Gary Struhl

Regulation of the Hedgehog and Wingless signalling pathways by the F- box/WD40-repeat protein Slimb

Members of the Hedgehog (Hh) and Wnt/Wingless (Wg) families of secreted proteins control many aspects of growth and patterning during animal development,. Hh signal transduction leads to increased stability of a transcription factor, Cubitus interruptus (Ci),, whereas Wg signal transduction causes increased stability of Armadillo (Arm/β-catenin), a possible co-factor for thetranscriptional regulator Lef1/TCF. Here we describe a new gene, slimb (for supernumerary limbs), which negatively regulates both of these signal transduction pathways. Loss of function of slimb results in a cell-autonomous accumulation of high levels of both Ci and Arm, and the ectopic expression of both Hh− and Wg− responsive genes. The slimb gene encodes a conserved F-box/WD40-repeat protein related to Cdc4p, a protein in budding yeast that targets cell-cycle regulators for degradation by the ubiquitin/proteasome pathway. We propose that Slimb protein normally targets Ci and Arm for processing or degradation by the ubiquitin/proteasomepathway, and that Hh and Wg regulate gene expression at least in part by inducing changes in Ci and Arm, which protect them from Slimb-mediated proteolysis.

DIRECT AND LONG-RANGE ACTION OF A DPP MORPHOGEN GRADIENT

During development of the Drosophila wing, the decapentaplegic (dpp) gene is expressed in a stripe of cells along the anteroposterior compartment boundary and gives rise to a secreted protein that exerts a long-range organizing influence on both compartments. Using clones of cells that express DPP, or in which DPP receptor activity has been constitutively activated or abolished, we show that DPP acts directly and at long range on responding cells, rather than by proxy through the short-range induction of other signaling molecules. Further, we show that two genes, optomotor-blind and spalt are transcriptionally activated at different distances from DPP-secreting cells and provide evidence that these genes respond to different threshold concentrations of DPP protein. We propose that DPP acts as a gradient morphogen during wing development.

Dual Roles for Patched in Sequestering and Transducing Hedgehog

Secreted proteins of the Hedgehog (Hh) family have diverse organizing roles in animal development. Recently, a serpentine protein Smoothened (Smo) has been proposed as a Hh receptor. Here, we present evidence that implicates another multiple-pass transmembrane protein, Patched (Ptc), in Hh reception and suggests a novel signal transduction mechanism in which Hh binds to Ptc, or a Ptc-Smo complex, and thereby induces Smo activity. Our results also show that Ptc limits the range of Hh action; we provide evidence that high levels of Ptc induced by Hh serve to sequester any free Hh and therefore create a barrier to its further movement.

Organizing activity of wingless protein in Drosophila

The adult appendages of Drosophila are formed from imaginal discs, sheets of epithelial cells that proliferate during larval development and differentiate during metamorphosis. wingless (wg, DWnt-1) protein, a putative signaling molecule, is expressed only in prospective ventral cells in each of the leg discs. To test the role of wg, we have generated randomly positioned clones of cells that express wg protein constitutively. Clones that arise in the prospective ventral portions of the leg discs develop normally. In contrast, dorsally situated clones give rise to ventrolateral patterns and exert a ventralizing influence on neighboring wild-type tissue. We propose that wg protein organizes leg pattern along the dorsoventral axis by conferring ventral positional information within the disc.

Presenilin is required for activity and nuclear access of Notch in Drosophila

Presenilins are membrane proteins with multiple transmembrane domains that are thought to contribute to the development of Alzheimers disease by affecting the processing of β-amyloid precursor protein. Presenilins also facilitate the activity of transmembrane receptors of the LIN-12/Notch family. After ligand-induced processing, the intracellular domain of LIN-12/Notch can enter the nucleus and participate in the transcriptional control of downstream target genes. Here we show that null mutations in the Drosophila Presenilin gene abolish Notch signal transduction and prevent its intracellular domain from entering the nucleus. Furthermore, we provide evidence that presenilin is required for the proteolytic release of the intracellular domain from the membrane following activation of Notch by ligand.

Nuclear Access and Action of Notch In Vivo

The Drosophila Notch (N) gene encodes a conserved single-pass transmembrane receptor that transduces extracellular signals controlling cell fate. Here, we present evidence that the intracellular domain of Notch gains access to the nucleus in response to ligand, possibly through a mechanism involving proteolytic cleavage and release from the remainder of the protein. In addition, our results suggest that signal transduction by Notch depends on the ability of the intracellular domain, particularly the portion containing the CDC10 repeats, to reach the nucleus and to participate in the transcriptional activation of downstream target genes.

Direct and Long-Range Action of a Wingless Morphogen Gradient

Wingless (Wg), a founding member of the Wingless/Int-1 (Wnt) family of secreted proteins, acts as a short-range inducer and as a long-range organizer during Drosophila development. Here, we determine the consequences of ectopically expressing (i) a wild-type form of Wg, (ii) a membrane-tethered form of Wg, and (iii) a constitutively active form of the cytosolic protein Armadillo (Arm), which normally acts to transduce Wg, and we compare them with the effects of removing endogenous Wg or Arm activity. Our results indicate that wild-type Wg acts at long range, up-regulating the transcription of particular target genes as a function of concentration and distance from secreting cells. In contrast, tethered Wg and Arm have only short-range or autonomous effects, respectively, on the transcription of these genes. We interpret these findings as evidence that Wg can act directly and at long range as a gradient morphogen during normal development.

The gradient morphogen bicoid is a concentration-dependent transcriptional activator.

The bicoid (bcd) protein is expressed in an anteroposterior gradient in early Drosophila embryos and controls the zygotic activation of the segmentation gene hunchback (hb) in a broad but precisely bounded anterior domain. Here we show that the hb gene contains multiple regulatory elements that mediate transcriptional activation in response to bcd protein. Further, we demonstrate that the resulting patterns of expression in vivo depend critically on both the bcd gradient profile and the number and quality of these hb elements. Finally, we show that these same elements mediate bcd-dependent transcriptional activation in yeast and that this interaction requires distinct DNA binding and activating regions in the bcd protein. Our results argue that bcd protein normally binds and activates the hb gene in a concentration-dependent fashion, thereby allowing the gradient of bcd protein to dictate where the hb gene is initially turned on in early embryos. They also suggest that the bcd gradient has the instructive capacity to activate other subordinate control genes by the same mechanism, each in a distinct spatial domain according to its affinity for bcd protein.

Morphogens, Compartments, and Pattern: Lessons from Drosophila?

We thank Javier Sampedro for planning Figure 4Figure 4 and Dan Barbash, Jose Casal, Jim Smith, and Jean-Paul Vincent for help with the manuscript. The advice and support of Hugh Pelham and Jim Smith is much appreciated.

Intrinsic activity of the lin-12 and Notch intracellular domains in vivo

The lin-12 gene of C. elegans and the Notch gene of D. melanogaster encode structurally related transmembrane proteins that mediate intercellular signaling. We show that truncated forms of these proteins consisting of only the intracellular domains cause cell fate transformations associated with constitutive activity in their respective organisms. This activity does not depend on endogenous gene function. Our results indicate that the intracellular domains of Lin-12 and Notch have intrinsic activity and that the principal role of the extracellular domains in the intact proteins is to regulate this activity. Our results also suggest that equivalent truncated forms of lin-12/Notch family members in vertebrates, including known oncogenes, are similarly active.