Teneurins: a novel family of neuronal cell surface proteins in vertebrates, homologous to the Drosophila pair-rule gene product Ten-m

We have characterized chicken teneurin-1 and teneurin-2, two homologues of the Drosophila pair-rule gene product Ten-m and Drosophila Ten-a. The high degree of conservation between the vertebrate and invertebrate proteins suggests that these belong to a novel family. We propose to name the vertebrate members of this family teneurins, because of their predominant expression in the nervous system. The expression of teneurin-1 and -2 was investigated by in situ hybridization. We show that teneurin-1 and -2 are expressed by distinct populations of neurons during the time of axonal growth. The most prominent site of expression of chicken teneurins is the developing visual system. Recombinant teneurin-2 was expressed to assay its molecular and functional properties. We show that it is a type II transmembrane protein, which can be released from the cell surface by proteolytic cleavage at a furin site. The expression of teneurin-2 in neuronal cells led to a significant increase in the number of filopodia and to the formation of enlarged growth cones. The expression pattern of teneurins in the developing nervous system and the ability of teneurin-2 to reorganize the cellular morphology indicate that these proteins may have an important function in the formation of neuronal connections.     

All four members of the Ten-m/Odz family of transmembrane proteins form dimers

Ten-m/Odz/teneurins are a new family of four distinct type II transmembrane molecules. Their extracellular domains are composed of an array of eight consecutive EGF modules followed by a large globular domain. Two of the eight modules contain only 5 instead of the typical 6 cysteine residues and have the capability to dimerize in a covalent, disulfide-linked fashion. The structural properties of the extracellular domains of all four mouse Ten-m proteins have been analyzed using secreted, recombinant molecules produced by mammalian HEK-293 cells. Electron microscopic analysis supported by analytical ultracentrifugation data revealed that the recombinant extracellular domains of all Ten-m proteins formed homodimers. SDS-PAGE analysis under nonreducing conditions as well as negative staining after partial denaturation of the molecules indicated that the globular COOH-terminal domains of Ten-m1 and -m4 contained subdomains with a pronounced stability against denaturing agents, especially when compared with the homologous domains of Ten-m2 and -m3. Cotransfection experiments of mammalian cells with two different extracellular domains revealed that Ten-m molecules have also the ability to form heterodimers, a property that, combined with alternative splicing events, allows the formation of a multitude of molecules with different characteristics from a limited set of genes.     

The murine Ten-m/Odz genes show distinct but overlapping expression patterns during development and in adult brain

The mouse TEN-M/ODZ proteins belong to a new family of type II transmembrane proteins with unknown function. The family consists of four members, which are expressed highly in brain and less in many other tissues. In the present study we have generated specific RNA probes and antibodies to characterize the expression of the 4 Ten-m/Odz genes in the developing and adult central nervous system (CNS) of mice. Ten-m/Odz3 and Ten-m/Odz4 mRNAs were first detectable at E7.5, Ten-m/Odz2 expression started at the 37 somite (E 10.5) stage, while Ten-m/Odz1 mRNA is not found before E15.5. In the adult mouse CNS mRNAs of the 4 Ten-m/Odzs were expressed in distinct patterns, which partially overlapped. Immunostaining and in situ hybridization localized proteins and mRNAs of Ten-m/Odzs in adjacent areas suggesting that TEN-M/ODZ proteins might be transported from the cell body along the axon or that they are shed from the cell surface and diffuse into distant regions.     

Teneurins: transmembrane proteins with fundamental roles in development

Teneurins are a novel family of transmembrane proteins expressed during pattern formation and morphogenesis. Originally discovered as ten-m and ten-a in Drosophila, four vertebrate teneurins as well as a Caenorhabditis elegans homologue were identified. The conserved domain architecture of teneurins includes an intracellular domain containing polyproline motifs. The long extracellular domain consists of eight EGF-like repeats, a region of conserved cysteines and unique YD-repeats. Vertebrate teneurins are most prominently expressed in the developing central nervous system, but are also expressed in developing limbs. In C. elegans, RNAi experiments and studies of mutants reveal that teneurins are required during fundamental developmental processes like cell migration and axon pathfinding. Cell culture experiments suggest that the intracellular domain of teneurins translocates to the nucleus following release from the membrane by proteolytic processing. Interestingly, the human teneurin-1 gene is located on the X-chromosome in a region where several families with X-linked mental retardation are mapped.     

