Tenascin major
Teneurin-2, a vertebrate homolog of the Drosophila pair-rule gene ten-m/odz, is revealed to be a membrane-bound transcription regulator. In the nucleus, the intracellular domain of teneurin-2 colocalizes with promyelocytic leukemia (PML) protein in nuclear bodies implicated in transcription control. Since Drosophila ten-m acts epistatically to another pair-rule gene opa, whether gene regulation by the mammalian opa homolog zic-1 was influenced by the intracellular domain of teneurin-2 was investigated. zic-mediated transcription from the apolipoprotein E promoter was inhibited. Release of the intracellular domain of teneurin-2 can be stimulated by homophilic interaction of the extracellular domain, and the intracellular domain is stabilized by proteasome inhibitors. Teneurin-2 is expressed by neurons belonging to the same functional circuit. Therefore, it is hypothesized that homophilic interaction enables neurons to identify their targets and that the release of the intracellular domain of teneurin-2 provides them with a signal to switch their gene expression program from growth towards differentiation once the proper contact has been made (Bagutti, 2003).
Studies in Drosophila have revealed the existence of two members of a new protein family, namely, Ten-a and Ten-m (Baumgartner, 1993; Baumgartner, 1994; Minet, 2000; Fascetti, 2002). Ten-m, also known as odz, is a pair-rule gene. This is surprising, since Ten-m is a cell-surface or secreted protein, and all other pair-rule genes are transcription factors. It has been proposed that Ten-m acts as a pair-rule gene by binding to a receptor, which in turn transmits the extracellular signal into the nucleus. Mutational analysis has indicated that Ten-m initiates a signal transduction cascade via or in concert with opa receptor (Baumgartner, 1994), another pair-rule gene that encodes a zinc finger transcription factor (Bagutti, 2003 and references therein).
During the later stages of development, Ten-a and Ten-m/Odz are predominantly expressed in the nervous system (Levine, 1997; Minet, 1999; Fascetti, 2002). The predominant neuronal expression is conserved in the vertebrate homologs ten-m1, 2, 3 and 4 in the mouse (Oohashi, 1999; Ben-Zur, 2000), neurestin in the rat (Otaki, 1999) and ten-m3 and ten-m4 in zebrafish (Mieda, 1999; Bagutti, 2003 and references therein).
Most of the functional studies have been performed on the avian ten-m family members. Three family members have been described in the chicken so far and have been termed teneurin-1 (Minet, 1999), teneurin-2 (Rubin, 1999) and teneurin-4 (Tucker, 2000). Teneurin-2 is a type II transmembrane protein containing a furin cleavage site in the extracellular domain (Rubin, 1999). Both teneurin-1 and -2 promote neurite outgrowth in vitro (Minet, 1999; Rubin, 1999). Teneurin-2 also acts as a homophilic adhesion protein and may play a role in the specification of neuronal circuits in the developing visual system (Rubin, 2002). In addition to being found in the nervous system, teneurin-2 and -4 are expressed in two important organizing centers of limb development: the apical ectodermal ridge and the zone of polarizing activity, respectively (Tucker, 2001; Tucker, 2000; Bagutti, 2003 and references therein).
Since all members of the teneurin family are type II transmembrane proteins (Rubin, 1999; Feng, 2002), one potential scenario by which such membrane-spanning proteins can fulfill their role as signaling molecules is by a mechanism recently described as regulated intramembrane proteolysis (RIP). RIP is a two-step mechanism that leads to the cleavage of transmembrane proteins at and in the lipid bilayer. The cleavage and release of the extracellular or intraluminal parts of the protein is a prerequisite for a second cleavage, which leads to the separation of the intracellular part from the membrane. The latter takes place within the transmembrane domain. The resulting soluble intracellular part translocates to the nucleus, where it participates in transcription. RIP was first proposed as a signaling model by which the sterol regulatory element binding protein (SREBP) regulates lipid metabolism. It is now known to control diverse cellular and developmental processes. The study of Notch, another protein exerting function by this mechanism, was crucial to discover important features of RIP. Also Ire1 and ATF6, both of which are involved in the unfolded secretory protein pathway (endoplasmatic reticulum stress response), signal through RIP. Amyloid precursor protein (APP; see Drosophila beta amyloid protein precursor-like), which is thought to be involved in Alzheimer's disease, is a prominent example of this mechanism. Not only does proteolysis of APP lead to the accumulation of the toxic APP peptide underlying Alzheimer disease, but RIP may be part of normal APP signaling. The most recently recognized and least described examples of RIP include CD44, ErbB-4, luman and E-cadherin (see Drosophila Shotgun). These diverse examples of RIP could well be just the tip of the iceberg of a large group of transmembrane proteins undergoing proteolytic cleavage to initiate a signal transduction cascade (Bagutti, 2003 and references therein).
It was the aim of the present work to determine whether a similar proteolytic mechanism is responsible for the signaling by teneurins, thus reconciling the enigma of Drosophila ten-m being a pair-rule gene and a bona fide transcription regulator despite its cell-surface location. Indeed the intracellular domain of teneurin-2 can be released from the cell membrane and it translocates to the nucleus where it is able to influence the transcription activity of zic, a vertebrate homolog of the Drosophila Opa (Bagutti, 2003).
