Fringe, Notch, and making developmental boundaries.

Multiple mechanisms are involved in positioning and restricting specialized dorsal-ventral border cells in the Drosophila wing, including modulation of Notch signaling by Fringe, autonomous inhibition by Notch ligands, and inhibition of Notch target genes by Nubbin. Recent studies have revealed that Fringe also modulates a Notch-mediated signaling process between dorsal and ventral cells in the Drosophila eye, establishing an organizer of eye growth and patterning along the dorsal-ventral midline. Fringe-dependent modulation of Notch signaling also plays a key role in Drosophila leg segmentation and growth. Lunatic Fringe has been shown to be required for vertebrate somitogenesis, where it appears to act as a crucial link between a molecular clock and the regulation of Notch signaling.     

msh specifies dorsal cell fate in the Drosophila wing

Drosophila limbs develop from imaginal discs that are subdivided into compartments. Dorsal-ventral subdivision of the wing imaginal disc depends on apterous activity in dorsal cells. Apterous protein is expressed in dorsal cells and is responsible for (1) induction of a signaling center along the dorsal-ventral compartment boundary (2) establishment of a lineage restriction boundary between compartments and (3) specification of dorsal cell fate. Here, we report that the homeobox gene msh (muscle segment homeobox) acts downstream of apterous to confer dorsal identity in wing development.     

Robustness and stability of the gene regulatory network involved in DV boundary formation in the Drosophila wing

Gene regulatory networks have been conserved during evolution. The Drosophila wing and the vertebrate hindbrain share the gene network involved in the establishment of the boundary between dorsal and ventral compartments in the wing and adjacent rhombomeres in the hindbrain. A positive feedback-loop between boundary and non-boundary cells and mediated by the activities of Notch and Wingless/Wnt-1 leads to the establishment of a Notch dependent organizer at the boundary. By means of a Systems Biology approach that combines mathematical modeling and both in silico and in vivo experiments in the Drosophila wing primordium, we modeled and tested this regulatory network and present evidence that a novel property, namely refractoriness to the Wingless signaling molecule, is required in boundary cells for the formation of a stable dorsal-ventral boundary. This new property has been validated in vivo, promotes mutually exclusive domains of Notch and Wingless activities and confers stability to the dorsal-ventral boundary. A robustness analysis of the regulatory network complements our results and ensures its biological plausibility.     

Two subunits of the Drosophila mediator complex act together to control cell affinity

The organizing centers for Drosophila imaginal disc development are created at straight boundaries between compartments; these are maintained by differences in cell affinity controlled by selector genes and intercellular signals. skuld and kohtalo encode homologs of TRAP240 and TRAP230, the two largest subunits of the Drosophila mediator complex; mutations in either gene cause identical phenotypes. We show here that both genes are required to establish normal cell affinity differences at the anterior-posterior and dorsal-ventral compartment boundaries of the wing disc. Mutant cells cross from the anterior to the posterior compartment, and can distort the dorsal-ventral boundary in either the dorsal or ventral direction. The Skuld and Kohtalo proteins physically interact in vivo and have synergistic effects when overexpressed, consistent with a skuld kohtalo double-mutant phenotype that is indistinguishable from either single mutant. We suggest that these two subunits do not participate in all of the activities of the mediator complex, but form a submodule that is required to regulate specific target genes, including those that control cell affinity.     

Compartments and organising boundaries in the Drosophila eye: the role of the homeodomain Iroquois proteins.

The Drosophila eye is patterned by a dorsal-ventral organising centre mechanistically similar to those in the fly wing and the vertebrate limb bud. Here we show how this organising centre in the eye is initiated - the first event in retinal patterning. Early in development the eye primordium is divided into dorsal and ventral compartments. The dorsally expressed homeodomain Iroquois genes are true selector genes for the dorsal compartment; their expression is regulated by Hedgehog and Wingless. The organising centre is then induced at the interface between the Iroquois-expressing and non-expressing cells at the eye midline. It was previously thought that the eye develops by a mechanism distinct from that operating in other imaginal discs, but our work establishes the importance of lineage compartments in the eye and thus supports their global role as fundamental units of patterning.     

