The vestigial gene product provides a molecular context for the interpretation of signals during the development of the wing in Drosophila

The vestigial (vg) gene of Drosophila plays a central role in the development and patterning of the wing: loss of vestigial results in failures in wing development and ectopic expression of vestigial leads to the development of ectopic wings. The wing-specific regulation of vestigial is mediated through two enhancers: (1) the Boundary Enhancer (vgBE) is early acting and becomes restricted to the wing margin, and (2) the Quadrant Enhancer (vgQE), acts later and is responsible for the expression of vestigial in the developing wing blade. These enhancers receive regulatory inputs from three signalling pathways: wingless, decapentaplegic and Notch/Suppressor of Hairless. Our experiments show that the vestigial gene product is also an input in the regulation of vestigial expression. In particular, Vestigial provides an important input for the regulation of the activity of the vgQE acting in concert with Wingless and Decapentaplegic. Our results suggest how interactions between vgBE and the vgQE mediated by Vestigial can explain the interactions between the wing margin and the wing blade during the growth of the wing. We further show that Vestigial and Notch collaborate with Wingless to subdivide and pattern the wing blade. These results lead us to propose a general role for Wingless during development in which it stabilizes cell fate decisions that have been implemented by other molecules.     

Interactions among Delta, Serrate and Fringe modulate Notch activity during Drosophila wing development

The Notch signalling pathway plays an important role during the development of the wing primordium, especially of the wing blade and margin. In these processes, the activity of Notch is controlled by the activity of the dorsal specific nuclear protein Apterous, which regulates the expression of the Notch ligand, Serrate, and the Fringe signalling molecule. The other Notch ligand, Delta, also plays a role in the development and patterning of the wing. It has been proposed that Fringe modulates the ability of Serrate and Delta to signal through Notch and thereby restricts Notch signalling to the dorsoventral boundary of the developing wing blade. Here we report the results of experiments aimed at establishing the relationships between Fringe, Serrate and Delta during wing development. We find that Serrate is not required for the initiation of wing development but rather for the expansion and early patterning of the wing primordium. We provide evidence that, at the onset of wing development, Delta is under the control of apterous and might be the Notch ligand in this process. In addition, we find that Fringe function requires Su(H). Our results suggest that Notch signalling during wing development relies on careful balances between positive and dominant negative interactions between Notch ligands, some of which are mediated by Fringe.     

Organising activities of engrailed, hedgehog, wingless and decapentaplegic in the genital discs of Drosophila melanogaster

The genes engrailed (en), hedgehog (hh), wingless (wg) and decapentaplegic (dpp) have been shown to play vital organising roles in the development and differentiation of thoracic imaginal discs. We have analysed the roles of these genes in organising the development and differentiation of the genital discs, which are bilaterally symmetrical and possess different primordia, namely, the male and female genital primordia and an anal primordium. Our results suggest that the organising activity of en in genital discs programs the normal development and differentiation of the genital disc by regulating the expression of hh. Hh in turn induces wg and dpp, the genes whose products act as secondary signalling molecules. Moreover, the complementary patterns of wg and dpp expression are essential for the bilateral symmetry and are maintained by mutual repression.     

Wingless and Notch regulate cell-cycle arrest in the developing Drosophila wing

In developing organs, the regulation of cell proliferation and patterning of cell fates is coordinated. How this coordination is achieved, however, is unknown. In the developing Drosophila wing, both cell proliferation and patterning require the secreted morphogen Wingless (Wg) at the dorsoventral compartment boundary. Late in wing development, Wg also induces a zone of non-proliferating cells at the dorsoventral boundary. This zone gives rise to sensory bristles of the adult wing margin. Here we investigate how Wg coordinates the cell cycle with patterning by studying the regulation of this growth arrest. We show that Wg, in conjunction with Notch, induces arrest in both the G1 and G2 phases of the cell cycle in separate subdomains of the zone of non-proliferating cells. Wg induces G2 arrest in two subdomains by inducing the proneural genes achaete and scute, which downregulate the mitosis-inducing phosphatase String (Cdc25). Notch activity creates a third domain by preventing arrest at G2 in wg-expressing cells, resulting in their arrest in G1.     