Pilot results with a yeast two-hybrid assay indicated transcriptional activity for the intracellular part of teneurin-2. Consistent with this the intracellular domain of teneurin-2 (referred to as construct I) is translocated to the nucleus if transfected into HT1080 cells. Surprisingly, transfection of construct I did not lead to a uniform nuclear accumulation but its expression instead was confined to discrete spots within the nucleus. The nuclear localization coincides with a very similar punctate pattern obtained by staining for PML protein (promyelocytic leukemia protein) and may represent nuclear bodies, termed promyelocytic oncogenic domains (PODs) or PML bodies. Double immunofluorescence staining of construct I transfected cells did show substantial, but not complete, overlap of PML with the intracellular domain of teneurin-2. Since PML bodies are involved in a number of functions associated with transcriptional control, it was of interest to determine whether there was a genuine colocalization of teneurin-2 and PML in nuclear bodies. Therefore construct I was cotransfected with an expression plasmid encoding PML in one case and with PML-RAR (PML-retinoic acid receptor fusion protein) in the other. Transfection of PML into cells containing endogenous PML protein leads to a massive enlargement of the nuclear bodies. In contrast, transfection of PML-RAR should result in a destruction of the PML body architecture. These effects were indeed seen. Interestingly, for teneurin-2 I an equivalent staining pattern was detected after cotransfection with PML, and I was pulled into the enlarged PML bodies. Furthermore, destruction of the PML body architecture after transfection with PML-RAR also changed the expression pattern of cotransfected I, which was no longer accumulated in discrete spots in the nucleus but seemed to be expressed homogeneously throughout the cells. These results indicate that the intracellular domain of teneurin-2 accumulates within nuclear bodies, thus supporting the hypothesis that the intracellular domain of teneurin-2 is involved in transcriptional regulation (Bagutti, 2003).
In Drosophila, Ten-m was postulated to modulate the activity of Opa protein (Baumgartner, 1994). It was therefore of interest to investigate whether the zinc finger transcription factor zic, a vertebrate homolog of Opa, would influence or would be influenced by the intracellular domain of teneurin-2.
When both proteins are expressed in COS-7 cells by transient transfections, a marked downregulation of the intracellular domain I of teneurin-2 was observed compared with its usual expression level. The zic-induced downregulation of the intracellular domain I of teneurin-2 is counteracted by the addition of the proteasome inhibitor lactacystin. Thus the nuclear intracellular domain of teneurin-2 seems to be subject to degradation by the proteasome pathway. By immunofluorescence staining of the transfected cells it was observed that zic-transfected cells reveal a relatively diffuse nuclear staining and in nuclei containing high amounts of zic protein, the punctate staining of teneurin-2 I disappears and becomes diffuse. Thus, the presence of zic prevents the association of the teneurin-2 intracellular domain with PML bodies and makes it amenable to proteasome-mediated degradation (Bagutti, 2003).
The intracellular domain of teneurin-2 appears to have an inhibiting effect on the transcriptional activity of zic, and this effect is more pronounced in the presence of the proteasome inhibitor ALLN, which stabilizes teneurin-2 I.
To be a functional regulator of transcription, wild-type transmembane teneurin-2 would have to be specifically cleaved in or at the plasma membrane, possibly upon a signal by ligand binding. In turn its intracellular part must be released and translocated to the nucleus in a manner similar to that established for proteins regulated by RIP. To test this hypothesis a sensitive method was developed to detect the released intracellular domain of teneurin-2 in the nucleus. Fusion proteins of full-length teneurin-2 (or of smaller transmembrane versions truncated in their extracellular domain) were fused to a Gal4 DNA-binding domain (BD) and a NFkappaB activation domain (AD). If cleavage and translocation to the nucleus occurred, BDAD-I could be detected by binding to specific Gal4 recognition sequences in the promotor of the cotransfected luciferase reporter plasmid, and subsequent initiation of luciferase gene expression activated by AD could be monitored (Bagutti, 2003).
For analysis of the luciferase activity induced by the teneurin-2 fusion constructs, HT1080 cells were cotransfected with the respective BDAD-teneurin-2 constructs, the luciferase reporter plasmid, as well as a ß-galactosidase construct for normalization of transfection efficiencies. BDAD constructs did indeed lead to an induction of luciferase activity above the negative control. Teneurin-2 has recently been shown to bind homophilically by its extracellular domain. It is therefore speculated that this interaction could induce cleavage and translocation of the intracellular domain of the BDAD-teneurin-2 fusion proteins, which in turn would be represented by enhanced luciferase activities. Interactions through the C-terminal half of the extracellular domain were shown to able to stimulate the release of the intracellular domain of teneurin-2 (Bagutti, 2003).
Taken together, it is concluded that the activity of the luciferase reporter gene originates from cleavage of the BDAD-teneurin-2 fusion proteins at (or in the vicinity of) the membrane. However, cleavage of full-length teneurin-2 leads to a significant induction of the luciferase gene only when processing is upregulated by homophilic binding of the extracellular C-terminal part of teneurin-2. Furthermore, the cleaved intracellular domain is subject to rapid degradation by the proteasome pathway (Bagutti, 2003).