A novel interaction between hedgehog and Notch promotes proliferation at the anterior-posterior organizer of the Drosophila wing

Notch has multiple roles in the development of the Drosophila melanogaster wing imaginal disc. It helps specify the dorsal-ventral compartment border, and it is needed for the wing margin, veins, and sensory organs. Here we present evidence for a new role: stimulating growth in response to Hedgehog. We show that Notch signaling is activated in the cells of the anterior-posterior organizer that produce the region between wing veins 3 and 4, and we describe strong genetic interactions between the gene that encodes the Hedgehog pathway activator Smoothened and the Notch pathway genes Notch, presenilin, and Suppressor of Hairless and the Enhancer of split complex. This work thus reveals a novel collaboration by the Hedgehog and Notch pathways that regulates proliferation in the 3-4 intervein region independently of Decapentaplegic.     

Roles for scalloped and vestigial in regulating cell affinity and interactions between the wing blade and the wing hinge

The scalloped and vestigial genes are both required for the formation of the Drosophila wing, and recent studies have indicated that they can function as a heterodimeric complex to regulate the expression of downstream target genes. We have analyzed the consequences of complete loss of scalloped function, ectopic expression of scalloped, and ectopic expression of vestigial on the development of the Drosophila wing imaginal disc. Clones of cells mutant for a strong allele of scalloped fail to proliferate within the wing pouch, but grow normally in the wing hinge and notum. Cells overexpressing scalloped fail to proliferate in both notal and wing-blade regions of the disc, and this overexpression induces apoptotic cell death. Clones of cells overexpressing vestigial grow smaller or larger than control clones, depending upon their distance from the dorsal-ventral compartment boundary. These studies highlight the importance of correct scalloped and vestigial expression levels to normal wing development. Our studies of vestigial-overexpressing clones also reveal two further aspects of wing development. First, in the hinge region vestigial exerts both a local inhibition and a long-range induction of wingless expression. These and other observations imply that vestigial-expressing cells in the wing blade organize the development of surrounding wing-hinge cells. Second, clones of cells overexpressing vestigial exhibit altered cell affinities. Our analysis of these clones, together with studies of scalloped mutant clones, implies that scalloped- and vestigial-dependent cell adhesion contributes to separation of the wing blade from the wing hinge and to a gradient of cell affinities along the dorsal-ventral axis of the wing.     

The Drosophila position-specific antigens are a family of cell surface glycoprotein complexes

Position-specific (PS)1 and PS2 monoclonal antibodies bind non-uniformly to the mature wing imaginal disc of Drosophila with respect to the boundary separating the dorsal and ventral developmental compartments. PS1 antibodies preferentially recognize dorsal cells, PS2 antibodies ventral cells. Antibodies of the two classes extract distinct sets of glycoproteins from an imaginal disc lysate. PS3 antibodies bind to both dorsal and ventral disc cells and extract both PS1 and PS2 glycoprotein sets together with an additional component. We show that the PS antigens are related multimeric glycoprotein complexes on the cell surface. PS3 antibodies recognize a glycoprotein present in all complexes, while PS1 and PS2 antibodies recognize unique components of their own complexes. Spatial and temporal correlations suggest the molecules may have a function in development.     

Multiple roles of the Dcdc42 GTPase during wing development in Drosophila melanogaster

The Rho sub-family of GTPases, comprising Rho, Rac and Cdc42. regulates many biological processes, including morphogenesis, cell polarity, migration, the cell cycle and gene expression. It is important to develop genetic approaches to allow the dissection, in vivo, of the mechanisms of GTPase regulation and signal transmission, and their biological consequences. In this regard, wing development in Drosophila melanogaster is an excellent model system. To investigate the functions of the Drosophila Cdc42 GTPase (Dcdc42), we generated phenotypes during wing development, by expression of the dominant-negative N17 and L89 mutants of Dcdc42. We have identified roles for Dcdc42 in wing growth, and in cell fate choice during the development of the wing veins and the peripheral nervous system. Reduction of Dcdc42 signalling following over-expression of Dcdc42N17 resulted in a broader but more diffuse domain characterised by wing-margin sensory bristles. This was correlated with a broadened stripe of wingless expression along the dorsal-ventral boundary of third-instar wing imaginal discs. Together with genetic interactions with loss- and gain-of-function Notch alleles, these data support a role for wild-type Dcdc42 as a negative regulator of Notch signalling.     

