Teneurin, the animal gene family, has members widely reported in triploblast-bilaterians (Tucker et al., 2012; Mosca, 2015), but never in diploblasts. Every sequenced bilaterian genome has at least one Teneurin, but no trace of them can be found in sequenced diploblast genomes to date. The only non-bilaterian Teneurin gene is found in one choanoflagellate, in the phylum considered to be the closest living phylum to that of the animal kingdom (Tucker et al., 2012). That exception is treated below as “footnote three” in section V.

Teneurins encode type II transmembrane proteins composed of four distinct domain component regions: an intracellular domain; a single trans-membrane spanning domain; a extracellular domain with EGF-like repeats and its associates; and carboxy-terminally, a roughly 2000 aa ‘multi-domain entity’ whose three dimensional structure has been solved (Figure 1, Jackson et al., 2018; Li et al., 2018). Prior to these independent, and highly concurring, 3D solutions of this 2000 aa “super-fold” (Jackson et al., 2018), extracellular Teneurin domains were described using varied borders and domain names. The 2000 aa super-fold can be treated as one discrete unit in an evolutionary discussion, in that: it was adopted, all domains en masse, from a precursor gene encoding a bacterial protein; and it is essentially invariant among all Teneurins.

It is therefore most likely that the first Teneurin arose from an ancient small number of gene fusions that brought together the four mentioned component regions. A pair of fusions of a gene encoding a trans-membrane helix: to one encoding EGF-like repeats, C-terminally; and to one encoding an intracellular stretch, N-terminally; led to the type II membrane-orientation now found for every Teneurin (Figure 1A,B). The intracellular and transmembrane domains are highly variable and are poorly conserved between Teneurins. Therefore no homology can be found, nor relationships inferred, between these domains and non-Teneurin proteins’ domains.

In contrast, the affinities between Teneurin EGF-like repeats and analogous domains in other proteins and protein families can be examined. EGF-like repeats are nearly exclusively an animal phenomenon (Wouters et al., 2005). A number of EGF-like-domain bearing proteins have family orthologs that populate and ‘straddle’ both protostome and deuterostome genomes, so were likely present in the ‘urBilaterian.’ From among those ancient ‘cross-bilaterian’ proteins, the EGF-like repeats of Teneurin are most like those of Delta, Serrate/Jagged, Notch, Wif1/Shifted, Eyes-shut, Crumbs, Integrin beta2, and Slit. They are a subset of proteins that contain hEGFs, rather than cEGFs (Wouters et al., 2005). The gene fusion to include these EGF-like repeats into urTeneurin must have taken place in the urBilaterian, most likely by adopting EGF-repeats from one of these proteins. The protein bearing the most similar EGF-like block is most likely the one whose gene Teneurin “adopted from.” The eight EGF-like repeat content of Teneurin probably arose from a combination of: the integration of several adopted EGF-like encoding repeats as-a-block, plus accretion of repeats via tandem duplications (and possible repeat loss). Proposing candidate protein families as the most likely donors of Teneurin’s EGF-repeats will likely require modeling and testing a combination of these processes. Note: it is formally possible that Teneurins adopted EGFs from some no longer existing family, rather than one among the cluster above.

There is only one set of EGF-like repeats more similar to those of Teneurins, rather than those of the proteins listed above: the Tenascins. Tenascins arose in chordates, and are absent in any other phyla of animals, or life (Tucker and Chiquet-Ehrismann, 2009; Adams et al., 2015). Since Tenascins’ EGFs are closest to those of Teneurins, Tenascin’s EGFs most likely arose from deuterostome-chordate, Teneurin EGF-like domains. Genes encoding these EGFs must have fused with those encoding fibronectin type-III, and fibrinogen, domains, during Tenascin’s genesis. Historically, Teneurins therefore take their name from their ‘offspring.’ Another conceivable scenario is that Tenascins adopted EGFs from some no longer existing family, or from proteins extant only in lower chordates. There will be further discussion of this below, in section III.

