Assembly, maintenance and function of synaptic junctions depend on extracellular matrix (ECM) proteins and their receptors. This study reports that Tenectin (Tnc), a Mucin-type protein with RGD motifs, is an ECM component required for the structural and functional integrity of synaptic specializations at the neuromuscular junction (NMJ) in Drosophila. Using genetics, biochemistry, electrophysiology, histology and electron microscopy, this study shows that Tnc is secreted from motor neurons and striated muscles and accumulates in the synaptic cleft. Tnc selectively recruits alphaPS2/betaPS integrin at synaptic terminals, but only the cis Tnc/integrin complexes appear to be biologically active. These complexes have distinct pre- and post-synaptic functions, mediated at least in part through the local engagement of the spectrin-based membrane skeleton: the presynaptic complexes control neurotransmitter release, while postsynaptic complexes ensure the size and architectural integrity of synaptic boutons. This study reveals an unprecedented role for integrin in the synaptic recruitment of spectrin-based membrane skeleton (Wang, 2018).

The extracellular matrix (ECM) and its receptors impact every aspect of neuronal development, from axon guidance and migration to formation of dendritic spines and neuromuscular junction synaptic junctions and function. The heavily glycosylated ECM proteins provide anchorage and structural support for cells, regulate the availability of extracellular signals, and mediate intercellular communications. Transmembrane ECM receptors include integrins, syndecans and the dystrophin-associated glycoprotein complex. Integrins in particular are differentially expressed and have an extensive repertoire, controlling multiple processes during neural development. In adults, integrins regulate synaptic stability and plasticity. However, integrin roles in synapse development have been obscured by their essential functions throughout development. How integrins are selectively recruited at synaptic junctions and how they engage in specific functions during synapse development and homeostasis remain unclear (Wang, 2018).

One way to confer specificity to ECM/integrin activities is to deploy specialized ECM ligands for the synaptic recruitment and stabilization of selective heterodimeric integrin complexes. For example, at the vertebrate NMJ, three laminins containing the β2 subunit (laminin 221, 421 and 521, that are heterotrimers of α2/4/5, β2 and γ1 subunits) are deposited into the synaptic cleft and basal lamina by skeletal muscle fibers and promote synaptic differentiation. However, only laminin 421 interacts directly with presynaptic integrins containing the α3 subunit and anchors a complex containing the presynaptic Cavα and cytoskeletal and active zone-associated proteins. Studies with peptides containing the RGD sequence, recognized by many integrin subtypes, have implicated integrin in the morphological changes and reassembly after induction of long-term potentiation (LTP). Several integrin subunits (α3, α5, α8, β1 and β2) with distinct roles in the consolidation of LTP have been identified, but the relevant ligands remain unknown (Wang, 2018).

Drosophila neuromuscular junction (NMJ) is a powerful genetic system to examine the synaptic functions of ECM components and their receptors. In flies, a basal membrane surrounds the synaptic terminals only in late embryos; during development, the boutons 'sink' into the striated muscle, away from the basal membrane. The synaptic cleft relies on ECM to withstand the mechanical tensions produced by the muscle contractions. The ECM proteins, including laminins, tenascins/teneurins (Ten-a and -m) and Mind-the-gap (Mtg), interact with complexes of five integrin subunits (αPS1, αPS2, αPS3, βPS, and βν). The αPS1, αPS2 and βPS subunits localize to pre- and post-synaptic compartments and have been implicated in NMJ growth. The αPS3 and βν are primarily presynaptic and control activity-dependent plasticity. The only known integrin ligand at the fly NMJ is Laminin A, which is secreted from the muscle and signals through presynaptic αPS3/βν and Focal adhesion kinase 56 (Fak56) to negatively regulate the activity-dependent NMJ growth. Teneurins have RGD motifs, but their receptor specificities remain unknown. Mtg secreted from the motor neurons influences postsynaptic βPS accumulation, but that may be indirectly due to an essential role for Mtg in the organization of the synaptic cleft and the formation of the postsynaptic fields. The large size of these proteins and the complexity of ECM-integrin interactions made it difficult to recognize relevant ligand-receptor units and genetically dissect their roles in synapse development (Wang, 2018).

