WNT control of patterning and cell lineage in limb development

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We propose to study the mechanism of action of Wnt signalling in pattern formation and cell lineage in the Drosophila melanogaster leg. The expression of the fly Wnt-1 homolog wingless (wg) is localized to the ventral most cells of the leg. Wg acts as a tissue organizer coordinating the development of the ventral quadrant of the leg. Wg is both absolutely necessary and completely sufficient to induce ventral patterns of development. Two other Wnts, Dwnt4 and Dwnt6 are co-expressed with wg in the ventral leg. We propose a series of studies with the goal of resolving and clarifying 1) the relative contribution of the three ventral Wnt genes, 2) the range of action and cellular mechanism employed by Wnts, and 3) the influence of Wnt on ventral cell-lineage mechanisms.******In Objective 1 we address a current controversy about whether Wg acts as a morphogen in a long-range direct manner or whether it signals only locally. The controversy arises because in some experiments a membrane-tethered version of Wg (Nrt-wg) is able to rescue Wg function in wing patterning but in other cases it cannot. The difficulties in resolving this conflict stems from the complex expression of Wg and the limited availability of downstream target genes and morphological markers for studying the readout of Wg singling in the wing. We argue that the fly leg offers a better system for asking how Wg signals because the expression is consistently restricted to the organizer region and because of better morphological markers and associated target genes. We will first address whether the other two Wnt molecules compensate for loss of wg. This would provide an explanation for some of the contradictory results described above. We will examine systematically how modulating Wg signaling affects both the gene expression patterns and morphology of the ventral leg and whether loss-or gain-of-function of Wg signaling indicates that the genes are activated in a long-range manner predicted for a morphogen.******In Objective 2 we study the role of Wg and Dpp, a fly BMP, in a novel lineage boundary we have identified between Wg-dependent ventral cells and Dpp-dependent dorsal cells. Lineage tracing shows that clones of ventral cells are not recovered in the dorsal domain and vice-versa. We will use lineage tracing of cells compromised for Wg and/or Dpp signaling or downstream target genes to determine which pathway is responsible for the lineage boundary. By observing behaviors of labeled clones and by manipulating known and candidate genes downstream of Wg and Dpp, we will test whether the lineage restriction is controlled by cell competition and cell death, cell adhesion or mechanical tension.