The genome sequence of the protostome Daphnia pulexencodes respective orthologues of a neurotrophin, a Trk and a p75NTR: Evolution of neurotrophin signaling components and related proteins in the bilateria
© Wilson; licensee BioMed Central Ltd. 2009
Received: 1 December 2008
Accepted: 6 October 2009
Published: 6 October 2009
Neurotrophins and their Trk and p75NTR receptors play an important role in the nervous system. To date, neurotrophins, Trk and p75NTR have only been found concomitantly in deuterostomes. In protostomes, homologues to either neurotrophin, Trk or p75NTR are reported but their phylogenetic relationship to deuterostome neurotrophin signaling components is unclear. Drosophila has neurotrophin homologues called Spätzles (Spz), some of which were recently renamed neurotrophins, but direct proof that these are deuterostome neurotrophin orthologues is lacking. Trks belong to the receptor tyrosine kinase (RTK) family and among RTKs, Trks and RORs are closest related. Flies lack Trks but have ROR and ROR-related proteins called NRKs playing a neurotrophic role. Mollusks have so far the most similar proteins to Trks (Lymnaea Trk and Aplysia Trkl) but the exact phylogenetic relationship of mollusk Trks to each other and to vertebrate Trks is unknown. p75NTR belongs to the tumor necrosis factor receptor (TNFR) superfamily. The divergence of the TNFR families in vertebrates has been suggested to parallel the emergence of the adaptive immune system. Only one TNFR representative, the Drosophila Wengen, has been found in protostomes. To clarify the evolution of neurotrophin signaling components in bilateria, this work analyzes the genome of the crustacean Daphnia pulex as well as new genetic data from protostomes.
The Daphnia genome encodes a neurotrophin, p75NTR and Trk orthologue together with Trkl, ROR, and NRK-RTKs. Drosophila Spz1, 2, 3, 5, 6 orthologues as well as two new groups of Spz proteins (Spz7 and 8) are also found in the Daphnia genome. Searching genbank and the genomes of Capitella, Helobdella and Lottia reveals neurotrophin signaling components in other protostomes.
It appears that a neurotrophin, Trk and p75NTR existed at the protostome/deuterostome split. In protostomes, a "neurotrophin superfamily" includes Spzs and neurotrophins which respectively form two paralogous families. Trks and Trkl proteins also form closely related paralogous families within the protostomian RTKs, whereby Trkls are absent in deuterostomes. The finding of p75NTR in several protostomes suggests that death domain TNFR superfamily proteins appeared early in evolution.
In mammals, four paralogous neurotrophins play a role in embryonic neural development, adult neuroplasticity[2, 3] and regeneration of the nervous system: Nerve Growth Factor (NGF), Brain Derived Neurotrophic Factor (BDNF), Neurotrophin 3 (NT3) and Neurotrophin 4/5 (NT4/5). The neurotrophin signaling system is also involved in the immune system. The biological effects of the neurotrophins are mediated by three paralogous Trks (TrkA, TrkB, TrkC) belonging to the Trk family of Receptor Tyrosine Kinases (RTK) as well as by the 75 kDa neurotrophin receptor p75NTR. Each Trk receptor preferably binds to a different neurotrophin, whereby TrkA, TrkB and TrkC can be activated by NGF, BDNF/NT4/5 and NT3 respectively. In contrast, the p75NTR receptor is non selective and has a similar affinity for all neurotrophins. Additional functional complexity results from formation of heterodimeric complexes between p75NTR and TrkA, TrkB or TrkC. These complexes alter the signaling properties of both partners of the complex. Paralogues of p75NTR, known as neurotrophin receptor homologue NRH1 in fish, birds and amphibians, and NRH2 in mammals also form complexes with, and alter signaling by TrkA, TrkB and TrkC. The multiplicity of neurotrophins and Trk receptors in vertebrates apparently arose as a result of two rounds of genome duplications that occurred at the root of vertebrate evolution, the first duplication occurring before the emergence of agnathan fish and the second occurring after the emergence of cartilaginous fish [7–9]. The NRH paralogues of p75NTR may have been generated by a similar mechanism. If the multiple vertebrate paralogues of the components of the signaling system arose by genome duplication events what was the evolutionary origin of the genes that were duplicated by those events?