This study reports the functional analysis of Tenectin (Tnc), an integrin ligand secreted from both motor neurons and muscles; Tnc accumulates at synaptic terminals and functions in cis to differentially engage presynaptic and postsynaptic integrin. tnc, which encodes a developmentally regulated RGD-containing integrin ligand (Fraichard, 2006; Fraichard, 2010), in a screen for ECM candidates that interact genetically with neto, a gene essential for NMJ assembly and function (Kim, 2012). This study found that Tnc selectively recruits the αPS2/βPS integrin at synaptic locations, without affecting integrin anchoring at muscle attachment sites. Dissection of Tnc functions revealed pre- and postsynaptic biologically active cis Tnc/integrin complexes that function to regulate neurotransmitter release and postsynaptic architecture. Finally, the remarkable features of this selective integrin ligand were explored to uncover a novel synaptic function for integrin, in engaging the spectrin-based membrane skeleton (Wang, 2018).

The ECM proteins and their receptors have been implicated in NMJ development, but their specific roles have been difficult to assess because of their early development functions and the complexity of membrane interactions they engage. This study has shown that Tnc is a selective integrin ligand that enables distinct pre- and post-synaptic integrin activities mediated at least in part through the local engagement of the spectrin-based cortical skeleton. First, Tnc depletion altered NMJ development and function and correlated with selective disruption of αPS2/βPS integrin and spectrin accumulation at synaptic terminals. Second, manipulation of Tnc and integrin in neurons demonstrated that presynaptic Tnc/integrin modulate neurotransmitter release; spectrin mutations showed similar disruptions of the presynaptic neurotransmitter release. Third, postsynaptic Tnc influenced the development of postsynaptic structures (bouton size and SSR complexity), similar to integrin and spectrin. Fourth, presynaptic Tnc/integrin limited the accumulation and function of postsynaptic Tnc/integrin complexes. Fifth, secreted Tnc bound integrin complexes at cell membranes, but only the cis complexes were biologically active; trans Tnc/integrin complexes can form but cannot function at synaptic terminals and instead exhibited dominant-negative activities. These observations support the model that Tnc is a tightly regulated component of the synaptic ECM that functions in cis to recruit αPS2/βPS integrin and the spectrin-based membrane skeleton at synaptic terminals and together modulate the NMJ development and function (Wang, 2018).

Tnc appears to fulfill unique, complementary functions with the other known synaptic ECM proteins at the Drosophila NMJ. Unlike Mtg, which organizes the active zone matrix and the postsynaptic domains, Tnc does not influence the recruitment of iGluRs and other PSD components. LanA ensures a proper adhesion between the motor neuron terminal and muscle and also acts retrogradely to suppress the crawling activity-dependent NMJ growth. The latter function requires the presynaptic βν integrin subunit and phosphorylation of Fak56 via a pathway that appears to be completely independent of Tnc. Several more classes of trans-synaptic adhesion molecules have been implicated in either the formation of normal size synapses, for example Neurexin/Neuroligin, or in bridging the pre- and post-synaptic microtubule-based cytoskeleton, such as Teneurins. However, genetic manipulation of Tnc did not perturb synapse assembly or microtubule organization, indicating that Tnc functions independently from these adhesion molecules. Instead, Tnc appears to promote expression and stabilization of αPS2/βPS complexes, which in turn engage the spectrin-based membrane skeleton (SBMS) at synaptic terminals. On the presynaptic side these complexes modulate neurotransmitter release. On the postsynaptic side, the Tnc-mediated integrin and spectrin recruitment modulates bouton morphology. A similar role for integrin and spectrin in maintaining tissue architecture has been reported during oogenesis; egg chambers with follicle cells mutant for either integrin or spectrin produce rounder eggs (Wang, 2018).

