Andrew Tomlinson

arrow encodes an LDL-receptor-related protein essential for Wingless signalling.

The Wnt family of secreted molecules functions in cell-fate determination and morphogenesis during development in both vertebrates and invertebrates (reviewed in ref. 1). Drosophila Wingless is a founding member of this family, and many components of its signal transduction cascade have been identified, including the Frizzled class of receptor. But the mechanism by which the Wingless signal is received and transduced across the membrane is not completely understood. Here we describe a gene that is necessary for all Wingless signalling events in Drosophila. We show that arrow gene function is essential in cells receiving Wingless input and that it acts upstream of Dishevelled. arrow encodes a single-pass transmembrane protein, indicating that it may be part of a receptor complex with Frizzled class proteins. Arrow is a low-density lipoprotein (LDL)-receptor-related protein (LRP), strikingly homologous to murine and human LRP5 and LRP6. Thus, our data suggests a new and conserved function for this LRP subfamily in Wingless/Wnt signal reception.

A LIM-homeodomain combinatorial code for motor-neuron pathway selection

Different classes of vertebrate motor neuron that innervate distinct muscle targets express unique combinations of LIM-homeodomain transcription factors, suggesting that a combinatorial code of LIM-homeodomain proteins may underlie the control of motor-neuron pathway selection. Studies of LIM-homeodomain genes in mouse, Drosophila melanogaster and Caenorhabditis elegans have revealed functions of these genes in neuronal survival, axon guidance, neurotransmitter expression and neuronal function, but, to our knowledge, none of these studies have addressed the issue of a functional code. Here we study two members of this gene family in Drosophila, namely lim3, the homologue of the vertebrate Lhx3 and Lhx4 genes, and islet, thehomologue of the vertebrate Isl1 and Isl2 genes. We show that Drosophila lim3 is expressed by a specific subset of islet-expressing motor neurons and that mutating or misexpressing lim3 switches motor-neuron projections predictably. Our results provide evidence that lim3 and islet constitute a combinatorial code that generates distinct motor-neuron identities.

Trimeric G Protein-Dependent Frizzled Signaling in Drosophila

Frizzled (Fz) proteins are serpentine receptors that transduce critical cellular signals during development. Serpentine receptors usually signal to downstream effectors through an associated trimeric G protein complex. However, clear evidence for the role of trimeric G protein complexes for the Fz family of receptors has hitherto been lacking. Here, we show roles for the Galpha(o) subunit (Go) in mediating the two distinct pathways transduced by Fz receptors in Drosophila: the Wnt and planar polarity pathways. Go is required for transduction of both pathways, and epistasis experiments suggest that it is an immediate transducer of Fz. While overexpression effects of the wild-type form are receptor dependent, the activated form (Go-GTP) can signal when the receptor is removed. Thus, Go is likely part of a trimeric G protein complex that directly transduces Fz signals from the membrane to downstream components.

Transducing the Dpp Morphogen Gradient in the Wing of Drosophila: Regulation of Dpp Targets by brinker

Dpp, a TGFbeta, organizes pattern in the Drosophila wing by acting as a graded morphogen, activating different targets above distinct threshold concentrations. Like other TGFbetas, Dpp appears to induce transcription directly via activation of a SMAD, Mad. However, here we demonstrate that Dpp can also control gene expression indirectly by downregulating the expression of the brinker gene, which encodes a putative transcription factor that functions to repress Dpp targets. The medial-to-lateral Dpp gradient along the anterior-posterior axis is complemented by a lateral-to-medial gradient of Brinker, and the presence of these two opposing gradients may function to allow cells to detect small differences in Dpp concentration and respond by activating different target genes.

Axis specification in the developing Drosophila appendage: the role of wingless, decapentaplegic, and the homeobox gene aristaless.

The wingless (wg) and decapentaplegic (dpp) genes of Drosophila encode homologs of secreted growth factors and are required for the correct patterning of the appendages. We show that the presumptive tips of both the leg and wing, the distal extreme of the proximodistal axis, are characterized by the close association of cells expressing wg, dpp, and the homeobox gene aristaless (al). Ectopic expression of wg can induce both ectopic al expression and a duplication of the proximodistal axis (the development of supernumerary legs), but only in regions expressing high levels of dpp. Ectopic al expression can induce a duplication of the proximodistal axis in the wing, suggesting that it may be directly involved in axis specification. The proximodistal axis may be specified via a mechanism involving a direct interaction between cells expressing wg, dpp, and possibly al.

