Notch signalling and the initiation of neural development in the Drosophila eye.

Neural determination in the Drosophila eye occurs progressively. A diffusible signal, Dpp, causes undetermined cells first to adopt a 'pre-proneural' state in which they are primed to start differentiating. A second signal is required to trigger the activation of the transcription factor Atonal, which causes the cells to initiate overt photoreceptor neurone differentiation. Both Dpp and the second signal are dependent on Hedgehog (Hh) signalling. Previous work has shown that the Notch signalling pathway also has a proneural role in the eye (as well as a later, opposite function when it restricts the number of cells becoming photoreceptors - a process of lateral inhibition). It is not clear how the early proneural role of Notch integrates with the other signalling pathways involved. We provide evidence that Notch activation by its ligand Delta is the second Hh-dependent signal required for neural determination. Notch activity normally only triggers Atonal expression in cells that have adopted the pre-proneural state induced by Dpp. We also report that Notch drives the transition from pre-proneural to proneural by downregulating two repressors of Atonal: Hairy and Extramacrochaetae.

A primary role for the epidermal growth factor receptor in ommatidial spacing in the Drosophila eye.

BACKGROUND: The differentiation of regularly spaced structures within an epithelium is a common feature of developmental pattern formation. The regular spacing of ommatidia in the Drosophila eye imaginal disc provides a good model for this phenomenon. The correct spacing of ommatidia is a central event in establishing the precise hexagonal pattern of ommatidia in the Drosophila compound eye. The R8 photoreceptors are the founder cells of each of the ommatidia that comprise the adult eye and are specified by a bHLH transcription factor, Atonal. RESULTS: We find that the epidermal growth factor receptor (Egfr) has a primary function in regulating R8 spacing. The receptor's activation within nascent ommatidia induces the expression of a secreted inhibitor that blocks atonal expression, and therefore ommatidial initiation, in nearby cells. The identity of the secreted inhibitor remains elusive but, contrary to previous suggestions, we show that it is not Argos. This Egfr-dependent inhibition acts in parallel to the inhibition of atonal by the secreted protein Scabrous. The activation of the Egfr pathway is dependent on Atonal function via the expression of Rhomboid-1. Our results also allow us to conclude that Egfr's role in promoting cell survival is largely independent of its role in photoreceptor recruitment; even when cell death is blocked, most photoreceptors fail to form. CONCLUSIONS: Based on our data and those of others, we propose a model for R8 spacing that comprises a self-organizing network of signaling molecules. This model describes how successive rows of ommatidia form out of phase with each other, leading to the hexagonal array of facets in the compound eye.

DER signaling restricts the boundaries of the wing field during Drosophila development.

Arthropod and vertebrate limbs develop from secondary embryonic fields. In insects, the wing imaginal disk is subdivided early in development into the wing and notum subfields. The activity of the Wingless protein is fundamental for this subdivision and seems to be the first element of the hierarchy of regulatory genes promoting wing formation. Drosophila epidermal growth factor receptor (DER) signaling has many functions in fly development. Here we show that antagonizing DER signaling during the second larval instar leads to notum to wing transformations and wing mirror-image duplications. DER signaling is necessary for confining the wing subregion in the developing wing disk and for the specification of posterior identity. To do so, DER signaling acts by restricting the expression of Wingless to the dorsal-posterior quadrant of wing discs, suppressing wing-organizing activities, and by cooperating in the maintenance of Engrailed expression in posterior compartment cells.

Relationships between extramacrochaetae and Notch signalling in Drosophila wing development.

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).

Notch signaling directly controls cell proliferation in the Drosophila wing disc.

Notch signaling is involved in cell differentiation and patterning during morphogenesis. In the Drosophila wing, Notch activity regulates the expression of several genes at the dorsal/ventral boundary, and this is thought to elicit wing-cell proliferation. In this work, we show the effect of clones of cells expressing different forms of several members of the Notch signaling pathway, which result in an alteration of Notch activity. The ectopic expression in clones of activated forms of Notch or of its ligands (Delta or Serrate) in the wing causes outgrowths associated with the appearance of ectopic wing margins. These outgrowths consist of mutant territories and of surrounding wild-type cells. However, the ectopic expression of Delta, at low levels in ventral clones, causes large outgrowths that are associated neither with the generation of wing margin structures nor with the expression of genes characteristic of the dorsal/ventral boundary. These results suggest that Notch activity is directly involved in cell proliferation, independently of its role in the formation of the dorsal/ventral boundary. We propose that the nonautonomous effects (induction of extraproliferation and vein differentiation in the surrounding wild-type cells) result from pattern accommodation to positional values caused by the ectopic expression of Notch.

A temporal switch in DER signaling controls the specification and differentiation of veins and interveins in the Drosophila wing.