The data are consistent with a local function for the Tnc/βPS-recruited SBMS at synaptic terminals; this is distinct from the role of spectrin in endomembrane trafficking and synapse organization. Embryos mutant for spectrins have reduced neurotransmitter release, a phenotype shared by larvae lacking presynaptic Tnc or βPS integrin. However, Tnc perturbations did not induce synapse retraction and axonal transport defects as seen in larvae with paneuronal α- or β- spectrin knockdown. Spectrins interact with ankyrins and form a lattice-like structure lining neuronal membranes in axonal and interbouton regions. This study found that Tnc manipulations did not affect the distribution of Ankyrin two isoforms (Ank2-L and Ank2-XL) in axons or at the NMJ; also loss of ankyrins generally induces boutons swelling, whereas Tnc perturbations shrink the boutons and erode bouton-interbouton boundaries. Like tnc, loss of spectrins in the striated muscle shows severe defects in SSR structure. Lack of spectrins also disrupts synapse assembly and the recruitment of glutamate receptors. In contrast, manipulations of tnc had no effect on PSD size and composition. Instead, tnc perturbations in the muscle led to boutons with altered size and individualization and resembled the morphological defects seen in spectrin tetramerization mutants, spectrin^R22S. spectrin^R22S mutants have more subtle defects than tnc, probably because spectrin is properly recruited at NMJs but fails to crosslink and form a cortical network. Spectrins are also recruited to synaptic locations by Teneurins, a pair of transmembrane molecule that form trans-synaptic bridges and influence NMJ organization and function. Drosophila Ten-m has an RGD motif; this study found that βPS levels were decreased by 35% at ten-mMB mutant NMJs. Thus, Ten-m may also contribute to the recruitment of integrin and SBMS at the NMJ, a function likely obscured by the predominant role both play in cytoskeleton organization (Wang, 2018).

Previous work has shown that α-Spectrin is severely disrupted at NMJs with suboptimal levels of Neto, such as neto¹⁰⁹- a hypomorph with 50% lethality. These mutants also had sparse SSR, reduced neurotransmitter release, as well as reduced levels of synaptic βPS. In this genetic background, lowering the dose of tnc should further decrease the capacity to accumulate integrin and spectrin at synaptic terminals and enhance the lethality. This may explain the increased synthetic lethality detected in the genetic screen (Wang, 2018).

In flies or vertebrates, the ECM proteins that comprise the synaptic cleft at the NMJ are not fully present when motor neurons first arrive at target muscles. Shortly thereafter, the neurons, muscles and glia begin to synthesize, secrete and deposit ECM proteins. At the vertebrate NMJ, deposition of the ECM proteins forms a synaptic basal lamina that surrounds each skeletal myofiber and creates a ~ 50 nm synaptic cleft. In flies, basal membrane contacts the motor terminal in late embryos, but is some distance away from the synaptic boutons during larval stages. Nonetheless, the NMJ must withstand the mechanical tensions produced by muscle contractions. The current data suggest that Tnc is an ideal candidate to perform the space filling, pressure inducing functions required to engage integrin and establish a dynamic ECM-cell membrane network at synaptic terminals. First, Tnc is a large mucin with extended PTS domains that become highly O-glycosylated, bind water and form gel-like complexes that can extend and induce effects similar to hydrostatic pressure. In fact, Tnc fills the lumen of several epithelial tubes and forms a dense matrix that acts in a dose-dependent manner to drive diameter growth. Second, the RGD and RGD-like motifs of Tnc have been directly implicated in αPS2/βPS-dependent spreading of S2 cells (Fraichard, 2010). Third, secreted Tnc appears to act close to the source, presumably because of its size and multiple interactions. In addition to the RGD motifs, Tnc also contains five complete and one partial vWFC domains, that mediate protein interactions and oligomerization in several ECM proteins including mucins, collagens, and thrombospondins. The vWFC domains are also found in growth factor binding proteins and signaling modulators such as Crossveinless-2 and Kielin/Chordin suggesting that Tnc could also influence the availability of extracellular signals. Importantly, Tnc expression is hormonally regulated during development by ecdysone (Fraichard, 2010). Tnc does not influence integrin responsiveness to axon guidance cues during late embryogenesis; unlike integrins, the tnc mutant embryos have normal longitudinal axon tracks. Instead, Tnc synthesis and secretion coincide with the NMJ expansion and formation of new bouton structures during larval stages. Recent studies have reported several mucin-type O-glycosyltransferases that modulate integrin signaling and intercellular adhesion in neuronal and non-neuronal tissues, including the Drosophila NMJ. Tnc is likely a substrate for these enzymes that may further regulate Tnc activities (Wang, 2018).