Tenascin is a large glycoprotein, expressed in a restricted pattern in the extracellular matrix
(ECM) of vertebrate embryos. Tenascin interferes with cell-fibronectin interactions in vitro, and may play a role in the control of cell migration and differentiation during development. In Xenopus, tenascin immunoreactivity is first detected at the early tailbud stage in the ECM of the most anterior somite. Thereafter, it is distributed dorsally along neural crest cell migration pathways. Tenascin mRNA is most abundant in dorsal mesoderm at the neurula stage and in somites at the early
tailbud stage, indicating that the initial accumulation of tenascin in the ECM is due to secretion from paraxial mesoderm. To understand how tenascin expression in somitic mesoderm is controlled, Xbra and the myogenic factors XMyoD and XMyf5 were expressed in blastula animal cap tissue. The tenascin gene is activated by all three transcription factors. Interestingly, expression of tenascin mRNA, and accumulation of the protein in the ECM, can occur without formation of muscle. These results suggest that tenascin regionalization in early Xenopus embryos depends on tenascin RNA expression in somitic mesoderm, where it is likely to be activated by myogenic factors (Umbhauer, 1994).
The spatiotemporal expression of the chick extracellular matrix protein cytotactin/tenascin during
somitogenesis suggests that it plays a role in the morphogenetic events that give rise to the pattern of
neural crest (NC) development. Prior to NC cell invasion, cytotactin mRNA is restricted to the caudal half of the newly formed
epithelial somites. As each epithelial somite matures, giving rise to a sclerotome and dermamyotome,
the mRNA is first restricted to the dermamyotome and later restricted to the rostral protion of the
sclerotome, consistent with the protein distribution. Analysis
of the distribution of cytotactin and NC cells in embryos with ablations that remove NC cells, or with
simple wounds that leave NC cells in place, demonstrate that the presence of NC cells is neither
necessary nor sufficient for the correct positioning of cytotactin. Cytotactin synthesized by sclerotomes in the absence of NC cells is of similar molecular mass to that
produced in their presence. These findings are in accord with the notion that the abnormalities of
cytotactin distribution are related to the wounding process. There is no causal link between the
presence of NC cells and the distribution and molecular mass of sclerotomal cytotactin (Tan, 1991).
Zebrafish ten-m3 and ten-m4 encode proteins highly
similar to the product of Drosophila pair-rule gene tenm/odd Oz
(odz). Their products contain eight epidermal growth factor (EGF)-like
repeats that resemble mostly those of tenascin, an extracellular matrix molecule.
During the segmentation period, ten-m3 is expressed in the
somites, notochord, pharyngeal arches, and the brain, while expression of
ten-m4 is mainly restricted to the brain. In the developing brain,
ten-m3 and ten-m4 expression delineates several
compartments. Interestingly, ten-m3 and ten-m4 show
expression patterns complementary to one another in the developing
forebrain and midbrain along both rostrocaudal and dorsoventral axes,
depending on developmental stages and locations (Mieda, 1999).
During development of the chick peripheral nervous system, the extracellular matrix molecule tenascin has been
found to be closely associated with growing axons. However, its origin and function in peripheral nerve
formation are far from clear. During outgrowth of sensory and motor axons, a high concentration of tenascin and its
mRNA surrounds sensory and motor axons in the newly formed spinal nerves. Neural crest removals do not
alter the distribution of tenascin protein or its mRNA surrounding the spinal nerves. Transplantation of
quail somites into chick embryos shows that, similar to the distribution of tenascin, there is a high
concentration of somitic cells surrounding the spinal nerves. Moreover, somite removals result in a
reduction of the tenascin and tenascin mRNA surrounding the spinal nerves. Taken together, these results
suggest that the majority of the tenascin surrounding the spinal nerves is of somitic origin. Possible
functions of tenascin associated with peripheral nerve formation were examined through injections of
tenascin or its antiserum into individual somites prior to or during axon outgrowth. Injections of tenascin
or its antiserum do not alter the trajectory of peripheral axons in the anterior half of the somite, nor
produce gross abnormalities in the morphology of peripheral nerves, suggesting that tenascin does not play a crucial role in the early formation of peripheral nerves (Yip, 1995).
Mutations in the gene for the transcription factor Pax6 (Drosophila homolog: Eyeless) induce marked developmental abnormalities in the CNS and the eye, but the cellular mechanisms that underlie the phenotype are unknown. The adhesive properties of cells from the developing forebrain in Small eye, the Pax6 mutant mouse, have been examined. The
segregation normally observed in aggregates of cortical and striatal cells in an in vitro
assay is lost in Small eye. This correlates with an alteration of in vivo expression of
the homophilic adhesion molecule, R-cadherin, which is expressed exclusively in the cortex. Moreover, the boundary between cortical and striatal regions of the telencephalon is dramatically altered in Small eye: radial glial fascicles do not form at the border, and the normal expression of R-cadherin and tenascin-C at the border is lost. These data suggest links between the transcription factor Pax6 and R-cadherin expression, cellular adhesion and boundary formation between developing forebrain regions (Stoykova, 1997).