The Drosophila EGF receptor (DER) is required for the specification of diverse cell fates throughout development. We have examined how the activation of DER controls the development of vein and intervein cells in the Drosophila wing. The data presented here indicate that two distinct events are involved in the determination and differentiation of wing cells. (1) The establishment of a positive feedback amplification loop, which drives DER signaling in larval stages. At this time, rhomboid (rho), in combination with vein, initiates and amplifies the activity of DER in vein cells. (2) The late downregulation of DER activity. At this point, the inactivation of MAPK in vein cells is necessary for the maintenance of the expression of decapentaplegic (dpp) and becomes essential for vein differentiation. Together, these temporal and spatial changes in the activity of DER constitute an autoregulatory network that controls the definition of vein and intervein cell types.

Dual role of extramacrochaetae in cell proliferation and cell differentiation during wing morphogenesis in Drosophila.

The Extramacrochaetae (emc) gene encodes a transcription factor with an HLH domain without the basic region involved in interaction with DNA present in other proteins that have this domain. EMC forms heterodimers with bHLH proteins preventing their binding to DNA, acting as a negative regulator. The function of emc is required in many developmental processes during the development of Drosophila, including wing morphogenesis. Mitotic recombination clones of both null and gain-of-function alleles of emc, indicate that during wing morphogenesis, emc participates in cell proliferation within the intervein regions (vein patterning), as well as in vein differentiation. The study of relationships between emc and different genes involved in wing development reveal strong genetic interactions with genes of the Ras signalling pathway (torpedo, vein, veinlet and Gap), blistered, plexus and net, in both adult wing phenotypes and cell behaviour in genetic mosaics. These interactions are also analyzed as variations of emc expression patterns in mutant backgrounds for these genes. In addition, cell proliferation behaviour of emc mutant cells varies depending on the mutant background. The results show that genes of the Ras signalling pathway are co-operatively involved in the activity of emc during cell proliferation, and later antagonistically during cell differentiation, repressing EMC expression.

Genetic interactions and cell behaviour in blistered mutants during proliferation and differentiation of the Drosophila wing.

In this work, we analyse the blistered function in wing vein development by studying genetic mosaics of mutant cells, genetic interactions with other genes affecting vein development and blistered expression in several mutant backgrounds. blistered encodes for a nuclear protein homologous to the mammalian Serum Response Factor and is expressed in presumptive intervein cells of third larval instar and pupal wing discs. Clones of blistered mutant cells proliferate normally but tend to grow along veins and always differentiate as vein tissue. These observations indicate that vein-determined wing cells show a particular behaviour that is responsible for their allocation to vein regions. We observe strong genetic interactions between blistered, veinlet and genes of the Ras signaling cascade. During disc proliferation, blistered expression is under the control of the Ras signal transduction pathway, but its expression is independent of veinlet. During the pupal period, blistered and veinlet expression become interdependent and mutually exclusive. These results link the activity of the Ras pathway to the process of early determination of intervein cells, by the transcriptional control of the blistered nuclear factor.

Wing surface interactions in venation patterning in Drosophila.

The adult wing of Drosophila consists of two wing surfaces apposed by their basal membranes which first came into contact following disc eversion at metamorphosis. Veins appear in these surfaces in a dorsal-ventral symmetric pattern, but are 'corrugated' (vein cells are more compacted and more pigmented) in a dorsal-ventral asymmetric pattern. We prevented dorsal-ventral contact apposition during wing imaginal disc morphogenesis by implanting fragments of discs into metamorphosing hosts. In these implants, longitudinal veins differentiate but with wider corrugation and in both surfaces. These results and those of genetic mosaics of mutants removing veins or causing ectopic veins reveal mutual dorso-ventral induction/inhibition at work to modulate the final vein differentiation pattern and corrugation.

Behavior of extramacrochaetae mutant cells in the morphogenesis of the Drosophila wing.

The gene extramacrochaetae (emc) encodes a non-basic Helix-loop-helix (HLH) protein that interacts and antagonises other basic-HLH proteins. The expression pattern of emc, and the phenotype of lethal emc alleles indicate that this gene is operative in several developmental processes. Here we study the requirements for emc during cell proliferation and vein differentiation in the wing. Mosaic analysis of hypomorphic conditions of emc reveals the tendency of mutant cells to proliferate along the veins as long stripes. Large clones abuting two adjacent veins obliterate the corresponding inter-vein, affecting the size and shape of the whole wing. Thus, the emc gene participates in the control of cell proliferation within inter-vein regions in the wing. Similar effects were found in the haltere and in the leg. The behavior of emc cells in genetic mosaics indicate that (1) proliferation is locally controlled within inter-vein sectors, (2) cells proliferate according to their genetic activity along preferential positions in the wing morphogenetic landscape and (3) cell proliferation in the wing is integrated by 'accommodation' between mutant and wild type cells.