Role of Jun N-terminal Kinase (JNK) signaling in the wound healing and regeneration of a Drosophila melanogaster wing imaginal disc

When a fragment of a Drosophila imaginal disc is cultured in growth permissive conditions, it either regenerates the missing structures or duplicates the pattern present in the fragment. This kind of pattern regulation is known to be epimorphic, i.e. the new pattern is generated by proliferation in a specialized tissue called the blastema. Pattern regulation is accompanied by the healing of the cut surfaces restoring the continuous epithelia. Wound healing has been considered to be the inductive signal to commence regenerative cell divisions. Although the general outlines of the proliferation dynamics in a regenerating imaginal disc blastema have been well studied, little is known about the mechanisms driving cells into the regenerative cell cycles. In this study, we have investigated the role of Jun N-terminal Kinase (JNK) signaling in the wound healing and regeneration of a Drosophila wing imaginal disc. By utilizing in vivo and in vitro culturing of incised and fragmented discs, we have been able to visualize the dynamics in cellular architecture and gene expression involved in the healing and regeneration process. Our results directly show that homotypic wound healing is not a prerequisite for regenerative cell divisions. We also show that JNK signaling participates in imaginal disc wound healing and is regulated by the physical dynamics of the process, as well as in recruiting cells into the regenerative cell cycles. A model describing the determination of blastema size is discussed.     

The LRR proteins capricious and Tartan mediate cell interactions during DV boundary formation in the Drosophila wing

Mechanisms to segregate cell populations play important roles in tissue patterning during animal development. Rhombomeres and compartments in the ectoderm and imaginal discs of Drosophila are examples in which initially homogenous populations of cells come to be separated by boundaries of lineage restriction. Boundary formation depends in part on signaling between the distinctly specified cell populations that comprise compartments and in part on formation of affinity boundaries that prevent intermingling of these cell populations. Here, we present evidence that two transmembrane proteins with leucine-rich repeats, known as Capricious and Tartan, contribute to formation of the affinity boundary between dorsal and ventral compartments during Drosophila wing development.     

GINsim: a software suite for the qualitative modelling, simulation and analysis of regulatory networks

This paper presents GINsim, a Java software suite devoted to the qualitative modelling, analysis and simulation of genetic regulatory networks. Formally, our approach leans on discrete mathematical and graph-theoretical concepts. GINsim encompasses an intuitive graph editor, enabling the definition and the parameterisation of a regulatory graph, as well as a simulation engine to compute the corresponding qualitative dynamical behaviour. Our computational approach is illustrated by a preliminary model analysis of the inter-cellular regulatory network activating Notch at the dorsal-ventral boundary in the wing imaginal disc of Drosophila. We focus on the cross-regulations between five genes (within and between two cells), which implements the dorsal-ventral border in the developing imaginal disc. Our simulations qualitatively reproduce the wild-type developmental pathway, as well as the outcome of various types of experimental perturbations, such as loss-of-function mutations or ectopically induced gene expression.     

Dynamics of decapentaplegic expression during regeneration of the Drosophila melanogaster wing imaginal disc

Regeneration of an imaginal disc involves highly ordered proliferation and pattern regulation of the newly formed tissue. Although the general principles of imaginal disc regeneration have been extensively studied, knowledge of the underlying molecular mechanisms is far from complete. Results from other model organisms suggest that regeneration is the result of local recapitulation of the normal patterning genes. To analyze the dynamics of one major Drosophila patterning gene, decapentaplegic (dpp), in wing imaginal disc regeneration, a vital GFP reporter together with iontophoretic cell labeling were used. Our observations reveal that the restoration of compartment-border-specific dpp expression is a common event in imaginal disc regeneration. However, we did not find evidence of an upregulation of dpp expression during the regeneration process.     

Refinement of wingless expression by a wingless- and notch-responsive homeodomain protein,defective proventriculus

Pattern formation during animal development is often induced by extracellular signaling molecules, known as morphogens, which are secreted from localized sources. During wing development in Drosophila, Wingless (Wg) is activated by Notch signaling along the dorsal-ventral boundary of the wing imaginal disc and acts as a morphogen to organize gene expression and cell growth. Expression of wg is restricted to a narrow stripe by Wg itself, repressing its own expression in adjacent cells. This refinement of wg expression is essential for specification of the wing margin. Here, we show that a homeodomain protein, Defective proventriculus (Dve), mediates the refinement of wg expression in both the wing disc and embryonic proventriculus, where dve expression requires Wg signaling. Our results provide evidence for a feedback mechanism that establishes the wg-expressing domain through the action of a Wg-induced gene product.     