Naturally processed and presented epitopes of the islet cell autoantigen IA-2 eluted from HLA-DR4

During immune responses, antigen-presenting cells (APCs) process antigens and present peptide epitopes complexed with human leukocyte antigen (HLA) molecules. CD4 cells recognize these naturally processed and presented epitopes (NPPEs) bound to HLA class II molecules. Epitope identification is important for developing diagnostic and therapeutic tools for immune-mediated diseases and providing insight into their etiology, but current approaches overlook effects of natural processing on epitope selection. We have developed a technique to identify NPPEs using mass spectrometry (MS) after antigen is targeted onto APCs using a lectin-based antigen delivery system (ADS). We applied the technique to identify NPPEs of the intracellular domain of the type 1 diabetes mellitus-associated (type 1 DM-associated) autoantigen insulinoma-associated-2 (IA-2ic), presented by HLA-DR4 (0401). IA-2ic-derived NPPEs eluted from HLA-DR4 constitute 6 sets of peptides nested around distinct core regions. Synthetic peptides based on these regions bind HLA-DR4 and elicit primary T-cell proliferation frequently in HLA-DR4-positive type 1 DM patients, but rarely in non-HLA-DR4 patients, and in none of the HLA-DR4 nondiabetic controls we tested. This flexible, direct approach identifies an HLA allele-specific map of NPPEs for any antigen, presented by any HLA class II molecule. This method should enable a greater understanding of epitope selection and lead to the generation of sensitive and specific reagents for detecting autoreactive T cells.

Drosophila Smoothened phosphorylation sites essential for Hedgehog signal transduction

The Hedgehog (Hh) signalling pathway is crucial for animal development and is aberrantly activated in several types of cancer. In Drosophila melanogaster, Hh signalling regulates target gene expression through the transcription factor Cubitus interruptus (Ci). Together, Protein Kinase A, Casein Kinase 1 and Glycogen Synthase Kinase 3 silence the pathway in the absence of ligand by phosphorylating Ci at a defined cluster of sites, thereby promoting its proteolytic conversion to a transcriptional repressor (Ci-75). In the presence of Hh, Ci-155 is no longer converted to Ci-75 and its ability to activate transcription is potentiated. All Hh responses require the seven transmembrane domain protein Smoothened, which itself becomes hyperphosphorylated during Hh signalling. Here we show that a cluster of protein kinase A and protein kinase A-primed casein kinase 1 phosphorylation sites in Smoothened, similarly distributed to those regulating Ci, are essential for Smoothened to transduce a Hh signal and for normal regulation of Smoothened protein levels.

The ALS-associated proteins FUS and TDP-43 function together to affect Drosophila locomotion and life span

The fatal adult motor neuron disease amyotrophic lateral sclerosis (ALS) shares some clinical and pathological overlap with frontotemporal dementia (FTD), an early-onset neurodegenerative disorder. The RNA/DNA-binding proteins fused in sarcoma (FUS; also known as TLS) and TAR DNA binding protein-43 (TDP-43) have recently been shown to be genetically and pathologically associated with familial forms of ALS and FTD. It is currently unknown whether perturbation of these proteins results in disease through mechanisms that are independent of normal protein function or via the pathophysiological disruption of molecular processes in which they are both critical. Here, we report that Drosophila mutants in which the homolog of FUS is disrupted exhibit decreased adult viability, diminished locomotor speed, and reduced life span compared with controls. These phenotypes were fully rescued by wild-type human FUS, but not ALS-associated mutant FUS proteins. A mutant of the Drosophila homolog of TDP-43 had similar, but more severe, deficits. Through cross-rescue analysis, we demonstrated that FUS acted together with and downstream of TDP-43 in a common genetic pathway in neurons. Furthermore, we found that these proteins associated with each other in an RNA-dependent complex. Our results establish that FUS and TDP-43 function together in vivo and suggest that molecular pathways requiring the combined activities of both of these proteins may be disrupted in ALS and FTD.

Delta/Notch and Boss/Sevenless Signals Act Combinatorially to Specify the Drosophila R7 Photoreceptor

The R7 photoreceptor, a unique cell type within the Drosophila ommatidium, was initially proposed to be specified by two distinct signals from neighboring cells, one from the R8 photoreceptor and another from the R1/6 photoreceptor pair. The R8-to-R7 signal is the transmembrane ligand Bride of Sevenless (Boss), which is received by the receptor tyrosine kinase Sevenless (Sev) and transduced via Ras activation within the presumptive R7 cell. However, the identity of the R1/6-to-R7 signal has remained elusive. Here, we present evidence that the transmembrane ligand Delta (Dl), expressed by the R1/6 pair, activates the receptor Notch (N) in the presumptive R7 cell and constitutes the postulated R1/6-to-R7 signal required in combination with the Boss/Sev signal to specify the R7 fate.

Patterning the peripheral retina of the fly: decoding a gradient.

The peripheral regions of the fly eye show a number of specializations. First, immediately interior to the circumscribing head capsule and completely encircling the rest of the eye lies a thick band of pigment cells (pigment rim; PR). Second, in the dorsal periphery of the eye directly interior to the PR lie the dorsal rim (DR) ommatidia that are specialized polarized light detectors. The equivalent position in the ventral eye is occupied by standard ommatidia. Third, ommatidia characteristically project mechanosensory hairs above their lenses, but in the most peripheral rows (including the DR) the ommatidia are bald. Wingless secreted from the head capsule appears to organize all these peripheral specializations. Higher Wg levels induce PR, intermediate levels induce DR, and lower levels induce baldness. The predisposition of dorsal cells to generate DR ommatidia appears to be endowed by the exclusive dorsal expression of Iroquois genes.