A screen for genes regulating the wingless gradient in Drosophila embryos

During the development of the Drosophila embryonic epidermis, the secreted Wingless protein initially spreads symmetrically from its source. At later stages, Wingless becomes asymmetrically distributed in a Hedgehog-dependent manner, to control the patterning of the embryonic epidermis. When Wingless is misexpressed in engrailed cells in hedgehog heterozygous mutant embryos, larvae show a dominant phenotype consisting of patches of naked cuticle in denticle belts. This dose-sensitive phenotype is a direct consequence of a change in Wg protein distribution. We used this phenotype to carry out a screen for identifying genes regulating Wingless distribution or transport in the embryonic epidermis. Using a third chromosome deficiency collection, we found several genomic regions that showed a dominant interaction. After using a secondary screen to test for mutants and smaller deficiencies, we identified three interacting genes: dally, notum, and brahma. We confirmed that dally, as well as its homolog dally-like, and notum affect Wingless distribution in the embryonic epidermis, directly or indirectly. Thus, our assay can be used effectively to screen for genes regulating Wingless distribution or transport.     

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.     

The EGF receptor and N signalling pathways act antagonistically in Drosophila mesothorax bristle patterning

An early step in the development of the large mesothoracic bristles (macrochaetae) of Drosophila is the expression of the proneural genes of the achaete-scute complex (AS-C) in small groups of cells (proneural clusters) of the wing imaginal disc. This is followed by a much increased accumulation of AS-C proneural proteins in the cell that will give rise to the sensory organ, the SMC (sensory organ mother cell). This accumulation is driven by cis-regulatory sequences, SMC-specific enhancers, that permit self-stimulation of the achaete, scute and asense proneural genes. Negative interactions among the cells of the cluster, triggered by the proneural proteins and mediated by the Notch receptor (lateral inhibition), block this accumulation in most cluster cells, thereby limiting the number of SMCs. Here we show that the proneural proteins trigger, in addition, positive interactions among cells of the cluster that are mediated by the Epidermal growth factor receptor (EGFR) and the Ras/Raf pathway. These interactions, which we denominate 'lateral co-operation', are essential for macrochaetae SMC emergence. Activation of the EGFR/Ras pathway appears to promote proneural gene self-stimulation mediated by the SMC-specific enhancers. Excess EGFR signalling can overrule lateral inhibition and allow adjacent cells to become SMCs and sensory organs. Thus, the EGFR and Notch pathways act antagonistically in notum macrochaetae determination.     

Different mechanisms initiate and maintain wingless expression in the Drosophila wing hinge

The Drosophila gene wingless encodes a secreted signalling molecule that is required for many patterning events in both embryonic and postembryonic development. In the wing wingless is expressed in a complex and dynamic pattern that is controlled by several different mechanisms. These involve the Hedgehog and Notch pathways and the nuclear proteins Pannier and U-shaped. In this report, we analyse the mechanisms that drive wingless expression in the wing hinge. We present evidence that wingless is initially activated by a secreted signal that requires the genes vestigial, rotund and nubbin. Later in development, wingless expression in the wing hinge is maintained by a different mechanism, which involves an autoregulatory loop and requires the genes homothorax and rotund. We discuss the role of wingless in patterning the wing hinge.     

Temporally dynamic response to Wingless directs the sequential elaboration of the proximodistal axis of the Drosophila wing

The Drosophila wing imaginal disc gives rise to three main regions along the proximodistal axis of the dorsal mesothoracic segment: the notum, proximal wing, and wing blade. Development of the wing blade requires the Notch and wingless signalling pathways to activate vestigial at the dorsoventral boundary. However, in the proximal wing, Wingless activates a different subset of genes, e.g., homothorax. This raises the question of how the downstream response to Wingless signalling differentiates between proximal and distal fate specification. Here, we show that a temporally dynamic response to Wingless signalling sequentially elaborates the proximodistal axis. In the second instar, Wingless activates genes involved in proximal wing development; later in the third instar, Wingless acts to direct the differentiation of the distal wing blade. The expression of a novel marker for proximal wing fate, zfh-2, is initially activated by Wingless throughout the "wing primordium," but later is repressed by the activity of Vestigial and Nubbin, which together define a more distal domain. Thus, activation of a distal developmental program is antagonistic to previously established proximal fate. In addition, Wingless is required early to establish proximal fate, but later when Wingless activates distal differentiation, development of proximal fate becomes independent of Wingless signalling. Since P-element insertions in the zfh-2 gene result in a revertable proximal wing deletion phenotype, it appears that zfh-2 activity is required for correct proximal wing development. Our data are consistent with a model in which Wingless first establishes a proximal appendage fate over notum, then the downstream response changes to direct the differentiation of a more distal fate over proximal. Thus, the proximodistal domains are patterned in sequence and show a distal dominance.     