Astrocytes are heterogeneous in expression of the ECM molecule tenascin. High-tenascin
astrocytes have a reduced ability to support neurite outgrowth. In addition, astrocytes treated with
exogenous basic fibroblast growth factor (See Drosophila Branchless) support reduced neuronal growth and adhesion. Basic FGF was added to cultures of rat cerebral cortical astrocytes at concentrations of up
to 30 ng/ml, concentrations shown to have a significant effect on neuronal adhesion. Tenascin levels begin to increase after 24-48 hr and continued to
increase throughout 8 days in culture. The increase in tenascin is concentration-dependent, with the
largest increase seen at 5 ng/ml bFGF. Tenascin production increases approximately 5.5-fold in
serum-containing medium but only about 2-fold in serum-free medium. When heparin is included along with bFGF in serum-free medium, tenascin production is further
enhanced. The bFGF treatment was discontinued after 8 days, and the cells were maintained for an
additional 8 days in culture. Tenascin levels returned to control values, demonstrating that the bFGF
effect is transient. The action of bFGF during injury may evoke the induction of
tenascin on astrocytes, thereby reducing regeneration in the central nervous system (Meiners, 1993).
O4(+) oligodendrocyte (OL) progenitors in the mammalian CNS are committed fully to terminal differentiation into myelin-forming
cells. In the absence of other cell types in vitro, OL differentiation reproduces the in vivo development with a correct timing,
suggesting the existence of an intrinsic regulatory mechanism (presently unknown). Examination has been made of the effect on the adhesion and maturation of OLs in vitro of two
isoforms of the extracellular matrix (ECM) molecule tenascin-R (TN-R); these isoforms are expressed by OLs during the process of
myelination. The substrate-bound molecules support the adhesion
of O4(+) OLs, independent of either the CNS region or the aged animals from which they were derived. At the molecular level this process is
mediated by protein binding to membrane surface sulfatides (Sulf), as indicated by the interference of O4 antibody and Sulf with the
attachment of OLs or other Sulf+ cells (erythrocytes) to TN-R substrates and by direct protein-glycolipid binding studies. In the
absence of platelet-derived growth factor (PDGF), exogenous TN-R induces myelin gene expression and the upregulation of its own
synthesis by cultured cells, resulting in a rapid terminal differentiation of O4(+) progenitors. These findings strongly suggest that TN-R
represents an intrinsic regulatory molecule that controls the timed OL differentiation by an autocrine mechanism and imply the
relevance of TN-R for CNS myelination and remyelination (Pesheva, 1997).
A novel cDNA encoding a putative transmembrane protein, neurestin, has been cloned from the rat olfactory bulb. Neurestin was identified based on a sequence
similar to that of the second extracellular loops of odorant receptors in the cysteine-rich CC box located immediately C-terminal to EGF-like motifs. Neurestin shows
homology to a neuregulin gene product, human gamma-heregulin, a Drosophila receptor-type pair-rule gene product, Odd Oz (Odz)/Ten(m), and Ten(a),
suggesting a possible function in synapse formation and morphogenesis. Homology to gamma-heregulin and Ten(a) is confined to the extracellular region of neurestin. The intracellular domain of neurestin contains CDC boxes conserved among neurestin, (Odz)/Ten(m), a C. elegans Odz homolog cOdz, and a cytosolic component of desmosome termed band-6-protein/plakophilin. Recently, a mouse neurestin homolog has independently been cloned as DOC4 from the
NIH-3T3 cell line. Northern blot analysis shows that neurestin is highly expressed in the brain and also in other tissues at much lower levels. During
development, neurestin is expressed in many types of neurons, including pyramidal cells in the cerebral cortex and tufted cells in the olfactory bulb. In adults, neurestin is mainly expressed in olfactory and hippocampal granule cells, which are known to be generated throughout adulthood.
Nonetheless, in adults the expression of neurestin is experimentally induced in external tufted cells during regeneration of olfactory sensory neurons. These results
suggest a role for neurestin in neuronal development and regeneration in the central nervous system (Otaki, 1999).
Teneurins are a family of type II transmembrane proteins originally discovered in Drosophila. The first member was Ten-a, which was found in a search for Drosophila homologs of tenascins and shares with this protein family the same type of EGF-like repeats. The second member of the teneurin family, Drosophila Ten-m/Odd oz (Odz), is expressed in seven
stripes during the blastoderm stage in early embryos. Later in development, teneurins are prominently expressed by specific subpopulations of neurons
in Drosophila, rat, chicken and zebrafish. Although teneurin 2 shows highest expression in the nervous system, it is also expressed at other locations known to be crucial regulatory sites of morphogenesis, such as the apical ectodermal ridge and the dorsomedial lip of the somite (Rubin, 2002 and references therein).
The domain organization of teneurins in invertebrates and vertebrates is highly conserved. All teneurins have a proline-rich cytoplasmic domain, and
extracellularly contain a series of EGF-like repeats and 26 YD repeats. The cytoplasmic domain may be
involved in a signal transduction cascade, since mutational analysis showed that ten-m/odz is a member of the 'pair-rule' gene family and has a central
role in determining the segmentation of the Drosophila embryo. A highly conserved dibasic furin-like cleavage site is
found between the transmembrane domain and the EGF-like repeats, meaning that teneurins may be proteolytically processed
in the same way as Notch. The function of the EGF-like repeats is unknown, but a recombinant murine teneurin forms
side-by-side dimers in vitro that appear to be linked via disulfide bridges between the EGF-like repeats. The YD repeats
bind heparin and are similar to those found in the rhs element of E. coli and in wall associated protein A of Bacillus subtilis,
where they may have appeared due to horizontal gene transfer from an ancestral teneurin (Rubin, 2002 and references therein).
