Merlin, the protein product of the Neurofibromatosis type-2 gene, acts as a tumour suppressor in mice and humans. Merlin is an adaptor protein with a FERM domain and it is thought to transduce a growth-regulatory signal. However, the pathway through which Merlin acts as a tumour suppressor is poorly understood. Merlin, and its function as a negative regulator of growth, is conserved in Drosophila, where it functions with Expanded, a related FERM domain protein. Here, we show that Drosophila Merlin and Expanded are components of the Hippo signalling pathway, an emerging tumour-suppressor pathway. We find that Merlin and Expanded, similar to other components of the Hippo pathway, are required for proliferation arrest and apoptosis in developing imaginal discs. Our genetic and biochemical data place Merlin and Expanded upstream of Hippo and identify a pathway through which they act as tumour-suppressor genes.

Notch signaling is broadly used to regulate cell-fate decisions. We have identified a gene, rumi, with a temperature-sensitive Notch phenotype. At 28 degrees C-30 degrees C, rumi clones exhibit a full-blown loss of Notch signaling in all tissues tested. However, at 18 degrees C only a mild Notch phenotype is evident. In vivo analyses reveal that the target of Rumi is the extracellular domain of Notch. Notch accumulates intracellularly and at the cell membrane of rumi cells but fails to be properly cleaved, despite normal binding to Delta. Rumi is an endoplasmic reticulum-retained protein with a highly conserved CAP10 domain. Our studies show that Rumi is a protein O-glucosyltransferase, capable of adding glucose to serine residues in Notch EGF repeats with the consensus C1-X-S-X-P-C2 sequence. These data indicate that by O-glucosylating Notch in the ER, Rumi regulates its folding and/or trafficking and allows signaling at the cell membrane.

Spinocerebellar ataxia type 1 (SCA1) is a neurodegenerative disease caused by an expanded glutamine tract in human Ataxin-1 (hAtx-1). The expansion stabilizes hAtx-1, leading to its accumulation. To understand how stabilized hAtx-1 induces selective neuronal degeneration, we studied Drosophila Atx-1 (dAtx-1), which has a conserved AXH domain but lacks a polyglutamine tract. Overexpression of hAtx-1 in fruit flies produces phenotypes similar to those of dAtx-1 but different from the polyglutamine peptide alone. We show that the Drosophila and mammalian transcription factors Senseless/Gfi-1 interact with Atx-1's AXH domain. In flies, overexpression of Atx-1 inhibits sensory-organ development by decreasing Senseless protein. Similarly, overexpression of wild-type and glutamine-expanded hAtx-1 reduces Gfi-1 levels in Purkinje cells. Deletion of the AXH domain abolishes the effects of glutamine-expanded hAtx-1 on Senseless/Gfi-1. Interestingly, loss of Gfi-1 mimics SCA1 phenotypes in Purkinje cells. These results indicate that the Atx-1/Gfi-1 interaction contributes to the selective Purkinje cell degeneration in SCA1.

During sensory organ precursor (SOP) specification, a single cell is selected from a proneural cluster of cells. Here, we present evidence that Senseless (Sens), a zinc-finger transcription factor, plays an important role in this process. We show that Sens is directly activated by proneural proteins in the presumptive SOPs and a few cells surrounding the SOP in most tissues. In the cells that express low levels of Sens, it acts in a DNA-binding-dependent manner to repress transcription of proneural genes. In the presumptive SOPs that express high levels of Sens, it acts as a transcriptional activator and synergizes with proneural proteins. We therefore propose that Sens acts as a binary switch that is fundamental to SOP selection.[Keywords: Neuronal specification; proneural genes; Enhancer of split; Drosophila; peripheral nervous system development] Sensory organs serve as transducers that convert various physical stimuli into electrical signals. In Drosophila, a variety of internal and external sensory organs cover the body of larvae and adults (Hartenstein 1988;Hartenstein and Posakony 1989;Jan and Jan 1993). The early phase of sensory organ development is usually marked by lowlevel expression of one or more members of a group of basic helix-loop-helix (bHLH) genes, the proneural genes. These genes are expressed in a cluster of cells, the proneural cluster, and provide these cells with the competence to become neuronal precursors (Villares and Cabrera 1987;Cubas et al. 1991;Skeath and Carroll 1991;Jarman et al. 1993Jarman et al. , 1994. The proneural competence is further refined to a smaller group of cells in each cluster, the proneural field, which accumulates relatively higher levels of proneural proteins (Cubas et al. 1991;Skeath and Carroll 1991). Ultimately, one cell of the proneural field will exhibit a yet higher level of proneural protein expression and become the SOP, whereas other cells will adopt an epidermal fate. The accumulation of large amounts of proneural proteins is thought to depend on the proneural gene activity via specific enhancers of proneural genes that show auto-and cross-regulatory characteristics (Van Doren et al. 1992;Modolell 1997;Culi and Modolell 1998).Although all of the cells in a proneural field have the potential to become an SOP, only one (or a few) cell(s) will realize their potential. This is achieved by inhibitory cell-cell interactions mediated by members of the Notch (N) signaling pathway (Artavanis-Tsakonas et al. 1999). Binding of the ligand Delta to the N receptor of the neighboring cells initiates a series of proteolytic cleavages that ultimately release the intracellular domain of N (N icd ), which translocates into the nucleus. Together with another protein called Suppressor of Hairless [Su(H)], N icd activates the transcription of the primary targets of N signaling, the genes of the Enhancer of split complex (E(spl)-C) (Delidakis and Artavanis-Tsakonas 1992; Knust et al. 1992;Schrons et al. 1992;Bailey and Posakony 1995;Lecourtois and Schweisguth 1995). Members...