odd-skipped genes and lines organize the notum anterior-posterior axis using autonomous and non-autonomous mechanisms

The growth and patterning of Drosophila wing and notum primordia depend on their subdivision into progressively smaller domains by secreted signals that emanate from localized sources termed organizers. While the mechanisms that organize the wing primordium have been studied extensively, those that organize the notum are incompletely understood. The genes odd-skipped (odd), drumstick (drm), sob, and bowl comprise the odd-skipped family of C(2)H(2) zinc finger genes, which has been implicated in notum growth and patterning. Here we show that drm, Bowl, and eyegone (eyg), a gene required for notum patterning, accumulate in nested domains in the anterior notum. Ectopic drm organized the nested expression of these anterior notum genes and downregulated the expression of posterior notum genes. The cell-autonomous induction of Bowl and Eyg required bowl, while the non-autonomous effects were independent of bowl. The homeodomain protein Bar is expressed along the anterior border of the notum adjacent to cells expressing the Notch (N) ligand Delta (Dl). bowl was required to promote Bar and repress Dl expression to pattern the anterior notum in a cell-autonomous manner, while lines acted antagonistically to bowl posterior to the Bowl domain. Our data suggest that the odd-skipped genes act at the anterior notum border to organize the notum anterior-posterior (AP) axis using both autonomous and non-autonomous mechanisms.     

Prepatterning the Drosophila notum: the three genes of the iroquois complex play intrinsically distinct roles

The Drosophila thorax exhibits 11 pairs of large sensory organs (macrochaetes) identified by their unique position. Remarkably precise, this pattern provides an excellent model system to study the genetic basis of pattern formation. In imaginal wing discs, the achaete-scute proneural genes are expressed in clusters of cells that prefigure the positions of each macrochaete. The activities of prepatterning genes provide positional cues controlling this expression pattern. The three homeobox genes clustered in the iroquois complex (araucan, caupolican and mirror) are such prepattern genes. mirror is generally characterized as performing functions predominantly different from the other iroquois genes. Conversely, araucan and caupolican are described in previous studies as performing redundant functions in most if not all processes in which they are involved. We have addressed the question of the specific role of each iroquois gene in the prepattern of the notum and we clearly demonstrate that they are intrinsically different in their contribution to this process: caupolican and mirror, but not araucan, are required for the neural patterning of the lateral notum. However, when caupolican and/or mirror expression is reduced, araucan loss of function has an effect on thoracic bristles development. Moreover, the overexpression of araucan is able to rescue caupolican loss of function. We conclude that, although retaining some common functionalities, the Drosophila iroquois genes are in the process of diversification. In addition, caupolican and mirror are required for stripe expression and, therefore, to specify the muscular attachment sites prepattern. Thus, caupolican and mirror may act as common prepattern genes for all structures in the lateral notum.     

Lateral inhibition and the development of the sensory bristles of the adult peripheral nervous system of Drosophila

Cells in the neurectoderm of Drosophila face a choice between neural and epidermal fates. On the notum of the adult fly, neural cells differentiate sensory bristles in a precise pattern. Evidence has accumulated that the bristle pattern arises from the spatial distribution of small groups of cells, proneural clusters, from each of which a single bristle will result. One class of genes, which includes the genes of the achaete-scute complex, is responsible for the correct positioning of the proneural clusters. The cells of a proneural cluster constitute an equivalence group, each of them having the potential to become a neural cell. Only one cell, however, will adopt the primary, dominant, neural fate. This cell is selected by means of cellular interactions between the members of the group, since if the dominant cell is removed, one of the remaining, epidermal, cells will switch fates and become neural. The dominant cell therefore prevents the other cells of the group from becoming neural by a phenomenon known as lateral inhibiton. They, then, adopt the secondary, epidermal, fate. A second class of genes, including the gene shaggy and the neurogenic genes mediate this process. There is some evidence that a proneural cluster is composed of a small number of cells, suggesting a contact-based mechanism of communication. The molecular nature of the protein products of the neurogenic genes is consistent with this idea.