Proceeding carboxy terminally, the remainder of Teneurin is the 2000 aa ‘super-fold.’ Since it is solved by cryo-EM and X-ray crystallography, the super-fold can now be coherently described using the nomenclature of these structure-solution papers. The terms used now are able to anchor the domains within the super-fold onto clearly-delineated 3D folds. However, the folds are named differently by the two groups: TTR-FN-plug/Ig-like; NHL/beta-propeller; YD-shell/Barrel, ABD, and Tox GHH [see Figure 1B, as superimposed basically on chicken/human Teneurin-2, (Jackson et al., 2018; Li et al., 2018)]. Subsequent to, or concomitant with, the gene fusions described above, this greater than 2000 aa structure was co-opted as-a-whole from bacteria, and was fused downstream of the EGF-like repeats. The wholesale adoption of this huge block of domains was not recognizable until recent bacterial genomes were sequenced. Some 100 newly sequenced genomes of bacterial species, collected mostly since 2016, encode proteins with this 2000 aa block. In the 100 cases, sequences of Teneurin (for instance of M. brevicollis or Ten-m of D. melanogaster) have about 70% of their length covered at more than 25% aa identity to the prokaryotic proteins. The homology starts just after the Teneurin EGF repeats, and covers nearly the entire post-EGF length of Teneurin. Five of the best hits are presented in Figure 1C. One of these hits (Figure 1C) was recognized as an analogous prokaryotic ‘super-fold’ containing protein in Bacillus subtilis strain CW14 in the structure paper (Jackson et al., 2018). The Teneurin extra-cellular regions’ structural homology to bacterial Tc-toxins is pivotal in the other structure paper (Li et al., 2018). A recognition that the homology between Teneurin and bacterial proteins extends to a full 2000 aa was also made in Ferralli et al. (2018). The Desulfurivibrio alkaliphilus YD protein discussed there also appears among the best hits shown here in Figure 1C. Note that before these recent abundant new bacterial genes were sequenced and recognized, Teneurins at best had RhS and YD-repeat portions with noticeable homology to bacteria. These previous alignments were shorter and localized (Minet and Chiquet-Ehrismann, 2000; Tucker et al., 2012). Those alignments did not suggest a wholesale adoption of the post-EGF portion of Teneurins as a block.

The genomes of protostomes contain a single Teneurin gene (Tucker et al., 2012) (Figure 2). The marked exception occurs in a single clade, the Arthropods, in all of its species’ genomes. The genomes of species in the Arthropod Phylum contain two Teneurin paralogs, Ten-a and Ten-m (Figure 2, 3B). Representatives shown for each sub-phylum (Figure 2, 3) have a Ten-a, and a Ten-m, gene: the fruit fly Drosophila melanogaster for insects in the sub-phylum of Hexapoda; the tick Ixodes scapularis for the class of arachnids, in the sub-phylum Chelicerata; the water flea Daphnia pulex for the class of branchiopoda, in the sub-phylum Crustacea; and the millipede Strigamia maritima for the class of millipedes, in the sub-phylum Myriapoda. A single duplication of an ancestral protostome Teneurin gene before divergence of the four sub-phyla, yielding Ten-a and Ten-m, is consistent with the trees’ geometries. The relative phylogenetic distances between Ten-a and Ten-m are appropriate to the accepted evolutionary history of the four sub-phyla (Borner et al., 2014; Chipman et al., 2014; Misof et al., 2014).

All genomes of species from other protostome phyla contain a single Teneurin (Figure 2, 3). This statement includes all lophotrochozoa. More importantly for this discussion, it also includes all non-arthropod genomes examined from among the ecdysozoan cluster phlya of protostomes. Ecdysozoan genomes are generally widely represented, despite the fact that there are still some ecdysozoan phyla with no sequenced genomes: Nematophora (horsehair worms); Kinorhyncha (mud dragons); and Loricifera. A ‘Teneurin-singleton’ only is found in every sequenced species of non-arthropod ecdysozoans, in: Priapulida; Nematodes; Onychophorans, and Tardigrades (Figure 3A,B).

The most diagnostic information for timing the Ten-a/Ten-m duplication comes from the closest, sister, phylum of Arthropods, Onychophora. It has one species with a sequenced genome, Euperipatoides rowelli (NCBI BioProject 203089, Georg Mayer at the Baylor i5K initiative, as described in Evans et al., 2013). This Onychophoran velvet worm E. rowelli has a single Teneurin gene, roughly equally distant from fly Ten-a and from Ten-m (Figure 3). Onychophorans have an estimated divergence date from Arthropods not long before the Arthropod splits to sub-phyla (Sanders and Lee, 2010). The generation of a Teneurin paralog type occurred once in the history of invertebrates, exclusively in Arthropods, at their inception as a phylum.