decapentaplegic overexpression affects Drosophila wing and leg imaginal disc development and wingless expression

We have used the GAL4-UAS expression system to increase the level of expression of the Drosophila gene decapentaplegic (dpp) in a pattern approximating its normal pattern in leg and wing imaginal discs. Intermediate increases of dpp expression have little effect in wing discs but high levels of dpp overexpression lead to reduction of the scutellum and duplication of posterior wing structures. In leg discs intermediate increases cause supernumerary outgrowths of ventral leg structures in the anterior-ventral region. Greater increases of dpp expression cause the loss of ventral leg structures with the concomitant fusion of left and right dorsal forelegs. The defects observed in both legs and wings appear to arise through dose-dependent effects of dpp on wingless (wg) expression. A high level of dpp overexpression in the wing disc causes reduction of wg expression in the presumptive scutellar region, consistent with the subsequent reduction of the scutellum. An intermediate increase of dpp expression in leg discs induces the expansion of wg expression into the ventral outgrowths. At higher dpp expression levels, ventral wg expression in leg discs is eliminated, consistent with the loss of ventral leg cuticle. In the leg disc end knob and in the wing margin primordium, where wg and dpp cooperate in producing distal outgrowth, dpp overexpression has no detectable effect either on patterning or on wg expression. We propose that a critical role for dpp in other regions of the leg and wing discs is to reduce or block the expression of wg. This role of dpp is supported by the observation that ectopic wg expression is detected in imaginal discs where dpp signaling is compromised by lowering the activity of one of its receptors, tkv. This antagonism between dpp and wg expression may be critical to assigning only one disc region as the distal organizer.     

Fringe is essential for mirror symmetry and morphogenesis in the Drosophila eye

An early event in Drosophila eye development is the division of the eye disc into dorsoventral domains. The dorsoventral pattern is displayed in the adult compound eye as a distinct mirror symmetry across the dorsoventral midline or equator. The dorsoventral axis is also implicated in organizing early development of the eye, as retinal differentiation is initiated at the posterior dorsoventral midline. Here we show that Fringe is expressed specifically in the ventral half of the undifferentiated eye disc, thus creating a dorsoventral boundary. Ectopic Fringe borders that are generated by clones of fringe cells can reverse the planar polarity of photoreceptor clusters, indicating that the Fringe boundary is crucial for the induction of mirror symmetry. Lack of a Fringe boundary disrupts equatorial expression of Notch signalling proteins and causes a complete failure of eye development. Our results indicate that the formation of the Fringe boundary and subsequent Notch signalling at the equator are essential for organizing mirror symmetry and eye morphogenesis.     

Intercellular signalling in Drosophila segment formation reconstructed in vitro

Genetic studies show that intercellular signalling is involved in key steps in Drosophila melanogaster development, but it has not previously been possible to investigate these processes in simplified in vitro systems. Analysis of engrailed (en) and wingless (wg) and other segment polarity genes suggests that two or more intercellular signalling processes may be involved in intrasegmental patterning. Expression of en and wg begins about three hours after egg laying, in adjacent rows of cells in the posterior half of each segmental primordium. In wg- embryos and in conditional mutants in which wg function is inactivated during a critical period between three and five hours after egg laying, early en expression begins normally but then disappears within several hours. The wg gene encodes a protein highly similar to the product of the mouse Wnt-1 proto-oncogene, a secreted glycoprotein; wg protein is proposed to function as an extracellular signal, maintaining en expression and activating other molecular and morphogenetic processes in nearby cells. Several lines of evidence support the model, including the secretion of wg protein in the embryo, genetic mosaic experiments and cell lineage studies. We tested this model using purified embryonic cells isolated by whole animal cell sorting, and validated three key predictions: (1) when en-expressing cells from early embryos are grown alone in culture, they rapidly and selectively lose en expression; (2) purified wg-expressing cells provide a locally active signal that prevents this loss; (3) heterologous cells engineered to express wg also show signalling activity, indicating that wg protein alone, or in conjunction with more generally expressed factors, is the signal.     