In flies as in vertebrates, integrins play essential roles in almost all aspects of synaptic development. Early in development, integrins have been implicated in axonal outgrowth, pathfinding and growth cone target selection. In adult flies, loss of αPS3 integrin activity is associated with the impairment of short-term olfactory memory. In vertebrates, integrin mediates structural changes involving actin polymerization and spine enlargement to accommodate new AMPAR during LTP, and 'lock in' these morphological changes conferring longevity for LTP. Thus far, integrin functions at synapses have been derived from compound phenotypes elicited by use of integrin mutants, RGD peptides, or enzymes that modify multiple ECM molecules. Such studies have been complicated by multiple targets for modifying enzymes and RGD peptides and by the essential functions of integrin in cell adhesion and tissue development (Wang, 2018).

In contrast, manipulations of Tnc, which affects the selective recruitment of αPS2/βPS integrin at synaptic terminals, have uncovered novel functions for integrin and clarified previous proposals. This study demonstrated that βPS integrin is dispensable for the recruitment of iGluRs at synaptic sites and for PSD maintenance. An unprecedented role was reveaked for integrin in connecting the ECM of the synaptic cleft with spectrin, in particular to the spectrin-based membrane skeleton. These Tnc/integrin/spectrin complexes are crucial for the integrity and function of synaptic structures. These studies uncover the ECM component Tnc as a novel modulator for NMJ development and function; these studies also illustrate how manipulation of a selective integrin ligand could be utilized to reveal novel integrin functions and parse the many roles of integrins at synaptic junctions (Wang, 2018).

Morphogenesis of the adult structures of holometabolous insects is regulated by ecdysteroids and juvenile hormones and involves cell-cell interactions mediated in part by the cell surface integrin receptors and their extracellular matrix (ECM) ligands. These adhesion molecules and their regulation by hormones are not well characterized. This study describes the gene structure of a newly described ECM molecule, tenectin, and demonstrate that it is a hormonally regulated ECM protein required for proper morphogenesis of the adult wing and male genitalia. Tenectin's function as a new ligand of the PS2 integrins is demonstrated by both genetic interactions in the fly and by cell spreading and cell adhesion assays in cultured cells. Its interaction with the PS2 integrins is dependent on RGD and RGD-like motifs. Tenectin's function in looping morphogenesis in the development of the male genitalia led to experiments that demonstrate a role for PS integrins in the execution of left-right asymmetry (Fraichard, 2010).

Tenectin is a protein localized to the ECM during Drosophila embryonic development. The presence of an integrin-binding RGD motif led to a speculation that tenectin could be a new integrin ligand. To study the function of tenectin during Drosophila development, tenectin knockdowns were generated by RNA interference. Two strains of tenectin knockdown flies were selected that gave visible hypomorphic phenotypes. Flies were also characterized that give phenotypes due to overexpression of the endogenous tenectin gene. Lowering mRNA level by RNAi partially rescued the effects of tenectin overexpression and overexpression of tenectin partially rescues tenectin knockdown phenotypes. Thus, the authors are confident that the tenectin knockdown phenotypes result specifically from reduced tenectin expression (Fraichard, 2010).