Navigating their way to the clinic: emerging roles for axon guidance molecules in neurological disorders and injury

The mechanisms underlying formation of the basic network of the nervous system are of fundamental interest in developmental neurobiology. During the wiring of the nervous system, newborn neurons send axons that travel long distances to their targets. These axons are directed by environmental cues, known as guidance cues, to their correct destinations. Through extensive studies in vertebrates and invertebrates many of the guidance cues and their receptors have been identified. Recently, guidance molecules have been suggested to have important roles in pathological conditions of the nervous system. Mutations in guidance receptors have been associated with hereditary neurological disorders, and deregulation of guidance cues might be associated with predisposition to epilepsy. In addition, it was suggested that guidance molecules play roles in the ability of the adult nervous system to recover and repair after injury. Thus, molecules that were first discovered as "developmental cues" are now emerging as important factors in neurological disease and injury in the adult.     

The brain within the tumor: new roles for axon guidance molecules in cancers

Slits, semaphorins and netrins are three families of proteins that can attract or repel growing axons and migrating neurons in the developing nervous system of vertebrates and invertebrates. Recent studies have shown that they are widely expressed outside the nervous system and that they may play important roles in cancers. Several of the genes encoding these proteins are localized on chromosomal region associated with frequent loss-of-heterozygosity in tumors and cancer cell lines and there is also significant hypermethylation of their promoter suggesting that they may act as tumor suppressors. In addition, proteins in all these families and their receptors appear to control the vascularization of the tumors. Last, many axon guidance molecules also regulate cell migration and apoptosis in normal and tumorigenic tissues. Overall, this suggests that molecules that could mimick or block the activity of axon guidance molecules may be used as therapeutic agents for the treatment of malignancy.     

Drosophila Ten-m and filamin affect motor neuron growth cone guidance

The Drosophila Ten-m (also called Tenascin-major, or odd Oz (odz)) gene has been associated with a pair-rule phenotype. We identified and characterized new alleles of Drosophila Ten-m to establish that this gene is not responsible for segmentation defects but rather causes defects in motor neuron axon routing. In Ten-m mutants the inter-segmental nerve (ISN) often crosses segment boundaries and fasciculates with the ISN in the adjacent segment. Ten-m is expressed in the central nervous system and epidermal stripes during the stages when the growth cones of the neurons that form the ISN navigate to their targets. Over-expression of Ten-m in epidermal cells also leads to ISN misrouting. We also found that Filamin, an actin binding protein, physically interacts with the Ten-m protein. Mutations in cheerio, which encodes Filamin, cause defects in motor neuron axon routing like those of Ten-m. During embryonic development, the expression of Filamin and Ten-m partially overlap in ectodermal cells. These results suggest that Ten-m and Filamin in epidermal cells might together influence growth cone progression.     

The expression of teneurin-4 in the avian embryo: potential roles in patterning of the limb and nervous system

Teneurins are type II transmembrane proteins that play important roles in pattern formation in Drosophila, axon fasciculation and organogenesis in Caenorhabidits elegans, and neuronal pathfinding in the visual system of the mouse. There is evidence that a peptide derived from a proteolytic event near the C-terminus of teneurins leads to formation of an active neuropeptide, while processing at and near the transmembrane domain leads to shedding of the extracellular domain into the extracellular matrix and the generation of an intracellular fragment that is transported to the nucleus. In vertebrates there are four teneurins. Here, we have studied the expression of teneurin-4 in the chicken embryo. An antiserum against part of the intracellular domain of teneurin-4 recognizes several low molecular weight bands on immunoblots of embryonic chicken brain homogenates, indicating that teneurin-4 is likely to be processed at one or more predicted proteolytic cleavage sites. Antisera against the EGF-like repeats of the extracellular domain label some mesenchyme in the early embryo, and near basement membranes this labeling partially overlaps with anti-laminin (gamma 1 chain) immunostaining. At embryonic day 7, anti-teneurin-4 labels bundles of axons in the nasal, but not temporal retina. Later in development, retinal expression switches so that teneurin-4 is found in the temporal, but not nasal, ganglion cell layer. Teneurin-4 immunolocalization was also compared with other teneurins in the developing limb, where each teneurin is expressed in distinctive regions. These patterns of expression suggest roles for teneurin-4 in patterning and neuronal pathfinding in the avian embryo.     

Eph receptors and ephrins

The Eph receptors are the largest known family of receptor tyrosine kinases. The Eph receptors and their membrane-attached ligands, ephrins, show diverse expression patterns during development. Recent studies have demonstrated that Eph receptors and ephrins play important roles in many developmental processes, including neuronal network formation, the patterning of the neural tube and the paraxial mesoderm, the guidance of cell migration, and vascular formation. In the nervous system, Eph receptors and ephrins have been shown to act as positional labels to establish topographic projections. They also play a key role in pathway finding by axons and neural crest cells. The crucial roles of Eph receptors and ephrins during development suggest involvement of these genes in congenital disorders affecting the nervous system and other tissues. It has also been suggested that Eph receptors and ephrins may be involved in carcinogenesis. It is therefore of clinical importance to further analyse the function of these molecules, as manipulation of their function may have therapeutic applications.     