Interactions between Wingless and DFz2 during Drosophila wing development

Drosophila Wingless (Wg) is a secreted signaling protein of the Wnt family. Mutations in the wg gene disrupt the patterning of embryonic segments and their adult derivatives. Wg protein has been shown in cell culture to functionally interact with DFz2, a receptor that is structurally related to the tissue polarity protein Frizzled (Fz). However, it has not been determined if DFz2 functions in the Wg signaling pathway during fly development. Here we demonstrate that overexpression of DFz2 increases Wg-dependent signaling to induce ectopic margin bristle formation in developing Drosophila wings. Overexpression of a truncated form of DFz2 acts in a dominant-negative manner to block Wg signaling at the wing margin, and this block is rescued by co-expression of full-length DFz2 but not full-length Fz. Our results suggest that DFz2 and not Fz acts in the Wg signaling pathway for wing margin development. However, a truncated form of Fz also blocks Wg signaling in embryo and wing margin development, and the truncated form of DFz2 affects ommatidial polarity during eye development. These observations suggest that a single dominant-negative form of Fz or DFz2 can block more than one type of Wnt signaling pathway and imply that truncated proteins of the Fz family lose some aspect of signaling specificity.     

Cooperativity in protein folding: from lattice models with sidechains to real proteins

Development of a multicellular organism requires that cells communicate with each other in order to regulate their growth, organize into tissues and coordinate their function. This cell-cell communication is mediated by signals cells receive (or send) between each other and from the environment. The signaling can be a short range remote signaling (through secreted signaling molecules), contact signaling (via plasma membrane bound molecules, gap junctions) or a long range signaling (through hormones). In this article, I have reviewed the recent advances on the role of cell-cell signaling in the development of the embryonic nervous system of the fruitfly Drosophila melanogaster and discussed some of the open questions raised by these studies. It discusses the contributions of the neurogenic genes Notch and Delta and the signaling pathways controlled by wingless, patched and hedgehog in neuroblast formation, neuroblast identity specification and neuroblast lineage elaboration.     

Molecular characterization of higher plant NAD-dependent isocitrate dehydrogenase: evidence for a heteromeric structure by the complementation of yeast mutants

A large number of observations suggest that, during Drosophila development there are close functional interactions between the activity of Notch receptor and that of a signaling molecule encoded by wingless gene. In this essay, I summarize these interactions and discuss the possibility that Wingless acts as a ligand for Notch as part of a switch that is iteratively involved in the assignation of cell fates during development.     

Role of dpp signalling in prepattern formation of the dorsocentral mechanosensory organ in Drosophila melanogaster

A proneural cluster of dorsocentral bristles forms adjacent to the dorsal side of wg-expressing cells in the notum region of the wing imaginal disc. It has been shown that wg activity is required for these structures to form. However, the restriction of this proneural cluster to the dorsal posterior side of the wg expression domain in the anterior compartment of the wing imaginal disc has suggested that Wg signalling itself is insufficient to establish the dorsocentral proneural cluster. Some factor(s) from the posterior side must participate in this action in cooperation with Wg signalling. We have examined the role of Dpp signalling in dorsocentral bristle formation by either ectopically activating or conditionally reducing Dpp signalling. Ubiquitous activation of Dpp signalling in the notum region of the wing imaginal disc induced additional dorsocentral proneural cluster all along the dorsal side of the wg expression domain, and altered wg expression. Conditional loss-of-function of Dpp signalling during disc development resulted in the inhibition of dorsocentral proneural cluster formation and expansion of the wg expression domain. These results suggest that Dpp signalling has two indispensable roles in dorsocentral bristle formation: induction of the dorsocentral proneural cluster in cooperation with Wg signalling and restriction of the wg expression domain in the notum region of the wing imaginal disc.     