Lethality is the most prevalent phenotype displayed by ubiquitous reduction in tenectin expression but this study focused on adult phenotypes to ascertain tenectin's function in morphogenetic processes of metamorphosis. The most striking adult phenotype observed in adult flies with reduced tenectin expression is deformed wings including blisters, nicks, lack of expansion and malformation. These phenotypes resemble those associated with mutations in integrin subunits, their extracellular ligands, and genes encoding intracellular proteins that interact with integrins. Three lines of evidence support tenectin functioning as a PS integrin ligand to facilitate wing morphogenesis. First, tenectin protein was found to localize between the dorsal and ventral epithelial cell layers in prepupal wings. Integrins function at this location to promote adhesion of these cell layers. Second, a mutation of mys, encoding the βPS subunit, interacts genetically to increase the frequency of blisters in flies with reduced tenectin expression. Finally, in vitro experiments demonstrate that tenectin, through multiple RGD motifs, can function to promote αPS2βPS-mediated cell spreading and adhesion. Taken together, these genetic and biochemical data provide strong evidence that tenectin is a new ligand of αPS2βPS integrin in the wing (Fraichard, 2010).

Perhaps relevant to tenectin's function in the wing, Syed (2008), using a bioinformatics approach, identified tenectin as being a mucin-related-protein. In an analysis of the tenectin protein this study also notice mucin like repeats. Mucins are highly hydrated O-glycosylated macromolecules that are important to the mucosal lining of mammalian organs. In addition to serving a protective function, various mucins interact with growth factors and cell surface receptors to modulate signaling. It has been shown in vertebrates that mucins also modulate cell adhesion. For example, MUC4 was found to sterically reduce the accessibility of integrins to extracellular matrix ligands and thereby interfere with adhesion. Interestingly, a mucin-type glycosyltransferase, PGANT3, glycosylates another PS2 integrin ligand, tiggrin. Moreover, mutation of the pgant3 gene results in a wing-blistering phenotype. In the developing wing disc PGANT3 glycosylates tiggrin and other matrix molecules, thus potentially modulating cell adhesion through integrin-ECM interactions. Future biochemical experiments will be needed to determine if tenectin is a bona fide mucin, glycosylated by PGANT3, and whether glycosylation down- or up-regulates its adhesive function (Fraichard, 2010).

The formation of the flat bi-layered wing from a folded imaginal disc involves several steps of apposition and separation of the ventral and dorsal epidermal sheets followed ultimately by an epithelial to mesenchymal transition and migration of the cells out of the wing. The resulting wing is predominantly two layers of cuticle cemented together by ECM. These studies point out the importance of regulating the adhesive properties of the wing epidermal cells by modulating the activity of integrins and their intracellular and extracellular binding partners. One mode of regulation is at the transcriptional level and several studies have demonstrated that the hormone 20E plays an important role in regulating at least some of these morphogenetic events including integrin expression levels. Consistent with tenectin's role in wing morphogenesis this study found that during metamorphosis tenectin mRNA expression correlates with the ecdysone titer profile. In vitro, imaginal disc cultures demonstrate that tenectin is a 20E target gene. The comparison of the developmental tenectin expression profile with those of early (E74A, E74B) and prepupal (β-Ftz-F1) genes defined more precisely the temporal expression pattern of tenectin. E74B is a class I transcript, induced in mid-third instar larvae in response to a low concentration of 20E and repressed at higher ecdysone concentrations. In contrast, the class II transcripts, including E74A, are induced by high 20E concentration and their expressions are unaffected by higher 20E concentrations. The temporal profile of tenectin is similar to those of E74A, with a slight delay in the peak levels of tenectin mRNA accumulation. This temporal delay in tenectin is similar to the delay observed in the early-late gene profiles. The early-late genes appear to share properties with both the early genes and late genes. Early-late genes respond directly to ecdysone even in the presence of protein synthesis inhibitors like cycloheximide but unlike early genes their full induction requires protein synthesis due to a requirement for other ecdysone induced gene products. It is proposed that tenectin is an early-late gene as its expression in cultured larval organs was induced by 20E in the presence of cycloheximide but maximal induction required protein synthesis. In the wing, it is proposed that 20E also regulates morphogenesis by regulation of tenectin mRNA levels, suggesting that ecdysone controls wing morphogenesis and cell adhesion not only by regulating integrin expression but also their ECM ligand expression. Just as E74A and E75B do not display identical expression profiles, the tenectin expression pattern is complicated and likely involves additional modes of regulation that will need to be elucidated (Fraichard, 2010).