神经迁移因子在血管系统中的表达与功能

神经迁移因子是近10年来在发育神经生物学中的研究热点,主要由ephrin、neuropilin、Slit和netrin四大家族成员构成,其主要功能是吸引或排斥神经元轴突的迁移,在神经系统中发挥着重要作用。现在,越来越多的实验证据表明：神经迁移因子的作用不仅仅局限在神经系统发育过程中,在血管发生或新生血管形成中同样具有不可替代的功能。     

Novel roles of the chemorepellent axon guidance molecule RGMa in cell migration and adhesion

The repulsive guidance molecule A (RGMa) is a contact-mediated axon guidance molecule that has significant roles in central nervous system (CNS) development. Here we have examined whether RGMa has novel roles in cell migration and cell adhesion outside the nervous system. RGMa was found to stimulate cell migration from Xenopus animal cap explants in a neogenin-dependent and BMP-independent manner. RGMa also stimulated the adhesion of Xenopus animal cap cells, and this adhesion was dependent on neogenin and independent of calcium. To begin to functionally characterize the role of specific domains in RGMa, we assessed the migratory and adhesive activities of deletion mutants. RGMa lacking the partial von Willebrand factor type D (vWF) domain preferentially perturbed cell adhesion, while mutants lacking the RGD motif affected cell migration. We also revealed that manipulating the levels of RGMa in vivo caused major migration defects during Xenopus gastrulation. We have revealed here novel roles of RGMa in cell migration and adhesion and demonstrated that perturbations to the homeostasis of RGMa expression can severely disrupt major morphogenetic events. These results have implications for understanding the role of RGMa in both health and disease.     

Identifying transient protein-protein interactions in EphB2 signaling by blue native PAGE and mass spectrometry

Receptor tyrosine kinases (RTKs) are proteins that upon ligand stimulation undergo dimerization and autophosphorylation. Eph receptors (EphRs) are RTKs that are found in different cell types, from both tissues that are developing and from mature tissues, and play important roles in the development of the central nervous system and peripheral nervous system. EphRs also play roles in synapse formation, neural crest formation, angiogenesis and in remodeling the vascular system. Interaction of EphRs with their ephrin ligands lead to activation of signal transduction pathways and formation of many transient protein-protein interactions that ultimately leads to cytoskeletal remodeling. However, the sequence of events at the molecular level is not well understood. We used blue native PAGE and MS to analyze the transient protein-protein interactions that resulted from the stimulation of EphB2 receptors by their ephrinB1-Fc ligands. We analyzed the phosphotyrosine-containing protein complexes immunoprecipitated from the cell lysates of both unstimulated (-) and ephrinB1-Fc-stimulated (+) NG108 cells. Our experiments allowed us to identify many signaling proteins, either known to be part of EphB2 signaling or new for this pathway, which are involved in transient protein-protein interactions upon ephrinB1-Fc stimulation. These data led us to investigate the roles of proteins such as FAK, WAVEs and Nischarin in EphB2 signaling.     

The role of astrocyte-secreted matricellular proteins in central nervous system development and function

Matricellular proteins, such as thrombospondins (TSPs1-4), SPARC, SPARC-like1 (hevin) and tenascin C are expressed by astrocytes in the central nervous system (CNS) of rodents. The spatial and temporal expression patterns of these proteins suggest that they may be involved in important developmental processes such as cell proliferation and maturation, cell migration, axonal guidance and synapse formation. In addition, upon injury to the nervous system the expression of these proteins is upregulated, suggesting that they play a role in tissue remodeling and repair in the adult CNS. The genes encoding these proteins have been disrupted in mice. Interestingly, none of these proteins are required for survival, and furthermore, there are no evident abnormalities at the gross anatomical level in the CNS. However, detailed analyses of some of these mice in the recent years have revealed interesting CNS phenotypes. Here we will review the expression of these proteins in the CNS. We will discuss a newly described function for thrombospondins in synapse formation in the CNS in detail, and speculate whether other matricellular proteins could play similar roles in nervous system development and function.     