The third milestone in the history of Teneurins, after their appearance in the urBilaterian, and after their duplication in the urArthropod, occurred in deuterostomes around the establishment of the chordate phylum. Most central and relevant to our topic was a quadruplication in vertebrates that led to the four long-recognized Teneurin paralog types in higher vertebrates: 1, 2, 3, and 4. The invariant existence of these four paralog types in all higher vertebrates has already been well reviewed (Tucker et al., 2012; Leamey and Sawatari, 2014; Mosca, 2015).

The window to reconstruct earlier events for deuterostome Teneurins has now opened yet further, due to recently completed genomes. This includes views of proto-chordates: Ambulacraria (Echinoderms plus Hemichordates) and cephalochordates (Figure 4A). This also includes Tunicates (or urochordates), and ‘lower and higher’ – jawed and unjawed – vertebrates. As described below in IIIC of this section, the duplications of bona fide Teneurins only began in jawless vertebrates. However, a Teneurin-derived distinct family, the TRIPs, arose in deuterostomes before the appearance of vertebrates. There are extant TRIPs only among hemi- and cephalo- chordates. This TRIP sidetrack occurred first, and its narrative is treated first, in sections IIIA and IIIB.

Beyond the two paralogs in arthropods and four paralogs in vertebrates, further Teneurin duplications are rare in the genomes sequenced to date, and only occur in a few very select Vertebrate and Arthropod lineages. The cases are limited to bony fish (teleosts) and amphibians, among vertebrates. None occur in cartilaginous fish, nor in the more modern vertebrates, Amniotes. Among Arthropods, they only occur in certain Chelicerata and Crustaceans. These duplications have arisen significantly more recently than the deep duplications described above, and always yield clearly identifiable paralog types 1, 2, 3, 4, a, or m.

The best known of these rare cases are in fish, modeled to have undergone teleost specific whole genome duplications (WGDs) (Pasquier et al., 2016). Well documented are the six Teneurins of the zebrafish Danio rerio, with a second Teneurin 2 paralog, and a second Teneurin 3 paralog (Howe et al., 2013). These duplicates share 81 and 74% aa identity, respectively, to their sister 2 and 3 genes, versus about 63% aa identity between paralogs Teneurin 2 and Teneurin 3. These extra paralog copies for Teneurins 2 and 3 are presumably those that persisted after a WGD, with loss of the Teneurin 1 and 4 duplicate-genes having occurred. Teneurin duplications are more extensive in Rainbow trout, whose genome is modeled as having undergone teleost specific, then salmonid lineage specific, WGDs (Berthelot et al., 2014). These two more recent lineage specific WGDs occurred long after the 2R genome quadruplication at the root of vertebrates. Rainbow trout have up to 16 Teneurins, all of identifiable types, Tenm1 – Tenm4. In amphibians, an even clearer case is seen when comparing diploid Xenopus tropicalis to the allotetraploid Xenopus laevis (Riadi et al., 2016). Like most vertebrates, X. tropicalis bears the four Teneurin vertebrate paralogs. X. laevis has clear second copies for each paralog (Figure 5A). This is especially compelling, as the eight X. laevis genes are nested in the expected syntenic surroundings, with pairs on the related chromosomes (allotetraploid pairings), as implicit in their chromosome names (Figure 5A).

Arthropods have the only other lineages showing duplications beyond those described in “Teneurin history steps two and three.” No further duplications are detectable in two of the four arthropod sub-phyla: neither in the one sequenced myriapod Strigamia maritima, nor in the hundreds of sequenced insects. Instead, additional duplications beyond the generation of the original urBilaterian Ten-a/Ten-m genes occur only in specific lineages of Chelicerates and Crustaceans.