Tenectin knockdown resulted in reduced rotation of male genitalia. Looping morphogenesis of the male genitalia occurs during the pupal stage as the genital disc undergoes a 360° dextral (clockwise) looping around the hindgut. A variety of genes expressed in larval posterior abdominal segments A8, A9 and A10 have been identified that affect male genital rotation. These include genes encoding a signaling protein (Pvf1), a transcription factor (Taf1, formerly TAF250), and a pro-apoptosis gene (hid). One adhesion molecule, fasciclin-2, was genetically demonstrated to be involved in genital rotation. However, the effect was indirect as Fas2^spin mutant alters the synapses connecting neurosecretory cells to the organ that produces juvenile hormone (the corpora allata), and genitalia under-rotation is due to an excess of juvenile hormone. The effects on genitalia rotation have been shown to be mediated by an excess of juvenile hormone, a retinoic-like molecule, establishing a parallel between vertebrate and invertebrate left right asymmetry, since the retinoic acid is involved in the control of asymmetry in vertebrates. In Drosophila, excessive juvenile hormone may result in the attenuation of ecdysone regulated processes required for male genital rotation as mutations in Broad-Complex, an ecdysone early-response gene, also result in malrotation of male genitalia. Mutations of the unconventional myosin 31DF gene (Myo31DF) have been shown to uniquely reverse the looping direction of genitalia. Knockdown of tenectin in imaginal discs, but not in neuronal cells, resulted in incomplete rotation of the genitalia but not in direction of looping. Thus, this study has for the first time identified a Drosophila ECM component required for genital looping morphogenesis (Fraichard, 2010).

The tenectin mutant phenotype in male genitalia prompted a re-examination ofe integrin hypomorphic mutations for a similar phenotype. Males bearing 3 different hypomorphic mutations in the gene encoding the βPS integrin subunit, mys^b13, mys^b47, and mys^b69 displayed under-rotated male genitalia when raised at elevated temperatures. A mutation has been described that was likely in myospheroid that produced under-rotated male genitalia when larvae and pupae were raised at elevated temperatures. Combining mys^b13 with the if³ mutation in the gene encoding the αPS2 integrin subunit caused a dramatic increase in the expressivity of the rotated genitalia phenotype. Therefore, tenectin's proposed cell surface adhesion receptor is also required for the execution of looping morphogenesis. In addition to adhesion, the PS integrins function in the regulation of intracellular signaling pathways and specifically the JNK pathway. JNK signaling pathway has also been suggested to function in apoptosis required for rotation of male genitalia. Thus, tenectin and PS integrin function in looping morphogenesis could be at the level of adhesion and/or signaling. Additional experiments are required to distinguish between these two models (Fraichard, 2010).

