From top to bottom: Cell polarity in Hedgehog and Wnt trafficking.

This is the final version of the article. Available from the publisher via the DOI in this record.Spatial organization of membrane domains within cells and cells within tissues is key to the development of organisms and the maintenance of adult tissue. Cell polarization is crucial for correct cell-cell signalling, which, in turn, promotes cell differentiation and tissue patterning. However, the mechanisms linking internal cell polarity to intercellular signalling are just beginning to be unravelled. The Hedgehog (Hh) and Wnt pathways are major directors of development and their malfunction can cause severe disorders like cancer. Here we discuss parallel advances into understanding the mechanism of Hedgehog and Wnt signal dissemination and reception. We hypothesize that cell polarization of the signal-sending and signal-receiving cells is crucial for proper signal spreading and activation of the pathway and, thus, fundamental for development of multicellular organisms.SS and LB funding was provided by the University of Exeter, UK (LSI Start-up Grant awarded to SS). ACG and DSH are funded on a Young Investigator Grant (BFU2015-73609-JIN) to ACG from MINECO (Spain). DSH was also funded by a collaboration short-term EMBO fellowship. The authors would like to thank Isabel Guerrero (University of Madrid) for her respective valuable comments that helped improve the quality of the manuscript

Regulation of hedgehog Ligand Expression by the N-End Rule Ubiquitin-Protein Ligase Hyperplastic Discs and the Drosophila GSK3β Homologue, Shaggy

Hedgehog (Hh) morphogen signalling plays an essential role in tissue development and homeostasis. While much is known about the Hh signal transduction pathway, far less is known about the molecules that regulate the expression of the hedgehog (hh) ligand itself. Here we reveal that Shaggy (Sgg), the Drosophila melanogaster orthologue of GSK3β, and the N-end Rule Ubiquitin-protein ligase Hyperplastic Discs (Hyd) act together to co-ordinate Hedgehog signalling through regulating hh ligand expression and Cubitus interruptus (Ci) expression. Increased hh and Ci expression within hyd mutant clones was effectively suppressed by sgg RNAi, placing sgg downstream of hyd. Functionally, sgg RNAi also rescued the adult hyd mutant head phenotype. Consistent with the genetic interactions, we found Hyd to physically interact with Sgg and Ci. Taken together we propose that Hyd and Sgg function to co-ordinate hh ligand and Ci expression, which in turn influences important developmental signalling pathways during imaginal disc development. These findings are important as tight temporal/spatial regulation of hh ligand expression underlies its important roles in animal development and tissue homeostasis. When deregulated, hh ligand family misexpression underlies numerous human diseases (e.g., colorectal, lung, pancreatic and haematological cancers) and developmental defects (e.g., cyclopia and polydactyly). In summary, our Drosophila-based findings highlight an apical role for Hyd and Sgg in initiating Hedgehog signalling, which could also be evolutionarily conserved in mammals

Parkin and relatives: The RBR family of ubiquitin ligases

Mutations in the parkin gene cause autosomal-recessive juvenile parkinsonism. Parkin encodes a ubiquitinprotein ligase characterized by having the RBR domain, composed of two RING fingers plus an IBR/DRIL domain. The RBR family is defined as the group of genes whose products contain an RBR domain. RBR family members exist in all eukaryotic species for which significant sequence data is available, including animals, plants, fungi, and several protists. The integration of comparative genomics with structural and functional data allows us to conclude that RBR proteins have multiple roles, not only in protein quality control mechanisms, but also as indirect regulators of transcription. A recently formulated hypothesis, based on a case of gene fusion, suggested that RBR proteins may be often part of cullin-containing ubiquitin ligase complexes. Recent data on Parkin protein agrees with that hypothesis. We discuss the involvement of RBR proteins in several neurodegenerative diseases and cancer

Functional Analysis of ESCRT-Positive Extracellular Vesicles in the Drosophila Wing Imaginal Disc

International audienceA large number of studies have shown that proteins of the Endosomal Sorting Complex Required for Transport (ESCRT) can trigger the biogenesis of different types of Extracellular Vesicles (EV). The functions that these vesicular carriers exert in vivo remain, however, poorly understood. In this chapter, we describe a series of experimental approaches that we established in the Drosophila wing imaginal disc to study the importance of ESCRT-positive EVs for the extracellular transport of signaling molecules, as exemplified by a functional analysis of the mechanism of secretion and propagation of the major developmental morphogen Hedgehog (Hh).Through the combined use of genetic, cell biological, and imaging approaches, we investigate four important aspects of exovesicle biology: (1) The genetic identification of ESCRT proteins that are specifically required for Hh secretion. (2) The imaging of ESCRT and Hh-positive EVs in the lumenal space of both living and fixed wing imaginal discs. (3) The receptor-mediated capture of Hh-containing EVs on the surface of Hh-receiving cells. (4) The effect of manipulations of ESCRT function on the extracellular pool of Hh ligands

Cytonemes are required for the establishment of a normal Hedgehog morphogen gradient in <em>Drosophila</em> epithelia

Hedgehog (Hh) signalling is important in development, stem cell biology and disease. In a variety of tissues, Hh acts as a morphogen to regulate growth and cell fate specification. Several hypotheses have been proposed to explain morphogen movement, one of which is transport via filopodia-like protrusions called cytonemes. Here, we analyse the mechanism underlying Hh movement in the wing disc and the abdominal epidermis of Drosophila. We show that, in both epithelia, cells generate cytonemes in regions of Hh signalling. These protrusions are actin-based and span several cell diameters. Various Hh signalling components localise to cytonemes, as well as to punctate structures that move along cytonemes and are probably exovesicles. Using in vivo imaging, we show that cytonemes are dynamic structures and that Hh gradient establishment correlates with cytoneme formation in space and time. Indeed, mutant conditions that affect cytoneme formation reduce both cytoneme length and Hh gradient length. Our results suggest that cytoneme-mediated Hh transport is the mechanistic basis for Hh gradient formation