In-Vivo Imaging of the Drosophila Wing Imaginal Disc over Time: Novel Insights on Growth and Boundary Formation

In developmental biology, the sequence of gene induction and pattern formation is best studied over time as an organism develops. However, in the model system of Drosophila larvae this oftentimes proves difficult due to limitations in imaging capabilities. Using the larval wing imaginal disc, we show that both overall growth, as well as the creation of patterns such as the distinction between the anterior(A) and posterior(P) compartments and the dorsal(D) and ventral(V) compartments can be studied directly by imaging the wing disc as it develops inside a larva. Imaged larvae develop normally, as can be seen by the overall growth curve of the wing disc. Yet, the fact that we can follow the development of individual discs through time provides the opportunity to simultaneously assess individual variability. We for instance find that growth rates can vary greatly over time. In addition, we observe that mechanical forces act on the wing disc within the larva at times when there is an increase in growth rates. Moreover, we observe that A/P boundary formation follows the established sequence and a smooth boundary is present from the first larval instar on. The division of the wing disc into a dorsal and a ventral compartment, on the other hand, develops quite differently. Contrary to expectation, the specification of the dorsal compartment starts with only one or two cells in the second larval instar and a smooth boundary is not formed until the third larval instar.     

Cell proliferation and DNA replication in the imaginal wing disc of Drosophila melanogaster

Cell proliferation in the imaginal wing disc of Drosophila has been analyzed by both pulse and chronic labeling with [3H]thymidine. We find neither spatial nor temporal variation in the fraction of S phase cells during the third instar. At or near the time of white prepupae formation the fraction of S phase cells falls sharply. Our chronic labeling experiments have demonstrated that almost all (and perhaps all) of the cells in a mid third instar wing disc are cycling. By examining sectioned material from such experiments we have found that the collumnar epithelial cell and the adepithetial cell populations become labeled with similar kinetics. The peripodial membrane cell population becomes labeled more slowly. We have also obtained estimates of cell cycle parameters for the imaginal wing disc cells.     

The missing link: implementation of morphogenetic growth control on the cellular and molecular level

In the wing imaginal disc of Drosophila melanogaster, the morphogen Dpp controls growth, probably in an instructive manner. Many models for growth control by Dpp have been proposed and have been extensively discussed elsewhere. In this review, we speculate on how instructive growth control could provide a link between Dpp signaling and cell growth and/or cell cycle progression and so implement morphogenetic growth control on the cellular and molecular levels.     

Transdetermination in the homeotic eye--antenna imaginal disc of Drosophila melanogaster

The effects of homeotic mutations on transdetermination in eye-antenna imaginal discs of Drosophila melanogaster were studied. After 12 days of culture in vivo, antenna discs transformed to ventral mesothorax by AntpNs or AntpZ, transdetermined to notum and wing structures four to five times more frequently than the corresponding wild-type antenna discs. Likewise, eye discs transformed to dorsal mesothorax by eyopt transdetermined to leg structures, also extremely frequently (90%). It seems that, during culture, homeotic antenna as well as homeotic eye discs tend to complete the structural inventory of the mesothoracic segment. Transdetermination in the homeotic disc parts is interpreted as a regeneration process which reestablishes an entire segment, i.e., the ventral mesothoracic portion (leg) in the antenna disc regenerates dorsal mesothoracic parts, and the dorsal mesothoracic portion in the eye disc (wing) regenerates ventral mesothoracic parts, respectively. This implies that antenna and leg discs (ventral qualities) as well as eye and wing discs (dorsal qualities) are serially homologous. The transdetermination frequency of the untransformed eye disc to notum and wing structures is enhanced by Antp to the same extent as is the transdetermination frequency of the antenna disc. The first allotypic wing disc structure formed by the eye disc is notum, followed by structures of the anterior wing compartment and finally by posterior wing structures. No evidence for such a sequence was found in the transdetermination pattern of the antenna disc.