In chelicerates, more than one paralog of Ten-m and Ten-a occur in specific mites, spiders, and horseshoe crabs. Both paralogs appear as multiples in all three sequenced scorpions and false scorpions. This is in contrast to the single Ten-a and Ten-m paralogs in the deer tick Ixodes scapularis, the Lyme disease vector known not to have undergone further WGDs. For the mite, spider, and horseshoe crab genomes with multiple Ten-m and Ten-a copies, the two paralog archetypes appear to always have the same duplication timing. This is based on the equivalent divergence tree profiles seen among the Ten-a and Ten-m duplicates, and likely indicates WGDs. An example is shown for the horseshoe crab Limulus polyphemus, that has two Ten-a and two Ten-m genes (Figure 5B). Compellingly, Limulus polyphemus and the other mentioned species have recently been fully sequenced, and are modeled as having had lineage specific WGDs (Nossa et al., 2014; Kenny et al., 2016; Schwager et al., 2017). The WGDs in horseshoe crabs, that occurred significantly after the original Ten-a/Ten-m split, are themselves still considerably ancient duplications (>135 Myr) (Nossa et al., 2014; Kenny et al., 2016). There is also a modeled deep specific WGD shared by spiders and scorpions (Schwager et al., 2017). These include the common house spider, Parasteatoda tepidariorum, with 6 Teneurins: three of each paralog (Schwager et al., 2017). A list of species examined is found online as Supplementary Table S1.

This story repeats itself in Crustaceans, with many distributed species showing more than one Ten-a and more than one Ten-m. It is notable that the class Branchiopoda of crustacea is one clade without any Teneurin duplications. In contrast, there are crustaceans with the two paralogs both duplicated or triplicated, with apparently the same timing. This strongly supports WGD. However, unlike for the case of Chelicerates, no crustacean genome sequenced to date has been modeled as having undergone WGD. This will require further investigation in the coming years to assess the full nature of these crustacean genomes and duplications.

Teneurins are a bilaterian-only metazoan gene family, to the exclusion of the Kingdoms of prokaryotes, plants, fungi, and protists. The intriguing only exception comes from among the unicellular, opisthokont, closest relatives of metazoans, the choanoflagellates. Full genome sequences exist for only two choanoflagellates (Hoffmeyer and Burkhardt, 2016). There is a Teneurin in Monosiga brevicollis, as has been reported (Tucker et al., 2012). In contrast, the genome of the closely related Salpingoeca rosetta has no Teneurin.

To put these two contrasting choanoflagellate findings in context, it must be recognized that outside of triploblast-bilaterians, no animal Teneurins exist. There are no Teneurins in any diploblast genomes, including even sponges - those metazoans closest to choanoflagellates. This makes M. brevicollis’ gene strikingly unique, and all the more intriguing. Perhaps its existence bespeaks horizontal transfer. One of the two most parsimonious explanations for M. brevicollis’ gene’s existence is: the original Teneurin was ‘born’ in the shared ancestor of M. brevicollis and metazoans, then was lost in every diploblast metazoan lineage. This is possible, but is not an entirely satisfying explanation, due to the need for serial and multiple losses to “clear-the-board” in every branch. Alternatively, and favored above, is a model where urTeneruin was ‘fusion-assembled’ in the ‘urBilaterian,’ then was subsequently acquired by a single choanoflagellate clade, via horizontal transfer. Recently, nineteen additional Choanoflagellates had their transcriptomes extensively sequenced and compared (Richter et al., 2018). Evidence of Teneurins occur in 3 of the 19 species, essentially all clustered in one clade. As a comparison, signatures for domains of Notch and its ligands are found in the 19 transcriptomes, as well as in the two genome-sequenced choanoflagellate species. The conclusion reached is that Notch appears to be ‘indigenous’ to choanoflagellates, with its creation predating the choanoflagellate/metazoan split (Richter et al., 2018). In contrast, Teneurin distribution supports the idea that a metazoan Teneurin entered M. brevicollis and its clade with three other species, by horizontal transfer.

The functional partnership of Teneurins and Latrophilins was discovered in rodents through the LPHN1-TENM2 interaction (Silva et al., 2011). Further work extends this to further family members, with demonstrations that all 3 latrophilins bind all 4 Teneurins, in Mouse (Boucard et al., 2014). A survey of where the interacting domains of Teneurins and Latrophilins co-exist within different organisms can give an indication of how widespread their functional cooperation might be across bilaterians. From protostomes to deuterostomes, how many of each exist?