Tenectin's RGD sequence in the 3rd von Willebrand factor type-C (VWC) domain is conserved in the beetle homolog, tenebrin, and supported PS2 integrin-mediated cell spreading. This result is expected given that RGD is a well known integrin-binding motif of the PS2 integrins. More novel is the presence in the identical location in the 5th VWC of the sequence RSD and elsewhere in this 5th repeat the occurrence of RDD and RYE sequences. The biological importance of the 5th VWC domain is supported by the extraordinary high degree of conservation in this domain between Drosophila tenectin and Tenebrio tenebrin. The two proteins share 92% (62/67) sequence identity in the 5th VWC repeat and this includes the RDD, RSD, and RYE sequences. To date, this domain is found conserved, with greater than 84% sequence identity, in mosquitoes, honey bees, crickets, wasps, the beetle, and aphids (not shown). While RGD is the best studied integrin-binding motif, experimental evidence is accumulating that variants of this sequence are also important. These variants include KQAGD, KGD, RSD, WGD, MVD and RYD found in fibrinogen, thrombospondin, tenascin-W, CD40, snake venom disintegrins, viral coat proteins, and ligand mimetic monoclonal antibodies. Cell adhesion assays demonstrate that VWC#5 as well as VWC#3 promotes cell adhesion mediated by PS2 integrins. Mutations of the individual RGD-variant motifs in VWF#5 suggest that they have differing effects on different integrins. The RDD is required for strong adhesion by both the PS2m8 and PS2c integrin isoforms as mutation of this sequence reduced adhesion of cells expressing either integrin. This is the first time the RDD tripeptide in an ECM protein has been found to function in integrin-mediated adhesion. It also appears that the RSD and RYE motifs may be inhibitory for adhesion mediated by the PS2c isoform as their mutations increased cell adhesion. With multiple integrin-binding domains, both positive and inhibitory, tenectin potentially functions in multiple processes in development and specifically in metamorphosis (Fraichard, 2010).

Future experiments will be required to address the many unanswered issues regarding tenectin-PS integrin interactions including: which PS integrin(s) interact with tenectin in vivo; how the function of the motifs may be affected by the context of other ECM proteins; and how other regions of tenectin and modifications, such as glycosylation or cleavage, influence the functionality of the putative integrin-binding motifs. The presence of multiple motifs also raises the possibility that tenectin can bridge integrins on neighboring cells, or on the surface of the same cell. Finally, the different motifs may be needed to bind different integrins at different times in development and this binding of different motifs may have different adhesive and/or signaling consequences (Fraichard, 2010).

An important step in epithelial organ development is size maturation of the organ lumen to attain correct dimen/sions. This study shows that the regulated expression of Tenectin (Tnc) is critical to shape the Drosophila melanogaster hindgut tube. Tnc is a secreted protein that fills the embryonic hindgut lumen during tube diameter expansion. Inside the lumen, Tnc contributes to detectable O-Glycans and forms a dense striated matrix. Loss of tnc causes a narrow hindgut tube, while Tnc over-expression drives tube dilation in a dose-dependent manner. Cellular analyses show that luminal accumulation of Tnc causes an increase in inner and outer tube diameter, and cell flattening within the tube wall, similar to the effects of a hydrostatic pressure in other systems. When Tnc expression is induced only in cells at one side of the tube wall, Tnc fills the lumen and equally affects all cells at the lumen perimeter, arguing that Tnc acts non-cell-autonomously. Moreover, when Tnc expression is directed to a segment of a tube, its luminal accumulation is restricted to this segment and affects the surrounding cells to promote a corresponding local diameter expansion. These findings suggest that deposition of Tnc into the lumen might contribute to expansion of the lumen volume, and thereby to stretching of the tube wall. Consistent with such an idea, ectopic expression of Tnc in different developing epithelial tubes is sufficient to cause dilation, while epidermal Tnc expression has no effect on morphology. Together, the results show that epithelial tube diameter can be modelled by regulating the levels and pattern of expression of a single luminal glycoprotein (Syed, 2012).

This study shows that the luminal glycoprotein Tnc promotes diameter expansion of the Drosophila hindgut in a dose-dependent manner. The domain organization of Tnc, its contribution to detectable O-glycans in the hindgut lumen and its ability to form a dense luminal matrix suggest that Tnc has mucin-like characteristics. A possible involvement of mucin-like molecules in tubulogenesis has previously been recognized. The Caenorhabditis elegans let-653 is a secreted protein with a PTS domain of around 90-200 amino acids, depending on the splice variant. In mutants for let-653, the single-celled excretory canals develop massively enlarged lumen by an as yet unknown mechanism. During cyst formation in Madin-Darby Canine Kidney (MDCK), it has been suggested that the initial separation of apical membranes involves de-adhesive properties conferred by large apically localized glycoproteins. Candidate molecules are mucin 1 (MUC1) and the sialomucin Podocalyxin, which localize to the nascent lumens in MDCK cysts and in vivo. Recently, it was indeed shown that Podocalyxin is required to separate apical membranes during initial lumen formation in developing blood vessels. Podocalyxin is membrane-bound, and its negatively charged sialic acids are thought to cause electrostatic repulsion of the apical surfaces. Tnc does however appear to function differently from these mucin-like molecules, since it is not required for lumen formation per se, but drives the subsequent step of tube diameter expansion (Syed, 2012).