Unlike Drosophila, which has two teneurin genes, vertebrates have up to four teneurin genes. Although different nomenclatures have been developed in laboratories using different animal models, the number designation at the end of each name can be used to identify the orthologous genes: murine ten-m1 is the same as murine odz1 and corresponds to avian teneurin 1, etc. Note that the murine Doc4 gene encodes ten-m4, and that the rat teneurin 2 ortholog has also been called neurestin. In addition, at least three alternatively
spliced variants of teneurin 2 have been identified, including one variant that lacks the YD repeats. The functional significance of these variations is unknown (Rubin, 2002 and references therein).
There is some experimental evidence that teneurins may play a role in neurite outgrowth and pathfinding. In vitro, the YD repeats of teneurin 2 support the outgrowth of neurites, and this outgrowth is abolished by heparin. Transfection of Nb2a neuroblastoma cells with chicken teneurin 2 expression constructs results in the formation of numerous teneurin 2-enriched filopodia and enlarged growth cones, suggesting an interaction of teneurin 2 with the cytoskeleton. In situ hybridization reveals non-overlapping neuronal expression of teneurin 1 and teneurin 2 in interconnected parts of the developing diencephalon and midbrain. The possibility of homophilic interactions between these teneurins is supported by the observations that labeled ten-m1 binds to ten-m1 on blots, and labeled ten-m1 binds to ten-m1-rich regions of tissue sections. Finally, the human teneurin 1 gene maps to the same part of the X-chromosome (Xq25) as an X-linked mental retardation syndrome characterized by sensory neuropathology (Rubin, 2002 and references therein).
Teneurin 2 is expressed by neurons in specific brain regions; these neurons are known to be part of a specific circuit, namely the thalamofugal visual system of the chicken. Teneurin 2 is expressed at the time when axons find their targets. Expression of the cytoplasmic domain is required for the induction of filopodia, the transport of teneurin 2 into neurites and the co-localization of teneurin 2 with the cortical actin cytoskeleton. In addition, expression of the extracellular domain of teneurin 2 by HT1080 cells induces cell aggregation, and the extracellular domain of teneurin 2 become concentrated at sites of cell-cell contact in neuroblastoma cells. These observations indicate that the homophilic binding of teneurin 2 may play a role in the development of specific neuronal circuits in the developing visual system (Rubin, 2002).
The complex spatial and temporal pattern of teneurin 2 expression largely coincides with the development of the thalamofugal visual
pathway. At early stages of development this was seen by teneurin 2 expression in the retina and dorsal thalamus. At later stages, the hyperstriatum accessorium (HA or visual Wulst) and its targets are major sites of teneurin 2 expression. Teneurin 1, another member of the same
protein family, is expressed in the retina, optic tectum and rotund nucleus at E14. Thus, teneurin 1 is expressed by
interconnected neurons in the tectofugal visual pathway, and teneurin 2 is expressed by interconnected neurons in the thalamofugal pathway. These observations led to a hypothesis that homophilic binding of teneurins may assist in the formation of appropriate synapses and fasciculation
in the developing visual system. This hypothesis was tested in vitro by examining the morphology and behavior of cells transfected with full-length and partial sequences encoding teneurin 2. It was found that teneurin 2 expressing HT1080 cells aggregated with each other. Furthermore, in transiently
transfected Nb2a cells, teneurin 2 accumulated at cell-cell contact sites. In the presence of the cytoplasmic domain of teneurin 2, F-actin became
concentrated at these cell-cell contact sites as well. This is intriguing as F-actin was shown to be required for the development and stabilization of
young synapsis in cultured hippocampal neurons. It will be interesting to analyze whether other synapse-specific proteins
are attracted to these sites and which cellular proteins can interact with the cytoplasmic domain of teneurin 2 (Rubin, 2002).
Although the principal sites of teneurin 2 expression were parts of the thalamofugal visual pathway, teneurin 2 was found in a few other parts of the
embryonic CNS as well. The most prominent of these are the expression seen in Hp, the SN and nucleus taenia. Although not considered part of
the thalamofugal visual pathway, these interconnected regions are speculated to play a role in spatial memory and pattern recognition. Another interesting site of expression is the IO, which sends processes back along the teneurin 2-positive optic tract to the retina. Both the Hp and the IO project to regions that also expressed teneurin 2, supporting the hypothesis that teneurin
2/teneurin 2 interactions may play a role in the development of appropriate synapses. Finally, two other regions that were positive for teneurin 2
expression at E18, the olfactory bulb and cerebellum, are connected to the teneurin 2-positive regions of the developing visual system. The latter receives fibers from the visual
Wulst, and the former receives fibers from a part of the hypothalamus that: (1) is a target of retinal projections and (2) also projects to the visual Wulst and hippocampus (Rubin, 2002).