Until recently, the neurotrophin/Trk/p75NTR signaling system was not found outside the vertebrates. Indeed, no component of this signaling system was present in the genome sequence of two protostomes, the insect Drosophila melanogaster and the nematode Caenorhabditis elegans . An invertebrate deuterostome, the tunicate Ciona intestinalis did not have the signaling system either, which led to hypothesize that neurotrophins, Trks and p75NTR were vertebrate innovations. This hypothesis was challenged, however, when the genome of two additional invertebrate deuterostomes, the cephalochordate Branchiostoma floridae (amphioxus)[12, 13] and the echinoderm Strongylocentrotus purpuratus (purple sea urchin)[14, 15] were shown to include the neurotrophin/Trk signaling system in their sequence. Analysis of genomic data from Strongylocentrotus as well as EST data from another invertebrate deuterostome, the hemichordate Saccoglossus kowalevskii (acorn worm) additionally identified a p75NTR in both of those species. This suggested that the neurotrophin/Trk/p75NTR system arose at least at the root of deuterostomes[8, 10]. Clues to an earlier origin of the neurotrophin/Trk/p75NTR system arise from proteins in some protostomes bearing homology to either one of the signaling components of the neurotrophin/Trk/p75NTR system. Nevertheless, none of the neurotrophin/Trk/p75NTR homologues have so far been found concomitantly in a protostomian species. Moreover, when a homologue of a component of the neurotrophin/Trk/p75NTR signaling system has occasionally been described, its phylogenetic relationship to either the neurotrophin or Trk, or p75NTR, has remained uncertain. For example, insects have structural neurotrophin homologues called Spätzle (Spz) proteins. In Drosophila, 6 Spz proteins have been identified and are named Spz (or Spz1), Spz2, 3 etc. Spz proteins share a sequence homologous to the characteristic cysteine knot which is known to induce the particular neurotrophin three dimensional fold. Because Spz, Spz2 and Spz5 proteins are organized in the same way as neurotrophins, with a signal peptide and a pro-domain that can be proteolytically removed from the mature protein containing the Cys knot, Zhu et al.  recently investigated their function in the Drosophila nervous system. The researchers demonstrated that Drosophila Spz, Spz2 and Spz5 have neurotrophic properties comparable to neurotrophins. In the Zhu paper, Drosophila Spz2 and Spz5 were renamed Drosophila Neurotrophin 1 (DNT1) and Drosophila Neurotrophin 2 (DNT2) respectively, but the exact phylogenetic relationship of Spz, DNT1 and DNT2 to deuterostome neurotrophins could not be resolved. Whether Spzs signal through Trk receptors, which would be expected if they were neurotrophin orthologues is also not known, and the sequence of a Trk receptor orthologue has so far not been found in flies. Spz, the representative of the Spz family is moreover known to signal through the Toll receptor, a receptor involved in the nervous and innate immune systems and in dorso-ventral patterning in the Drosophila embryo[20, 21].
Trks are modular protein receptors consisting of a conserved extracellular part, a transmembrane domain and a cytoplasmic part. The cytoplasmic part contains a tyrosine kinase domain which is common to many representatives of the RTK family, to which Trks belong. Among RTKs, Trks and RORs share the most similar tyrosine kinase domain. In insects where Trks have not been found, RORs (Drosophila ROR and a Drosophila ROR-like protein called Neurospecific Receptor Kinase (NRK)) have been shown to have a neurotrophic role. As for protostomian Trk-related sequences, the closest related Trk receptors have been found in mollusks. In the mollusk Aplysia californica, a protein called ApTrkl was found to have a tyrosine kinase domain most similar to that of Trk receptors than to any other tyrosine kinase domain. ApTrkl is also expressed, like Trks, in sensory neurons. Nonetheless, the ApTrkl ectodomain and long intracytoplasmic C-terminal extension are totally unrelated to the Trk family of neurotrophic receptors. Accordingly, ApTrkl is not responsive to mammalian neurotrophin stimulation but to serotonin. In another mollusk, the snail Lymnaea stagnalis, a Trk related receptor called LTrk, which is specifically expressed in the central nervous system, was found to have most of the characteristics of vertebrate Trk receptors[25, 26]. Like ApTrkl, LTrk has a tyrosine kinase domain more related to Trks than to any other RTK. In addition, the LTrk extracellular part displays characteristics of a vertebrate Trk including a leucine rich repeat (LRR) motif flanked by two cysteine clusters. In this extracellular part, however, LTrk lacks two immunoglobulin (Ig) domains of the C2 type, which are a major interface for neurotrophin binding in vertebrates. LTrk has instead an Ig-like domain of the C1 type, which is exclusively common to molecules involved in immune system function, such as immunoglobulins, major histocompatibility complex molecules and T cell receptors. The phylogenetic relationship of Lymnaea Trk and Aplysia Trkl to each other and to deuterostome Trks is not well understood and neurotrophin like ligands have not been found so far in mollusks.