First, what defines the Latrophilins, and where do they occur? Latrophilins exist as an animal-only gene subfamily within the greater seven-transmembrane GPCR family. They are part of the Adhesion GPCRs, of which there are 33 in humans. Adhesion GPCRs are one of five main groups according to the GRAFS classification (see Hamann et al., 2015). The Adhesion GPCRs are ancient, and are believed to have evolved from the cAMP receptor family, arising approximately 1,275 million years ago, before the split of Unikonts to animals and fungi (Schioth et al., 2010; Nordstrom et al., 2011; Krishnan et al., 2012). Among the adhesion-GPCRs, Latrophilins constitute family I out of 9 (I-IX) (Hamann et al., 2015). Family I LPHNs 1 through 3 are formally known as ADGR1-3, with the less related ELTD1 known as ADGRL4. There are no HRM, Olfaction, or RB-Lectin domains in ELTD1/ADGRL4, making it a distinct outlier. LPHN1 was the first Latrophilin cloned (Lelianova et al., 1997). Again, the interaction with Teneurin 2 was discovered as: LPHN1 to Teneurin2 in 2011, as mentioned above (Silva et al., 2011).

LPHNs are animal only genes that are distributed more broadly than Teneurins. In diploblasts, there is a strong possibility that homologs exist that truly act as LPHNs (Krishnan et al., 2014). Examples of these homologs include sponge Amphimedon queenslandica Aq715659, and anemone Nematostella vectensis Nv24490. Whatever the function of these proteins, it must be Teneurin-independent (Table 1).

Protostomes all seem to have one Latrophilin only (Table 1). Exceptions are a duplicate in the Ixodes tick, and a possible two for Caenorhabditis elegans, Lat-2, and perhaps also Lat-1, For insects, a singleton clear Latrophilin exists. Therefore a LPHN duplication does not appear to co-occur with the Ten-a plus Ten-m generated paralogs in insects. Some insect LPHNs seem to have a more complete domain content than others. Nonetheless, the Drosophila melanogaster homolog Cirl, evidently missing many expected domains, has clearcut functions in flies (Scholz et al., 2015; Scholz et al., 2017).

In non-vertebrate Deuterostomes, there are to one to two LPHNs, although generally only one has all domains to make a firm case for likely function (Krishnan et al., 2013). However, from chordates to vertebrates, a co-increase between Teneurins and Latrophilins can be tracked (Table 1). Most intriguingly, the clades of vertebrates and arthropods that underwent WGD and duplication of Teneurin paralogs have a definite trend of equivalent increases for the LPHNs. This can be seen for Xenopus laevis, Zebrafish, Rainbow trout, and Chelicerates (Table 1).

The LPHN genes’ duplications at the root of vertebrates/chordates could have co-arisen with the vertebrate whole genome 2R quadruplication proposed by Ohno (see Krishnan et al., 2015). WGDs in restricted arthropods and vertebrates appear to co-copy, and co-retain, their balanced collections of Teneurins and Latrophilins.

There are three key steps to the Teneurin evolution story, the birth-by-fusions, then the very ancient duplication events. Three important footnotes serve either to bolster the character of those steps (e.g., conserved, specific additional duplications), or are not central to the generation of Teneurins as we know them (e.g., the TRIP spinoff, and a choanoflagellate’s homolog).

Teneurins are rigidly and extraordinarily conserved, both in their unchanging structure, and in their gene copy number per genome. The lineages where new paralogs arose are in the two most successful phyla on the planet: arthropods, and our own – chordate vertebrates. Is it possible that the addition of Teneurin paralogs to the gene toolkit contributed to the special success of these phyla? Can the overall success of triploblasts-bilaterians, compared to diploblasts, partially be attributed to the presence of Teneurin? Does a relatively fixed ratio of Teneurin to Latrophilin gene copies, conserved even when further arthropod and vertebrate WGDs occur, attest to the special advantage that this ‘team’ jointly contributes to a metazoan? These are all questions that could be further probed in the future, now that these balances have been uncovered.

The basic outline of this story should now be complete, and is even arguably comprehensive. Given the major gaps now filled in for the tree of animal life, large brushstrokes expected to change this story in the future seem unlikely. Albeit, some chapters of this story need improvement. A better-defined indication of the source of EGF-like repeats that were incorporated into Teneurin in the urBilaterian would be informative. An answer on whether Teneurins versus TRIPs provided Tenascins with their EGF-like domains should be more clear-cut with better modeling. Establishing a clearer relationship between agnathans’ two Teneurins and higher vertebrates’ four Teneurins is attainable with further work. Genome sequences of additional Choanoflagellates, and other unicellular opisthokonts, should shed more light on the significance of the Monosiga brevicollis Teneurin. The question: “just how rigidly conserved is the Teneurin family, relative to other gene families?”, should be interesting to model and probe. However, for all of these questions, it can only be expected that new genomes, and further connections within the omniome, will deliver new surprises.