A striking feature of the CNS is the precise wiring of its neuronal connections. During vertebrate visual system development, different subtypes of retinal ganglion cells (RGCs) form specific connections with their corresponding synaptic partners. However, the underlying molecular mechanisms remain to be fully elucidated. This study reports that the cell-adhesive transmembrane protein Teneurin-3 is required by zebrafish RGCs for acquisition of their correct morphological and functional connectivity in vivo. Teneurin-3 is expressed by RGCs and their presynaptic amacrine and postsynaptic tectal cell targets. Knockdown of Teneurin-3 leads to RGC dendrite stratification defects within the inner plexiform layer, as well as mistargeting of dendritic processes into outer portions of the retina. Moreover, a subset of RGC axons exhibits tectal laminar arborization errors. Finally, functional analysis of RGCs targeting the tectum reveals a selective deficit in the development of orientation selectivity after Teneurin-3 knockdown. These results suggest that Teneurin-3 plays an instructive role in the functional wiring of the vertebrate visual system (Antinucci, 2013).
Brain functions rely on specific patterns of connectivity. Teneurins are evolutionarily conserved transmembrane proteins that instruct synaptic partner matching in Drosophila and are required for vertebrate visual system development. The roles of vertebrate teneurins in connectivity beyond the visual system remain largely unknown and their mechanisms of action have not been demonstrated. This study shows that mouse teneurin-3 is expressed in multiple topographically interconnected areas of the hippocampal region, including proximal CA1, distal subiculum, and medial entorhinal cortex. Viral-genetic analyses reveal that teneurin-3 is required in both CA1 and subicular neurons for the precise targeting of proximal CA1 axons to distal subiculum. Furthermore, teneurin-3 promotes homophilic adhesion in vitro in a splicing isoform-dependent manner. These findings demonstrate striking genetic heterogeneity across multiple hippocampal areas and suggest that teneurin-3 may orchestrate the assembly of a complex distributed circuit in the mammalian brain via matching expression and homophilic attraction (Berns, 2018).
A striking feature of neural development is the formation of highly precise connections between neurons. Sensory and motor circuits have been extensively used to characterize the molecular control of wiring specificity, but relatively little is known about how neurons in complex high-order circuits find appropriate partners. This study has shown that Ten3 acts in both pre- and postsynaptic neurons in the hippocampus to control the assembly of a precise topographic projection. Loss-of-function phenotypes support a homophilic attraction mechanism: when Ten3 is lost from CA1 neurons, proximal CA1 axons spread throughout the entire subiculum, instead of projecting only to distal, Ten3-high targets; when Ten3 is lost from a subset of distal subicular cells, Ten3-high proximal CA1 axons do not target these areas and instead innervate nearby Ten3-high regions. In vitro data further show that Ten3 can interact homophilically in trans, supporting a model in which Ten3 on CA1 axons interacts with Ten3 on subicular targets, leading to contact-mediated attraction or stabilization of proximal CA1 axons by distal subicular target cells. This mechanism of action resembles that of the Drosophila teneurins in the development of olfactory and neuromuscular connections, suggesting an evolutionarily conserved mode of teneurin function in neural circuit assembly from insects to mammals. However, whereas Drosophila teneurins instruct matching of discrete types of pre- and postsynaptic cell, the graded expression in both CA1 and subiculum suggests that mouse Ten3 directs continuous topographic mapping along the proximal-distal axis (Berns, 2018).
This model does not exclude the possibility that interactions of Ten3 with heterophilic partners have additional roles in circuit assembly. While in many cases Ten3 expression was observed in both pre- and postsynaptic partners of specific connections, there were also cases where Ten3 was only observed in axons but not targets (for example, MEC->dentate gyrus-CA3). Further, the A0B0 isoform did not exhibit homophilic interactions but did interact heterophilically with latrophilin-3. These observations suggest that interactions between Ten3 and latrophilins or other potential heterophilic partners may also contribute to wiring specificity (Berns, 2018).
The results highlight small regions of Ten3 that are critical for trans interactions. Splice site A corresponds to the most C-terminal of the eight EGF-like repeats, which are thought to mediate cis-dimerization of teneurin proteins. The results suggest that the EGF-like repeats may also participate in trans interactions, or that teneurin cis interactions may influence trans interactions. Splice site B is within the NHL-repeat region, which was implicated in homophilic teneurin interactions using single-cell force spectroscopy. This result supports the importance of the NHL repeats, and identifies specific residues that are required for homophilic interactions (Berns, 2018).
What controls the distal CA1 -> proximal subiculum projection? Since none of the other three mouse teneurins exhibited differential expression along the proximal-distal axis of the hippocampal formation, other differentially expressed proteins might act in parallel with Ten3 to control the distal CA1->proximal subiculum projection. Axon-axon competition could also contribute, as in retinotopic map development. Indeed, the enhanced severity of the CA1-specific conditional knockout phenotype compared with the whole-animal Ten3KO phenotype may result from Ten3-expressing CA1 axons out-competing mutant axons for space in distal subiculum, supporting a role of axon-axon competition in determining CA1→subiculum targeting specificity (Berns, 2018).
Finally, the findings reveal genetic heterogeneity within many areas of the hippocampal region. Although genetic analyses focused on the CA1→subiculum projection, Ten3-high to Ten3-high connectivity was also observed in the entorhinal -> hippocampal projections, and probably exists in additional hippocampus-associated projections. The matching expression of Ten3 in multiple topographically connected subregions, combined with loss-of-function and in vitro data, suggests that Ten3 may control the assembly of a widely distributed circuit in mammalian brain (Berns, 2018).
