The Ly6 superfamily, present in most metazoan genomes, codes for different cell-surface proteins and secreted ligands containing an extracellular motif called a Ly6 domain or three-finger domain. This study reports the identification of 36 novel genes coding for proteins of this family in Drosophila. One of these fly Ly6 proteins, coded by the gene boudin (bou), is essential for tracheal morphogenesis in the fly embryo and contributes to the maintenance of the paracellular barrier and the organisation of the septate junctions in this tissue. Bou, a glycosylphosphatidylinositol anchored membrane protein, is also required for septate junction organisation in epithelial tissues and in the chordotonal organ glial cells, but not in the central nervous system. This study reveals interesting parallelisms between the Ly6 proteins of flies and vertebrates, such as the CD59 antigen. Similarly to this human protein, Bou travels from cell to cell associated with extracellular particles and, consistently, this study shows that it is required in a non-cell-autonomous fashion. This work opens the way for future studies addressing the function of Ly6 proteins using Drosophila as a model system (Hijazi, 2009).

Ly6 proteins share an extracellular motif spanning about 100 residues known as a three-finger domain, three-finger snake toxin motif or Ly6/uPAR domain. This structure, first identified in the sea-snake erabutoxin b, features a simple inner core stabilised by disulphide bridges, which supports three protruding loops or fingers. Besides a diagnostic set of 8 or 10 cysteines found in stereotyped positions, Ly6 primary sequences are poorly conserved, but they adopt remarkably similar three-dimensional structures (Kini, 2002; Ploug, 1994). The Ly6 module is a structural domain involved in protein-protein interactions, tolerating an unusual degree of variation and binding with high specificity to a broad spectrum of targets (Hijazi, 2009).

The human genome codes for 45 members of the Ly6 superfamily (Galat, 2008). These include 12 TGFβ receptors, the ectodomains of which adopt the three-finger fold, but also many glycosylphosphatidylinositol (GPI)-anchored proteins and soluble ligands. Only a few of these proteins have been studied in detail, such as the urokinase plasminogen activator receptor (uPAR; PLAUR - Human Gene Nomenclature Database), which plays important roles in cell adhesion, proliferation and migration (Blasi, 2002), and CD59, an inhibitor of complement activity (Davies, 1989). Other members, such as Lynx1 (Miwa, 2006) or the soluble SLURP proteins (Grando, 2008), act as regulators of nicotinic acetylcholine receptors, and are likely to be the ancestors of the snake neurotoxins. However, although they are often used as lymphocyte and tumoural markers (Bamezai, 2004), many Ly6 human and murine proteins have unknown roles (Hijazi, 2009).

A systematic search for members of the Ly6 superfamily in Drosophila identified 36 previously uncharacterised genes coding for one or more Ly6 motifs. The function of boudin (bou) was investigated. Phenotypic analysis of bou mutants shows that this Ly6 protein participates in the formation of paracellular barriers in epithelial and neural tissues, physiological fences that regulate the passage of solutes between cells in both epithelial and glial sheaths. bou is required for the organisation of septate junctions (SJs), invertebrate adhesion structures fulfilling an equivalent role to the vertebrate tight junctions. Differing from known SJ constituents, bou requirements are non-cell-autonomous, and, accordingly, it was found that Bou can be released in extracellular particles and become incorporated into neighbouring cells. Altogether, these results indicate that Drosophila could be an attractive system in which to study the function and general properties of Ly6 proteins in a developmental context (Hijazi, 2009).

The results reveal that Bou plays an essential role in the organisation of SJs and the maintenance of paracellular barriers in Drosophila epithelia and chordotonal organs. Although some vertebrate members of the Ly6 family are known to participate in cell-adhesion processes (Bamezai, 2004), this is the first example showing that they are required for the formation of this type of cellular junction. As bou is well conserved in other insect genomes, its role in SJ organisation could have been maintained during evolution. Invertebrate SJs and vertebrate tight junctions are considered analogous structures because both participate in the establishment of paracellular barriers, although they present a different organisation. However, vertebrates have adhesion structures functionally, morphologically and molecularly similar to insect pleated SJs: the so-called paranodal septate junctions, which are formed by neural axons and Schwann cells, at the level of the Ranvier's nodes. This study shows that Bou is necessary for SJ organisation in the embryonic peripheral nervous system, indicating that its activity is required in some neural tissues. Thus, these observations raise the possibility that some vertebrate Ly6 proteins could be involved in the formation of paranodal septate junctions, which are essential for axonal insulation and propagation of action potentials (Hijazi, 2009).

In insects, the epithelial and neural SJs share many components, so the observation that bou is not required for blood-brain barrier maintenance in the ventral nerve cord came as a surprise, revealing the existence of tissular and molecular heterogeneities in the organisation of these junctions. It will be interesting to establish whether these differences also determine different barrier selective properties. It is speculated that other Ly6 proteins expressed in the nervous system could contribute to blood-brain barrier formation in the subperineural glia (Hijazi, 2009).