A differential scanning calorimetric study of Newcastle disease virus: identification of proteins involved in thermal transitions

The spatial and temporal pattern of expression of enhancer trap lines reporting on the wingless (wg) and engrailed (en) genes was characterized in the adult antenna of Drosophila melanogaster. The time courses of expression seen for wg and en, although different from each other, reveal a complex well-controlled pattern of temporal expression, providing evidence that regulatory mechanisms are preserved throughout the life span of the adult fly. Altering the life span demonstrates that the temporal patterns of expression of both wg and en are linked to life span. These studies suggest that the expression of wg and en in the adult antenna is controlled by age-dependent mechanisms.     

Lhx2, a vertebrate homologue of apterous, regulates vertebrate limb outgrowth

apterous specifies dorsal cell fate and directs outgrowth of the wing during Drosophila wing development. Here we show that, in vertebrates, these functions appear to be performed by two separate proteins. Lmx-1 is necessary and sufficient to specify dorsal identity and Lhx2 regulates limb outgrowth. Our results suggest that Lhx2 is closer to apterous than Lmx-1, yet, in vertebrates, Lhx2 does not specify dorsal cell fate. This implies that in vertebrates, unlike Drosophila, limb outgrowth can be dissociated from the establishment of the dorsoventral axis.     

Mother- and father-rated competence, child-perceived competence, and cognitive distortions: unique relations with children''s depressive symptoms

The development of external sensory organs on the notum of Drosophila is promoted by the proneural genes achaete and scute. Their activity defines proneural cell clusters in the wing imaginal disc. Ectopic expression, under control of the GAL4 system, of the proneural gene lethal of scute (l'sc) causes the development of ectopic bristles. Persistent ectopic expression of l'sc is not sufficient to impose a neural fate on any given cell. This implies that mutual inhibition, mediated by the Notch signaling pathway, occurs among the cells of the ectopic proneural cluster. Consequently, the dominant, quantifiable phenotype associated with ectopic expression of l'sc is modified by mutations in genes known to be involved in neurogenesis. This phenotype has been utilized to screen for dominant enhancers and suppressors that modify the number of ectopic bristles. In this way, about 100,000 progeny of EMS or X-ray-treated flies have been analyzed to identify autosomal genes involved in regulation of the neural fate. In addition 1200 chromosomes carrying lethal P-element insertions were screened for modifiers. Besides mutations in genes expected to modify the phenotype, we have isolated mutations in six genes not known so far to be involved in neurogenesis.     

Identification and localization of a sea urchin Notch homologue: insights into vegetal plate regionalization and Notch receptor regulation

The specifications of cell types and germ-layers that arise from the vegetal plate of the sea urchin embryo are thought to be regulated by cell-cell interactions, the molecular basis of which are unknown. The Notch intercellular signaling pathway mediates the specification of numerous cell fates in both invertebrate and vertebrate development. To gain insights into mechanisms underlying the diversification of vegetal plate cell types, we have identified and made antibodies to a sea urchin homolog of Notch (LvNotch). We show that in the early blastula embryo, LvNotch is absent from the vegetal pole and concentrated in basolateral membranes of cells in the animal half of the embryo. However, in the mesenchyme blastula embryo LvNotch shifts strikingly in subcellular localization into a ring of cells which surround the central vegetal plate. This ring of LvNotch delineates a boundary between the presumptive secondary mesoderm and presumptive endoderm, and has an asymmetric bias towards the dorsal side of the vegetal plate. Experimental perturbations and quantitative analysis of LvNotch expression demonstrate that the mesenchyme blastula vegetal plate contains both animal/vegetal and dorsoventral molecular organization even before this territory invaginates to form the archenteron. Furthermore, these experiments suggest roles for the Notch pathway in secondary mesoderm and endoderm lineage segregation, and in the establishment of dorsoventral polarity in the endoderm. Finally, the specific and differential subcellular expression of LvNotch in apical and basolateral membrane domains provides compelling evidence that changes in membrane domain localization of LvNotch are an important aspect of Notch receptor function.