Asymmetric division of sensory organ precursors (SOPs) in Drosophila generates different cell types of the mature sensory organ. In a genetic screen designed to identify novel players in this process, we have isolated a mutation in Drosophila sec15, which encodes a component of the exocyst, an evolutionarily conserved complex implicated in intracellular vesicle transport. sec15(-) sensory organs contain extra neurons at the expense of support cells, a phenotype consistent with loss of Notch signaling. A vesicular compartment containing Notch, Sanpodo, and endocytosed Delta accumulates in basal areas of mutant SOPs. Based on the dynamic traffic of Sec15, its colocalization with the recycling endosomal marker Rab11, and the aberrant distribution of Rab11 in sec15 clones, we propose that a defect in Delta recycling causes cell fate transformation in sec15(-) sensory lineages. Our data indicate that Sec15 mediates a specific vesicle trafficking event to ensure proper neuronal fate specification in Drosophila.

SUMMARYProtein O-glucosylation is a conserved post-translational modification that occurs on epidermal growth factor-like (EGF) repeats harboring the C 1 -X-S-X-P-C 2 consensus sequence.

Altered regulation of 5-HT1A receptors is implicated in mood disorders such as anxiety and major depression. To provide insight into its transcriptional regulation, we previously identified a novel DNA element [14 bp 5'-repressor element (FRE)] of the 5-HT1A receptor gene that mediates repression in neuronal and non-neuronal cells (Ou et al., 2000). We have now cloned a novel DNA binding protein [five' repressor element under dual repression binding protein-1 (Freud-1)] that binds to FRE to mediate repression of the 5-HT1A receptor or heterologous promoters. Freud-1 is evolutionarily conserved and contains two DM-14 basic repeats, a predicted helix-loop-helix DNA binding domain, and a protein kinase C conserved region 2 (C2)/calcium-dependent lipid binding (CalB) calcium/phospholipid binding domain. An intact CalB domain was required for Freud-1-mediated repression. In serotonergic raphe cells, overexpression of Freud-1 repressed the 5-HT1A promoter and decreased 5-HT1A receptor protein levels, whereas transfection of antisense to Freud-1 derepressed the 5-HT1A gene and increased 5-HT1A receptor protein expression. Calcium-dependent signaling blocked Freud-1-FRE binding and derepressed the 5-HT1A promoter. Treatment with inhibitors of calmodulin or CAM-dependent protein kinase reversed calcium-mediated inhibition of Freud-1. Freud-1 RNA and protein were present in raphe nuclei, hippocampus, cortex, and hypothalamus, and Freud-1 protein was colocalized with 5-HT1A receptors, suggesting its importance in regulating 5-HT1A receptors in vivo. Thus, Freud-1 represents a novel calcium-regulated repressor that negatively regulates basal 5-HT1A receptor expression in neurons and may play a role in the altered regulation of 5-HT1A receptors associated with anxiety or major depression.

The Notch signaling pathway consists of multiple types of receptors and ligands, whose interactions can be tuned by Fringe glycosyltransferases. A major challenge is to determine how these components control the specificity and directionality of Notch signaling in developmental contexts. Here, we analyzed same-cell (cis) Notch-ligand interactions for Notch1, Dll1, and Jag1, and their dependence on Fringe protein expression in mammalian cells. We found that Dll1 and Jag1 can cis-inhibit Notch1, and Fringe proteins modulate these interactions in a way that parallels their effects on trans interactions. Fringe similarly modulated Notch-ligand cis interactions during Drosophila development. Based on these and previously identified interactions, we show how the design of the Notch signaling pathway leads to a restricted repertoire of signaling states that promote heterotypic signaling between distinct cell types, providing insight into the design principles of the Notch signaling system, and the specific developmental process of Drosophila dorsal-ventral boundary formation.DOI: http://dx.doi.org/10.7554/eLife.02950.001