The results show that bou inactivation specifically perturbs the organisation of SJs. Since these structures are large extracellular complexes including different transmembrane and GPI-anchored proteins, one hypothesis is that Bou could be a membrane SJ component. Consistently, HA-Bou is found at lateral contact areas in tracheal, salivary gland and wing disc epithelia, overlapping with the membrane domains that contain SJ. However, this protein does not significantly accumulate in these membrane regions and is also seen in the most apical part of the cells, opening up the possibility that it could operate in other membrane areas or act as a signalling molecule. Indeed, studies in vertebrates indicate that Ly6 proteins can assume roles in both cell signalling and cell adhesion (Bamezai, 2004). Clearly, identification of the Bou molecular partners will be a crucial step in understanding how this protein exerts its activity (Hijazi, 2009).

In contrast with other genes required for SJ formation, bou functions in a non-cell-autonomous way. Accordingly, the Bou protein is found in extracellular particles and can be captured by neighbouring cells, suggesting that its diffusion is responsible for the phenotypic non-autonomy. Although it is possible that Bou could act as a secreted ligand after release of its GPI anchor, a parallelism with other members of the family suggests that the full molecule could instead become incorporated into the membrane of neighbouring cells (Neumann, 2007). In fact, the mammalian Ly6 member CD59, a cell-surface antigen protecting host cells from the complement attack, travels coupled to membranous vesicles called prostasomes with its intact GPI. These specialised vesicles are secreted into the seminal fluid by prostatic glands, and allow CD59 transfer to the sperm cells, which can then elude complement attack (Rooney, 1993). GPI-bound CD59 has also been found associated with human HDL apolipoproteins (Vakeva, 1994). However, the Bou particles are not lipophorin vesicles, the insect equivalent to vertebrate apolipoproteins (Rodenburg, 2005). Therefore, the fly wing epithelium could produce a different type of vesicle, possibly similar to prostasomes, which are proposed to be called `boudosomes'. Unfortunately, it could not be determined whether the Bou GPI anchor is required for incorporation into these particles, because the C-terminus of the protein seems essential for prior exit from the ER. Thus, future work will be needed to characterise the biochemical features of boudosomes and their function (Hijazi, 2009).

Little is known about how epithelial cells coordinate their activity to form efficient fences. As many SJ components are required in a cell-autonomous manner, their simultaneous expression by each individual cell seems a prerequisite for barrier assembly. A component and/or SJ regulator shared by different cells could be an element coordinating the organisation of efficient barriers in a dynamic epithelium. Alternatively, Bou extracellular traffic could be a specialised feature of this GPI-anchored protein and not have functional relevance for SJ assembly during normal development (Hijazi, 2009).

Besides Bou and the TGFβ receptors, the only member of the Ly6 fly family with a characterised role is the Rtv protein, which is also expressed in epidermal derivatives. Both bou and rtv mutants affect the organisation of the tracheal chitin luminal cable, although rtv mutants exhibit stronger phenotypes. However, SJ integrity is a prerequisite for proper assembly of the chitin cable, and this study shows that rtv is neither required for paracellular barrier integrity nor for SJ organisation. Thus, whereas these observations confirm that chitin cable deposition relies on the organisation of SJs, they demonstrate that these Ly6 proteins act in different processes (Hijazi, 2009).

This study has carried out the first description of the Ly6 superfamily in the genome of an insect, identifying 36 new genes bearing this domain in Drosophila. The conservation of these proteins among the drosophilids indicates that the family was established before the evolutive radiation of this group. By contrast, only 14 genes coding for Ly6 domains were identified in the honeybee genome. Most of these genes have fly orthologues, like bou and rtv, pointing out the existence in higher insects of a core of ancestral genes with potentially conserved roles. Thus, repeated events of gene duplication followed by rapid divergence of coding and regulatory sequences occurred in the drosophilid lineage. Indeed, the presence of genomic clusters grouping together different Ly6 genes is a novel evolutive acquisition, since the conserved genes tend to be in isolated positions (Hijazi, 2009).

It seems that genes coding for a Ly6 motif are prone to sudden phases of extensive duplication and diversification in different phylogenetic groups. In fact, an interesting parallelism can be drawn with the evolution of three-finger elapid snake venoms. This large group of Ly6 secreted proteins operates using diverse strategies, such as forming membrane pores, targeting the activity of acetylcholine receptors, inactivating acetylcholine esterase or blocking platelet aggregation (Tsetlin, 1999). Moreover, crystallographic analysis has revealed that three-finger toxins can interact with their targets via virtually any part of their solvent exposed surfaces (Kini, 2002). Yet, most of them share a common ancestor (Fry, 2003). Given the broad diversity of expression patterns exhibited by the different Drosophila Ly6 members, it is likely that gene duplication has been followed by acquisition of new developmental and physiological functions. Analysis of this insect family from an evolutive perspective could be a way to enhance understanding of the mechanisms underlying the generation of evolutive innovations (Hijazi, 2009).