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The function of extramacrochaetae is required during the development of the Drosophila wing in processes such as cell proliferation and vein differentiation. extramacrochaetae encodes a transcription factor of the HLH family, but unlike other members of this family, Extramacrochaetae lacks the basic region that is involved in interaction with DNA. Some phenotypes caused by extramacrochaetae in the wing are similar to those observed when Notch signalling is compromised. Furthermore, maximal levels of extramacrochaetae expression in the wing disc are restricted to places where Notch activity is higher, suggesting that extramacrochaetae could mediate some aspects of Notch signalling during wing development. We have studied the relationships between extramacrochaetae and Notch in wing development, with emphasis on the processes of vein formation and cell proliferation. We observe strong genetic interaction between extramacrochaetae and different components of the Notch signalling pathway, suggesting a functional relationship between them. We show that the higher level of extramacrochaetae expression coincides with the domain of expression of Notch and its downstream gene Enhancer of split-m(beta). The expression of extramacrochaetae at the dorso/ventral boundary and in boundary cells between veins and interveins depends on Notch activity. We propose that at least during vein differentiation and wing margin formation, extramacrochaetae is regulated by Notch and collaborates with other Notch-downstream genes such as Enhancer of split-m(beta).

The Notch signalling pathway regulates cell fate choices during both vertebrate and invertebrate development. In the Drosophila wing disc, the activation of Notch by its ligands Delta and Serrate is required to make the dorsoventral boundary, where several genes, such as wingless and cut, are expressed in a 2- to 4-cell-wide domain. The interactions between Notch and its ligands are modulated by Fringe via a mechanism that may involve post-transcriptional modifications of Notch. The ligands themselves also help to restrict Notch activity to the dorsoventral boundary cells, because they antagonise the activation of the receptor in the cells where their expression is high. This function of the ligands is critical to establish the polarity of signalling, but very little is known about the mechanisms involved in the interactions between Notch and its ligands that result in suppression of Notch activity. The extracellular domain of Notch contains an array of 36 EGF repeats, two of which, repeats 11 and 12, are necessary for direct interactions between Notch with Delta and Serrate. We investigate here the function of a region of the Notch extracellular domain where several missense mutations, called Abruptex, are localised. These Notch alleles are characterised by phenotypes opposite to the loss of Notch function and also by complex complementation patterns. We find that, in Abruptex mutant discs, only the negative effects of the ligands and Fringe are affected, resulting in the failure to restrict the expression of cut and wingless to the dorsoventral boundary. We suggest that Abruptex alleles identify a domain in the Notch protein that mediates the interactions between Notch, its ligands and Fringe that result in suppression of Notch activity.

The nuclear proteins Spalt and Spalt-related belong to a conserved family of transcriptional regulators characterised by the presence of double zinc-finger domains. In the wing, they are regulated by the secreted protein Decapentaplegic and participate in the positioning of the wing veins. Here, we identify regulatory regions in the spalt/spalt-related gene complex that direct expression in the wing disc. The regulatory sequences are organised in independent modules, each of them responsible for expression in particular domains of the wing imaginal disc. In the thorax, spalt and spalt-related are expressed in a restricted domain that includes most proneural clusters of the developing sensory organs in the notum, and are regulated by the signalling molecules Wingless, Decapentaplegic and Hedgehog. We find that spalt/spalt-related participate in the development of sensory organs in the thorax, mainly in the positioning of specific proneural clusters. Later, the expression of at least spalt is eliminated from the sensory organ precursor cells and this is a requisite for the differentiation of these cells. We postulate that spalt and spalt-related belong to a category of transcriptional regulators that subdivide the thorax into expression domains (prepattern) required for the localised activation of proneural genes.

The differentiation of the veins in the Drosophila wing involves the coordinate activities of several signal transduction pathways, including those mediated by the transmembrane receptors Torpedo and Notch. In this report, the role of the signalling molecule Decapentaplegic during vein differentiation has been analysed. It is shown that decapentaplegic is expressed in the pupal veins under the control of genes that establish vein territories in the imaginal disc. Decapentaplegic, acting through its receptor Thick veins, activates vein differentiation and restricts expression of both veinlet and the Notch-ligand Delta to the developing veins. Genetic combinations between mutations that increase or reduce Notch, veinlet and decapentaplegic activities suggest that the maintenance of the vein differentiation state during pupal development involves cross-regulatory interactions between these pathways.

