Notch regulates wingless expression and is not required for reception of the paracrine wingless signal during wing margin neurogenesis in Drosophila.

In the developing wing margin of Drosophila, wingless is normally expressed in a narrow stripe of cells adjacent to the proneural cells that form the sensory bristles of the margin. Previous work has shown that this wingless is required for the expression of the proneural achaete-scute complex genes and the subsequent formation of the sensory bristles along the margin; recently, it has been proposed that the proneural cells require the Notch protein to properly receive the wingless signal. We have used clonal analysis of a null allele of Notch to test this idea directly. We found that Notch was not required by prospective proneural margin cells for the expression of scute or the formation of sensory precursors, indicating Notch is not required for the reception of wingless signal. Loss of Notch from proneural cells produced cell-autonomous neurogenic phenotypes and precocious differentiation of sensory cells, as would be expected if Notch had a role in lateral inhibition within the proneural regions. However, loss of scute expression and of sensory precursors was observed if clones substantially included the normal region of wingless expression. These 'anti-proneural' phenotypes were associated with the loss of wingless expression; this loss may be partially or wholly responsible for the anti-proneural phenotype. Curiously, Notch- clones limited to the dorsal or ventral compartments could disrupt wingless expression and proneural development in the adjacent compartment. Analysis using the temperature-sensitive Notch allele indicated that the role of Notch in the regulation of wingless expression precedes the requirement for lateral inhibition in proneural cells. Furthermore, overexpression of wingless with a heat shock-wingless construct rescued the loss of sensory precursors associated with the early loss of Notch.

Pathway specificity by the bifunctional receptor frizzled is determined by affinity for wingless.

The Frizzled (Fz) protein in Drosophila is a bifunctional receptor that acts through a GTPase pathway in planar polarity signaling and as a receptor for Wingless (Wg) using the canonical Wnt pathway. We found that the ligand-binding domain (CRD) of Fz has an approximately 10-fold lower affinity for Wg than the CRD of DFz2, a Wg receptor without polarity activity. When the Fz CRD is replaced by the high-affinity CRD of DFz2, the resulting chimeric protein gains Wg signaling activity, yet also retains polarity signaling activity. In contrast, the reciprocal exchange of the Fz CRD onto DFz2 is not sufficient to confer polarity activity to DFz2. This suggests that Fz has an intrinsic capacity for polarity signaling and that high-affinity interaction with Wg couples it to the Wnt pathway.

The function and regulation of cut expression on the wing margin of Drosophila: Notch, Wingless and a dominant negative role for Delta and Serrate.

We have investigated the role of the Notch and Wingless signaling pathways in the maintenance of wing margin identity through the study of cut, a homeobox-containing transcription factor and a late-arising margin-specific marker. By late third instar, a tripartite domain of gene expression can be identified about the dorsoventral compartment boundary, which marks the presumptive wing margin. A central domain of cut- and wingless-expressing cells are flanked on the dorsal and ventral side by domains of cells expressing elevated levels of the Notch ligands Delta and Serrate. We show first that cut acts to maintain margin wingless expression, providing a potential explanation of the cut mutant phenotype. Next, we examined the regulation of cut expression. Our results indicate that Notch, but not Wingless signaling, is autonomously required for cut expression. Rather, Wingless is required indirectly for cut expression; our results suggest this requirement is due to the regulation by wingless of Delta and Serrate expression in cells flanking the cut and wingless expression domains. Finally, we show that Delta and Serrate play a dual role in the regulation of cut and wingless expression. Normal, high levels of Delta and Serrate can trigger cut and wingless expression in adjacent cells lacking Delta and Serrate. However, high levels of Delta and Serrate also act in a dominant negative fashion, since cells expressing such levels cannot themselves express cut or wingless. We propose that the boundary of Notch ligand along the normal margin plays a similar role as part of a dynamic feedback loop that maintains the tripartite pattern of margin gene expression.

wingless refines its own expression domain on the Drosophila wing margin.

The imaginal discs of Drosophila, which give rise to the adult appendages, are patterned during a period of intense cell proliferation. The specification of differing regions occurs in some cases by subdividing the disc epithelium into lineage compartments. However, in most cases precise boundaries are formed between different cell types without early compartmentalization. One such boundary occurs between the wingless (wg)-expressing cells of the wing margin and the adjacent proneural cells, which give rise to margin sensory bristles. Here we show that this boundary arises in part by a mechanism of 'self-refinement', by which wingless protein (Wg) represses wg expression in adjacent cells. Cells unable to receive the Wg signal do not resolve the boundary between wg-expressing and proneural cells.

Wingless repression of Drosophila frizzled 2 expression shapes the Wingless morphogen gradient in the wing.

In Drosophila wing imaginal discs, the Wingless (Wg) protein acts as a morphogen, emanating from the dorsal/ventral (D/V) boundary of the disc to directly define cell identities along the D/V axis at short and long range. Here, we show that high levels of a Wg receptor, Drosophila frizzled 2 (Dfz2), stabilize Wg, allowing it to reach cells far from its site of synthesis. Wg signaling represses Dfz2 expression, creating a gradient of decreasing Wg stability moving toward the D/V boundary. This repression of Dfz2 is crucial for the normal shape of Wg morphogen gradient as well as the response of cells to the Wg signal. In contrast to other ligand-receptor relationships where the receptor limits diffusion of the ligand, Dfz2 broadens the range of Wg action by protecting it from degradation.