Phylogenetic analysis of teneurin genes and comparison to the rearrangement hot spot elements of E. coli

Teneurins are a novel family of transmembrane proteins conserved between invertebrates and vertebrates. There are two members in Drosophila, one in C. elegans and four members in mouse. Here, we describe the analysis of the genomic structure of the human teneurin-1 gene. The entire human teneurin-1 (TEN1) gene is contained in eight PAC clones representing part of the chromosomal locus Xq25. Interestingly, many X-linked mental retardation syndromes (XLMR) and non-specific mental retardation (MRX) are mapped to this region. The location of the human TEN1 together with the neuronal expression makes TEN1 a candidate gene for XLMR and MRX. We also identified large parts of the human teneurin-2 sequence on chromosome 5 and sections of human teneurin-4 at chromosomal position 11q14. Database searches resulted in the identification of ESTs encoding parts of all four human members of the teneurin family. Analysis of the genomic organization of the Drosophila ten-a gene revealed the presence of exons encoding a long form of ten-a, which can be aligned with all other teneurins known. Sequence comparison and phylogenetic trees of teneurins show that insects and vertebrates diverged before the teneurin ancestor was duplicated independently in the two phyla. This is supported by the presence of conserved intron positions between teneurin genes of man, Drosophila and C. elegans. It is therefore not possible to class any of the vertebrate teneurins with either Drosophila Ten-a or Ten-m. The C-terminal part of all teneurins harbours 26 repetitive sequence motifs termed YD-repeats. YD-repeats are most similar to the repeats encoded by the core of the rearrangement hot spot (rhs) elements of Escherichia coli. This makes the teneurin ancestor a candidate gene for the source of the rhs core acquired by horizontal gene transfer.     

Expression of Drosophila Ten-a,a dimeric receptor during embryonic development

Ten-a and Ten-m are the two Drosophila members of the newly discovered Ten-m family of dimeric type II transmembrane proteins. Here, we report complete cDNA cloning and protein expression patterns of Ten-a. The Ten-a protein, a dimeric receptor of about 500 kDa is mainly expressed on axons of the embryonic central nervous system and on muscle attachment sites.     

Longitudinal axon guidance

Our knowledge about molecular mechanisms underlying axon guidance along the antero-posterior axis in contrast to the dorso-ventral axis of the developing nervous system is very limited. During the past two years in vitro and in vivo studies have indicated that morphogens have a role in longitudinal axon guidance. Morphogens are secreted proteins that act in a concentration-dependent manner on susceptible groups of precursor cells and induce their differentiation to a specific cell fate. Thus, gradients of morphogens are responsible for the appropriate patterning of the nervous system during early phases of neural development. Therefore, it was surprising to find that gradients of two of these morphogens, Wnt4 and Shh, can be re-used for longitudinal axon guidance during later stages of nervous system development.     

Semaphorins: a new class of immunoregulatory molecules

The immune and nervous systems play distinct roles in maintaining physiological homeostasis. Recent data indicates that these systems influence one another and share many proteins and pathways that are essential for their normal function and development. Molecules originally shown to be critical for the development of proper immune responses have recently been found to function in the nervous system. Conversely, neuronal guidance cues can modulate immune functions. Although semaphorins were originally identified as axon guidance factors active during neuronal development, several recent studies have identified indispensable functions for these molecules in the immune system. This review provides an overview of the rapidly emerging functions of semaphorins and their receptors in the immune system.     

Drosophila Plexin B is a Sema-2a receptor required for axon guidance

Plexin receptors play a crucial role in the transduction of axonal guidance events elicited by semaphorin proteins. In Drosophila, Plexin A (PlexA) is a receptor for the transmembrane semaphorin semaphorin-1a (Sema-1a) and is required for motor and central nervous system (CNS) axon guidance in the developing embryonic nervous system. However, it remains unknown how PlexB functions during neural development and which ligands serve to activate this receptor. Here, we show that plexB, like plexA, is robustly expressed in the developing CNS and is required for motor and CNS axon pathfinding. PlexB and PlexA serve both distinct and shared neuronal guidance functions. We observe a physical association between these two plexin receptors in vivo and find that they can utilize common downstream signaling mechanisms. PlexB does not directly bind to the cytosolic semaphorin signaling component MICAL (molecule that interacts with CasL), but requires MICAL for certain axonal guidance functions. Ligand binding and genetic analyses demonstrate that PlexB is a receptor for the secreted semaphorin Sema-2a, suggesting that secreted and transmembrane semaphorins in Drosophila use PlexB and PlexA, respectively, for axon pathfinding during neural development. These results establish roles for PlexB in central and peripheral axon pathfinding, define a functional ligand for PlexB, and implicate common signaling events in plexin-mediated axonal guidance.