Bidirectional signaling by cell adhesion molecules is thought to mediate synapse formation, but the mechanisms involved remain elusive. This study found that the adhesion G protein-coupled receptors latrophilin-2 and latrophilin-3 (see Drosophila Cirl) selectively direct formation of perforant-path and Schaffer-collateral synapses, respectively, to hippocampal CA1-region neurons. Latrophilin-3 binds to two transcellular ligands: fibronectin leucine-rich repeat transmembrane proteins (FLRTs) and teneurins (see Drosophila Tenascin major). In transgenic mice in vivo, both binding activities were required for input-specific synapse formation, which suggests that coincident binding of both ligands is necessary for synapse formation. In cultured neurons in vitro, teneurin or FLRT alone did not induce excitatory synapse formation, whereas together they potently did so. Thus, postsynaptic latrophilins promote excitatory synapse formation by simultaneous binding of two unrelated presynaptic ligands, which is required for formation of synaptic inputs at specific dendritic localizations (Sando, 2019).
How synapses form, how they are maintained, and what molecular processes establish specificity in synaptic connections remain fundamental unanswered questions in neuroscience. This study provides three findings that reveal mechanisms involved in input-specific synapse formation in the brain and suggest an explanation for synapse specificity (Sando, 2019).
First, this study has shown that Lphn3 is specifically targeted to the dendritic domains of the S. oriens and S. radiatum of hippocampal CA1 pyramidal neurons, whereas the highly homologous Lphn2 is specifically targeted to the S. lacunosum-moleculare in the same neurons. Both Lphn2 and Lphn3 are essential for subsets of excitatory synapses on the dendritic domain to which they are targeted, suggesting that different isoforms of the same postsynaptic protein family differentially function in distinct synapses. These findings thus provide an explanation for the evolution of homologous adhesion GPCRs and their coexpression in the same neuron, and reveal that different isoforms of a postsynaptic cell recognition molecule can be targeted to distinct dendritic domains (Sando, 2019).
Second, this study has shown that autoproteolysis mediated by the GAIN domain-a canonical feature of adhesion GPCRs-is not required for Lphn3 function, suggesting that their activation does not involve the exposure of an intrinsic tethered agonist that is rendered competent for receptor binding by removal of the extracellular domains of Lphn3 (Sando, 2019).
Third, this study has shown that individual inactivation of FLRT binding or of teneurin binding to Lphn3 blocked its function in synapse formation, and that in the in vitro synapse formation paradigm, teneurin-2 and FLRT3 induced excitatory synapse formation only when they were coexpressed. Even when FLRT3 and teneurin-2 were coexpressed, only the teneurin-2 splice variant capable of binding to latrophilins was active in synapse formation. These results suggest that the requirement for two simultaneous ligands enables a higher specificity in synapse formation. More generally, the coincidence signaling by multiple ligands as described in this study contributes to the emerging realization that signal integration and coincidence detection are a key feature in synaptic plasticity and neural circuit. The results suggest that input-specific synapse formation requires integration of multiple transsynaptic signals acting on latrophilin adhesion GPCRs (Sando, 2019).
These results are at odds with several previous results. It has been proposed that latrophilins are presynaptic and FLRTs are postsynaptic, but this conclusion was largely based on the notion that latrophilins as α-latrotoxin receptors should be presynaptic. Moreover, a recent study arguing for a postsynaptic localization of FLRT2 is confounded by the use of an antibody targeting the FLRT2 extracellular region for localization analysis, which is presumably localized in the synaptic cleft, and the use of short hairpin RNA-mediated knockdowns, which are difficult to control. It was also proposed that teneurins act in establishing synaptic connectivity not as heterophilic but as homophilic cell adhesion molecules, but in assays described in this paper, teneurins do not engage as homophilic cell adhesion molecules, and teneurins act exclusively as presynaptic cell adhesion molecules (Sando, 2019).
The results raise multiple questions. For example, what is the nature of the postsynaptic signal that is activated by latrophilins during synapse formation? How is the specificity of Lphn2 and Lphn3 for different dendritic domains in the same pyramidal neuron determined, and is this due to intrinsic sequence determinants or to differential ligand-binding affinities? What postsynaptic ligands mediate teneurin action in inhibitory synapse formation? FLRT3 can simultaneously bind to Lphn3 and to Unc5 (a Netrin receptor protein involved in axon guidance during development) in a trans configuration, which suggests that the transsynaptic teneurin-latrophilin-FLRT complex may be even larger. As a result, this complex may include postsynaptic Unc5, which in turn could bind to yet another presynaptic adhesion molecule. These large, multiprotein transsynaptic complexes may be modular and may differ in distinct synapse subtypes to increase specificity and generate functional diversity. Thus, the overall portrait of synapse formation emerging from these data is that different latrophilin isoforms are targeted to defined postsynaptic dendritic domains, where they mediate specific excitatory synapse formation by binding to presynaptic FLRTs and teneurins on incoming axons (Sando, 2019).q
The trans-synaptic interaction of the cell-adhesion molecules teneurins (TENs; see Drosophila Ten-m) with latrophilins (LPHNs/ADGRLs; see Drosophila Cirl) promotes excitatory synapse formation when LPHNs simultaneously interact with FLRTs. Insertion of a short alternatively-spliced region within TENs abolishes the TEN-LPHN interaction and switches TEN function to specify inhibitory synapses. How alternative-splicing regulates TEN-LPHN interaction remains unclear. This study reports the 2.9 Å resolution cryo-EM structure of the TEN2-LPHN3 complex and describes the trimeric TEN2-LPHN3-FLRT3 complex. The structure reveals that the N-terminal lectin domain of LPHN3 binds to the TEN2 barrel at a site far away from the alternatively spliced region. Alternative-splicing regulates the TEN2-LPHN3 interaction by hindering access to the LPHN-binding surface rather than altering it. Strikingly, mutagenesis of the LPHN-binding surface of TEN2 abolishes the LPHN3 interaction and impairs excitatory but not inhibitory synapse formation. These results suggest that a multi-level coincident binding mechanism mediated by a cryptic adhesion complex between TENs and LPHNs regulates synapse specificity (Li, 2020).
