Dpp receptors in the trachea the number of cells expressing fusion cell-specific marker genes is reduced and fusion of the dorsal branches is defective. Ectopic expression of Dpp or the activated form of the Dpp receptor Tkv in all tracheal cells induces ectopic fusions of the tracheal lumen and ectopic expression of fusion gene markers in all tracheal branches. Among the fusion marker genes that are activated in the trachea in response to ectopic Dpp signaling is Delta. In conditional Notch loss of function mutants additional tracheal cells adopt the fusion cell fate and ectopic expression of an activated form of the Notch receptor in fusion cells results in suppression of fusion cell markers and disruption of the branch fusion. The number of cells that express the fusion cell markers in response to ectopic Dpp signaling is increased in Notch(ts1) mutants, suggesting that the two signaling pathways have opposing effects in the selection of the fusion cells in the dorsal branches.     

Distinct functions of the Drosophila genes Serrate and Delta revealed by ectopic expression during wing development

 The Drosophila gene Serrate encodes a transmembrane protein with 14 epidermal growth factor-(EGF)-like repeats in its extracellular portion. It has been suggested to act as a signal in the developing wing from the dorsal side to induce the organising centre at the dorsal/ventral compartment boundary, which is required for growth and patterning of the wing. Ectopic expression of Serrate during wing development induces ectopic outgrowth of ventral wing tissue and the formation of an additional wing margin. Here we present data to suggest that both events are mediated by genes that are required for normal wing development, including Notch as receptor. In order for Serrate to elicit these responses the concomitant expression of wingless seems to be required. The lack of wings in flies devoid of Serrate function can be partially restored by Gal4-mediated expression of Serrate, whilst expression of wingless is not sufficient. Ectopic expression of Delta, which encodes a structurally very similar transmembrane protein with EGF-like repeats, provokes wing outgrowth and induction of a new margin under all conditions tested here, both on the dorsal and ventral side. Our data further suggest that Serrate can act as an activating ligand for the Notch receptor only under certain circumstances; it inhibits Notch function under other conditions. Received: 26 april 1996 / Accepted: 24 May 1996     

Notch activation regulates the segregation and differentiation of rhombomere boundary cells in the zebrafish hindbrain

During segmentation of the vertebrate hindbrain, a distinct population of boundary cells forms at the interface between each segment. Little is known regarding mechanisms that regulate the formation or functions of these cells. We have investigated a potential role of Notch signaling and find that in the zebrafish hindbrain, radical fringe is expressed in boundary cells and delta genes are expressed adjacent to boundaries, consistent with a sustained activation of Notch in boundary cells. Mosaic expression experiments reveal that activation of the Notch/Su(H) pathway regulates cell affinity properties that segregate cells to boundaries. In addition, Notch signaling correlates with a delayed neurogenesis at hindbrain boundaries and is required to inhibit premature neuronal differentiation of boundary cells. These findings reveal that Notch activation couples the regulation of location and differentiation in hindbrain boundary cells. Such coupling may be important for these cells to act as a stable signaling center.     

Son of Notch, a winged-helix gene involved in boundary formation in the Drosophila wing

A P element enhancer trap screen was conducted to identify genes involved in dorsal-ventral boundary formation in Drosophila. The son of Notch (son) gene was identified by the son(2205) enhancer trap insertion, which is a partial loss-of-function mutation. Based on son(2205) mutant phenotypes and genetic interactions with Notch and wingless mutations, we conclude that son participates in wing development, and functions in the Notch signaling pathway at the dorsal-ventral boundary in the wing. Notch signaling pathway components activate son enhancer trap expression in wing cells. son enhancer trap expression is regulated positively by wingless, and negatively by cut in boundary cells. Ectopic Son protein induces wingless and cut expression in wing discs. We hypothesize that there is positive feedback regulation of son by wingless, and negative regulation by cut at the dorsal-ventral boundary during wing development.     

Roles for Zebrafish Focal Adhesion Kinase in Notochord and Somite Morphogenesis

We have cloned zebrafish focal adhesion kinase (Fak) and analyzed its subcellular localization. Fak protein is localized at the cortex of notochord cells and at the notochord-somite boundary. During somitogenesis, Fak protein becomes concentrated at the basal region of epithelial cells at intersomitic boundaries. Phosphorylated Fak protein is seen at both the notochord-somite boundary and intersomitic boundaries, consistent with a role for Fak in boundary formation and maintenance. The localization of Fak protein to the basal region of epithelial cells in knypek;trilobite double mutant embryos shows that polarization of Fak distribution in the somite border cells is independent of internal mesenchymal cells. In addition, we show that neither Notch signaling through Suppressor of Hairless (SuH) nor deltaD is necessary for the wild-type segmental pattern of fak mRNA expression in the anterior paraxial mesoderm. However, nonsegmental expression of fak mRNA occurs with ectopic activation of Notch signaling through SuH and also in fused somite and beamter mutant embryos, indicating that there are multiple regulators of fak mRNA expression. Our results suggest that Fak plays a central role in notochord and somite morphogenesis.     

Hephaestus encodes a polypyrimidine tract binding protein that regulates Notch signalling during wing development in Drosophila melanogaster

We describe the role of the Drosophila melanogaster hephaestus gene in wing development. We have identified several hephaestus mutations that map to a gene encoding a predicted RNA-binding protein highly related to human polypyrimidine tract binding protein and Xenopus laevis 60 kDa Vg1 mRNA-binding protein. Polypyrimidine tract binding proteins play diverse roles in RNA processing including the subcellular localization of mRNAs, translational control, internal ribosome entry site use, and the regulation of alternate exon selection. The analysis of gene expression in imaginal discs and adult cuticle of genetic mosaic animals supports a role for hephaestus in Notch signalling. Somatic clones lacking hephaestus express the Notch target genes wingless and cut, induce ectopic wing margin in adjacent wild-type tissue, inhibit wing-vein formation and have increased levels of Notch intracellular domain immunoreactivity. Clones mutant for both Delta and hephaestus have the characteristic loss-of-function thick vein phenotype of DELTA: These results lead to the hypothesis that hephaestus is required to attenuate Notch activity following its activation by Delta. This is the first genetic analysis of polypyrimidine tract binding protein function in any organism and the first evidence that such proteins may be involved in the Notch signalling pathway.