The function of Tnc also differs from that of the chitinous matrix in the tracheal lumen, as the latter is not needed to increase the luminal volume during diameter expansion, but to shape a uniform diameter. A difference in action between the two luminal components is further supported by the slightly shorter tracheal tubes in tnc mutants, while loss of chitin causes too long tracheal tubes. It is proposed that Tnc-driven tube dilation represents a mechanism for shaping an epithelial tube, where the extent of tube wall extension and lumen volume expansion can be controlled by the intraluminal accumulation of a single protein (Syed, 2012).

During wing development, Tnc is found basal to the epithelium and is proposed to act as a ligand for PS2 integrin via RGD motifs in the vWC-like domains. It is therefore possibly that luminal Tnc might cause tube wall remodelling by signalling through an apical cognate receptor(s). However, the results do not indicate a signalling function for Tnc: First, over-expression of Tnc in the hindgut causes an increase in tube diameter according to the levels of Tnc expression. Thus, a signaling function of Tnc would imply that Tnc is the limiting factor in the pathway. This is unlikely, since Tnc is abundant and fills the lumen of the wild type hindgut. Second, when Tnc was expressed at one side of the tube wall, all cells at the lumen perimeter were similarly affected. If Tnc signals via an apical receptor, the effects should be higher at the site of its secretion, given its strictly dose-dependent function. Third, the observed lumen-dependent function of Tnc implies that a putative receptor would have to be present in many epithelia in which Tnc is not normally expressed, but yet not ubiquitously, as Tnc had no effect on the epidermis (Syed, 2012).

Tnc-driven lumen expansion causes an increase in inner and outer tube diameter, associated with epithelial flattening. It is known that luminal volume expansion upon a hydrostatic pressure causes similar effects, for example during inflation of the zebrafish brain ventricle, expansion of the mouse blastocyst and in vitro growth of renal cysts. The results would therefore comply with a mechanism whereby luminal accumulation of Tnc forces an increase in lumen volume and, thereby, expansion of the surrounding tube wall. Since luminal Tnc appears to be a major O-glycan with low mobility in the lumen, an attractive hypothesis is that Tnc forms supra-molecular complexes that cause volume expansion due to hydration of the attached O-glycans. Secretion of Tnc into a confined luminal space would then cause a pressure on the tube wall and lumen dilation. In an attempt to further evaluate if the effect of Tnc requires O-glycosylation of the PTS domains, hindgut morphology and the size of Tnc was examined in mutants that lack different glycosyl transferases. However, the results were inconclusive, showing effects on both Tnc levels and secretion (Syed, 2012).

The current study also show that Tnc can steer regional differences in tube diameter expansion along the tube axis, according to its pattern of expression. Such a regional effect of Tnc presumably occurs during normal hindgut development, where the amount of Tnc produced by the small intestine is larger than the amount produced by large intestine. As a likely consequence, the small intestine undergoes a higher degree of diameter expansion than large intestine, and it also shows a larger reduction in diameter upon loss of Tnc (Syed, 2012).

In summary, this study has shown that Tnc forms a lumen-spanning complex that drives expansion of the surrounding tube wall. The local and dose-dependent effect of Tnc on tube dilation illustrates that a single protein can model differential lumen diameter along a tube. A model is suggested were Tnc causes a luminal pressure upon secretion and promotes tube dilation according to its voluminous expansion. Since the lumen of different epithelial organs have been shown to exhibit dynamic patterns of glycan distribution during development, it is possible that glycan-rich luminal components have a broad importance in shaping developing epithelial organs (Syed, 2012).