Drosophila imaginal discs are divided into units called compartments. Cells belonging to the same compartment are related by lineage and express a characteristic set of 'selector genes'. The borders between compartments act as organizing centres that influence cell growth within compartments. Thus, in the cells immediately anterior to the anterior-posterior compartment boundary the presence of the hedgehog product causes expression of decapentaplegic, which, in turn, influences the growth and patterning of the wing disc. The normal growth of the disc requires that posterior-specific genes, such as hedgehog and engrailed are not expressed in cells of the anterior compartment. Here we show that hedgehog can activate engrailed in the anterior compartment and that both hedgehog and engrailed are specifically repressed in anterior cells by the activity of the neurogenic gene groucho. In groucho mutant discs, hedgehog and engrailed are expressed at the dorsoventral boundary of the anterior compartment, leading to the ectopic activation of decapentaplegic and patched and to a localised increase in cell growth associated with pattern duplications. The presence of engrailed in the anterior compartment causes the transformation of anterior into posterior structures.

The ventral veinless gene (vvl) encodes the previously identified Cf1a protein, a transcription factor containing a POU-domain. During embryonic development vvl function is required for the formation of the tracheal tree and in the patterning of the ventral ectoderm. During imaginal development vvl is required for cell proliferation and the differentiation of the wing veins. vvl expression is restricted to the regions where its function is required, and is dependent on the coordinate activities of signalling molecules such as decapentaplegic, wingless and hedgehog. vvl interacts with other genes involved in vein differentiation, including veinlet, thick veins, torpedo, decapentaplegic and Notch suggesting that vvl function may affect several cell-to-cell communication pathways. We propose that the gene vvl integrates information from different signalling molecules and regulates the expression of specific cell differentiation genes during tracheal development and vein differentiation.

The cells along the dorsoventral boundary of the Drosophila wing imaginal disc have distinctive properties and their specification requires Notch activity. Later in development, these cells will form the wing margin, where sensory organs and specialised trichomes appear in a characteristic pattern. We find that Notch is locally activated in these cells, as demonstrated by the restricted expression of the Enhancer of split proteins in dorsal and ventral cells abutting the D/V boundary throughout the third larval instar. Furthermore other genes identified by their involvement in Notch signaling during neurogenesis, such as Delta and Suppressor of Hairless, also participate in Notch function at the dorsoventral boundary. In addition, Serrate, a similar transmembrane protein to Delta, behaves as a ligand required in dorsal cells to activate Notch at the boundary. Notch gain-of-function alleles in which Notch activity is not restricted to the dorsoventral boundary cause miss-expression of cut and wingless and overgrowth of the disc, illustrating the importance of localised Notch activation for wing development.

Notch function is required at the dorsoventral boundary of the developing Drosophila wing for its normal growth and patterning. We find that clones of cells expressing either Notch or its ligands Delta and Serrate in the wing mimic Notch activation at the dorsoventral boundary producing non-autonomous effects on proliferation, and activating expression of the target genes E(spl), wingless and cut. The analysis of these clones reveals several mechanisms important for maintaining and delimiting Notch function at the dorsoventral boundary. First, Notch activation in the wing leads to increased production of Delta and Serrate generating a positive feedback loop that maintains signalling. We propose that during normal development, wingless co-operates with Notch to reinforce this positive feedback and Cut, which is activated by Notch at late stages, acts antagonistically to prevent Delta and Serrate expression. Second, high levels of Delta and Serrate have a dominant negative effect on Notch, so that at late stages Notch can only be activated in cells next to the ligand-producing cells. Thus the combined effects of Notch and its target genes cut and wingless regulate the expression of Notch ligands which restrict Notch activity to the dorsoventral boundary.

The veins in the Drosophila wing have a characteristic width, which is regulated by the activity of the Notch pathway. The expression of the Notch-ligand Delta is restricted to the developing veins, and coincides with places where Notch transcription is lower. We find that this asymmetrical distribution of ligand and receptor leads to activation of Notch on both sides of each vein within a territory of Delta-expressing cells, and to the establishment of boundary cells that separate the vein from adjacent interveins. In these cells, the expression of the Enhancer of split gene m beta is activated and the transcription of the vein-promoting gene veinlet is repressed, thus restricting vein differentiation. We propose that the establishment of vein thickness utilises a combination of mechanisms that include: (1) independent regulation of Notch and Delta expression in intervein and vein territories, (2) Notch activation by Delta in cells where Notch and Delta expression overlaps, (3) positive feedback on Notch transcription in cells where Notch has been activated and (4) repression of veinlet transcription by E(spl)m beta and maintenance of Delta expression by veinlet/torpedo activity.