Latrophilin-1 (LPHN1; see Drosophila Cirl) was isolated as the main high-affinity receptor for alpha-latrotoxin from black widow spider venom, a powerful presynaptic secretagogue. As an adhesion G-protein-coupled receptor, LPHN1 is cleaved into two fragments, which can behave independently on the cell surface, but re-associate upon binding the toxin. This triggers intracellular signaling that involves the Galphaq/phospholipase C/inositol 1,4,5-trisphosphate cascade and an increase in cytosolic Ca(2+), leading to vesicular exocytosis. This study isolated its endogenous ligand, teneurin-2/Lasso (see Drosophila Ten-m). Both LPHN1 and Ten2/Lasso are expressed early in development and are enriched in neurons. LPHN1 primarily resides in axons, growth cones and presynaptic terminals, while Lasso largely localizes on dendrites. LPHN1 and Ten2/Lasso form a trans-synaptic receptor pair that has both structural and signaling functions. However, Lasso is proteolytically cleaved at multiple sites and its extracellular domain is partially released into the intercellular space, especially during neuronal development, suggesting that soluble Lasso has additional functions. This study discovered that the soluble fragment of Lasso can diffuse away and bind to LPHN1 on axonal growth cones, triggering its redistribution on the cell surface and intracellular signaling which leads to local exocytosis. This causes axons to turn in the direction of spatio-temporal Lasso gradients, while LPHN1 knockout blocks this effect. These results suggest that the LPHN1-Ten2/Lasso pair can participate in long- and short-distance axonal guidance and synapse formation (Ushkaryov, 2019).
Teneurins are ancient metazoan cell adhesion receptors that control brain development and neuronal wiring in higher animals. The extracellular C terminus binds the adhesion GPCR Latrophilin, forming a trans-cellular complex with synaptogenic functions. However, Teneurins (see Drosophila Ten-m), Latrophilins (see Drosophila Cirl), and FLRT proteins are also expressed during murine cortical cell migration at earlier developmental stages. This study presents crystal structures of Teneurin-Latrophilin complexes that reveal how the lectin and olfactomedin domains of Latrophilin bind across a spiraling beta-barrel domain of Teneurin, the YD shell. Structure-based protein engineering was coupled to biophysical analysis, cell migration assays, and in utero electroporation experiments to probe the importance of the interaction in cortical neuron migration. Binding of Latrophilins to Teneurins and FLRTs directs the migration of neurons using a contact repulsion-dependent mechanism. The effect is observed with cell bodies and small neurites rather than their processes. The results exemplify how a structure-encoded synaptogenic protein complex is also used for repulsive cell guidance (Del Toro, 2020).
In the spinal cord, the axonal tracts with various caliber sizes are myelinated by oligodendrocytes and function as high-velocity ways for motor and sensory nerve signals. In some neurological disorders, such as multiple sclerosis, demyelination of small caliber axons is observed in the spinal cord. While type I/II oligodendrocytes among the four types are known to myelinate small diameter axons, their characteristics including identification of regulating molecules have not been understood yet. This study found that in the wild-type mouse spinal cord, type I/II oligodendrocytes, positive for carbonic anhydrase II (CAII), were located in the corticospinal tract, fasciculus gracilis, and the inside part of ventral funiculus, in which small diameter axons existed. The type I/II oligodendrocytes started to appear between postnatal day (P) 7 and 11. The type I/II oligodendrocytes were further examined in the mutant mice, whose small diameter axons were hypomyelinated due to the deficiency of teneurin-4 (see Drosophila Tenascin major). In the teneurin-4 deficient mice, type I/II oligodendrocytes were significantly reduced, and the onset of the defect was at P11. These results suggest that CAII-positive type I/II oligodendrocytes myelinate small caliber axons in the spinal cord and teneurin-4 is the responsible molecule for the generation of type I/II oligodendrocytes (Hayashi, 2020).
Return: see Tenascin major Evolutionary Homologs part 1/2
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Tenascin major:
Biological Overview
| Regulation
| Developmental Biology
| Effects of Mutation
| References
Society for Developmental Biology's Web server.