@9cIntroduction@21T issues exhibit an impressive ability to respond to a myriad of insults by repairing and regenerating complex structures. The elegant and orderly process of regeneration provides clues to the mechanisms of pattern formation but also offers the hope that the process might one day be manipulated to replace damaged body parts. To manipulate the process, it will be necessary to understand the genetic basis of the process. In the case of the insect leg, we are coming close to such a level of understanding and many of the lessons learned are relevant to vertebrate systems. A dynamic web of gene regulatory networks appears to create a robust self-organizing system that is at once extremely intricate but also perhaps simple in its reliance on a few key signaling pathways and a few simple processes, e.g. autoactivation and lateral inhibition. Here we will summarize what has been learned about the networks of gene regulation present in the Drosophila leg discs and then we will explore how the regenerative responses to different insults can be understood as predictable responses to these networks. Each of the regulatory networks could themselves serve as the subject of a detailed review and that is beyond the scope of this discussion. Here we will focus on the interplay between the regulatory networks in patterning the tissue.

The finding that Wingless (WG) and Decapentaplegic (DPP) suppress each others transcription provides a mechanism for creating developmental territories in fields of cells. Here, we address the mechanism of that antagonism. The dishevelled (dsh) and shaggy (sgg) genes encode intracellular proteins generally thought of as downstream of WG signaling. We have investigated the effects of changing either DSH or SGG activity on both cell fate and wg and dpp expression. At the level of cell fate in discs, DSH antagonizes SGG activity. At the level of gene expression, SGG positively regulates dpp expression and negatively regulates wg expression while DSH activity suppresses dpp expression and promotes wg expression. Sharp borders of gene expression correlating precisely with clone boundaries suggest that the effects of DSH and SGG on transcription of wg and dpp are not mediated by secreted factors but rather act through intracellular effectors. The interactions described here suggest a model for the antagonism between WG and DPP that is mediated via SGG. The model incorporates autoactivation and lateral inhibition, which are properties required for the production of stable patterns. The regulatory interactions described exhibit extensive ability to organize new pattern in response to manipulation or injury.

The dishevelled gene of Drosophila is required to establish coherent arrays of polarized cells and is also required to establish segments in the embryo. Here, we show that loss of dishevelled function in clones, in double heterozygotes with wingless mutants and in flies bearing a weak dishevelled transgene leads to patterning defects which phenocopy defects observed in wingless mutants alone. Further, polarized cells in all body segments require dishevelled function to establish planar cell polarity, and some wingless alleles and dishevelled; wingless double heterozygotes exhibit bristle polarity defects identical to those seen in dishevelled alone. The requirement for dishevelled in establishing polarity in cell autonomous. The dishevelled gene encodes a novel intracellular protein that shares an amino acid motif with several other proteins that are found associated with cell junctions. Clonal analysis of dishevelled in leg discs provides a unique opportunity to test the hypothesis that the wingless dishevelled interaction species at least one of the circumferential positional values predicted by the polar coordinate model. We propose that dishevelled encodes an intracellular protein required to respond to a wingless signal and that this interaction is essential for establishing both cell polarity and cell identity.

Drosophila appendages develop from imaginal discs which become subdivided into distinct regions during normal patterning. At least 3 axes of asymmetry are required to produce a chiral appendage such as a leg. The A/P compartments provide one axis of asymmetry in all discs. In leg and antennal discs, the anterior compartment becomes asymmetric in the D/V axis with decapentaplegic (dpp) expression defining dorsal anterior leg, and wingless (wg) expression defining ventral anterior leg. However, unlike wing discs, no D/V compartment has been demonstrated in legs or antennae. How are the dorsal anterior and ventral anterior territories defined and maintained? Here we show that wg inhibits dpp expression and dpp inhibits wg expression in leg and eye/antennal discs. This mutual repression provides a mechanism for maintaining separate regions of wg and dpp expression in a developing field. We propose the term 'territory' to describe regions of cells that are under the domineering influence of a particular morphogen. Territories differ from compartments in that they are not defined by lineage but are dynamically maintained by continuous morphogen signaling. We propose that the anterior compartment of the leg disc is divided into dorsal and ventral territories by the mutual antagonism between WG and DPP signaling.

In this paper, we consider a mathematical model for the formation of spatial morphogen territories of two key morphogens: Wingless (Wg) and Decapentaplegic (DPP), involved in leg development of Drosophila. We define a gene regulatory network (GRN) that utilizes autoactivation and cros-sinhibition (modeled by Hill equations) to establish and maintain stable boundaries of gene expression. By computational analysis we find that in the presence of a general activator, neither autoactivation, nor cross-inhibition alone are sufficient to maintain stable sharp boundaries of morphogen production in the leg disc. The minimal requirements for a self-organizing system are a coupled system of two morphogens in which the autoactivation and cross-inhibition have Hill coefficients strictly greater than one. In addition, the GRN modeled here describes the regenerative responses to genetic manipulations of positional identity in the leg disc.

Background

Spatially restricted morphogen expression drives many patterning and regeneration processes, but how is the pattern of morphogen expression established and maintained? Patterning of Drosophila leg imaginal discs requires expression of the DPP morphogen dorsally and the wingless (WG) morphogen ventrally. We have shown that these mutually exclusive patterns of expression are controlled by a self-organizing system of feedback loops that involve WG and DPP, but whether the feedback is direct or indirect is not known.

Methods/findings

By analyzing expression patterns of regulatory DNA driving reporter genes in different genetic backgrounds, we identify a key component of this system by showing that WG directly represses transcription of the dpp gene in the ventral leg disc. Repression of dpp requires a tri-partite complex of the WG mediators armadillo (ARM) and dTCF, and the co-repressor Brinker, (BRK), wherein ARM.dTCF and BRK bind to independent sites within the dpp locus.

Conclusions/significance

Many examples of dTCF repression in the absence of WNT signaling have been described, but few examples of signal-driven repression requiring both ARM and dTCF binding have been reported. Thus, our findings represent a new mode of WG mediated repression and demonstrate that direct regulation between morphogen signaling pathways can contribute to a robust self-organizing system capable of dynamically maintaining territories of morphogen expression.