Finally, no p75NTR has been reported in protostomes. Despite being a neurotrophin receptor, p75NTR does not belong, as do Trk receptors, to the RTK family. Instead p75NTR is a member of the Tumor Necrosis Factor Receptor (TNFR) Superfamily (SF) (TNFRSF) . To date, the only TNFRSF representative in protostomes is the Drosophila TNFR Wengen. Wengen has some degree of similarity to p75NTR but it lacks a so-called "death domain" in the intracellular part of the protein. The death domain is present in p75NTR orthologues and in a few vertebrate TNFRs. In this study, the genome of Daphnia pulex was investigated for components of the neurotrophin/Trk/p75NTR signaling system. The search was subsequently extended to other protostomes by surveying genbank nucleotide and EST data, as well as the recent genome sequences of Capitella sp. I, Lottia gigantea and Helobdella robusta. The Daphnia genome encodes a neurotrophin, a p75NTR and a Trk orthologue together with Trkl, ROR and NRK-RTKs. Drosophila Spz1, 2, 3, 5, 6 orthologues as well as two new groups of Spz proteins (Spz7 and Spz8) are also present in the Daphnia genome. Neurotrophin signaling components are also found in other protostomes and the evolution of these components in the bilateria is discussed.
Results and discussion
Neurotrophin and Spz genes in Daphniaand protostomes: Evolution in the bilateria
Daphnia Spz genes
Daphnia Spz genes
Catalogue of Spätzle genes found in the Daphnia pulexgenome.
[GenBank:FE349417.1, GenBank:FE349418.1, GenBank:FE355503.1, GenBank:FE355502.1]
[GenBank:FE361273.1, GenBank:FE325461.1, GenBank:FE325462.1, GenBank:FE413050.1, GenBank:FE413051.1]
[GenBank:FE334520.1, GenBank:FE334519.1, GenBank:FE389946.1, GenBank:FE389945.1, GenBank:FE380627.1, GenBank:FE380626.1, GenBank:FE376962.1]
[GenBank:FE399977.1, GenBank:FE399976.1, GenBank:FE359418.1, GenBank:FE359417.1, GenBank:FE407285.1, GenBank:FE407284.1]
Daphnia magna: [GenBank:DW724455.1]
[GenBank:FE363720.1, GenBank:FE363719.1, GenBank:FE357035.1]
[GenBank:FE380921.1, GenBank:FE380920.1, GenBank:FE309270.1, GenBank:FE309269.1]
[GenBank:FE319054.1, GenBank:FE319053.1, GenBank:FE385764.1, GenBank:FE385763.1, GenBank:FE355807.1]
[GenBank:FE352671.1, GenBank:FE299965.1, GenBank:FE299964.1]
Daphnia magna: [GenBank:BJ926602.1, GenBank:BJ925927.1, GenBank:BJ925817.1, GenBank:BJ928192.1, GenBank:BJ926968.1, GenBank:FD467132.1, GenBank:DW724571.1]
Daphnia magna: [GenBank:EG565383.1, GenBank:BJ928378.1]
[GenBank:FE328688.1, GenBank:FE328687.1, GenBank:FE322916.1, GenBank:FE322915.1, GenBank:FE416776.1, GenBank:FE416775.1, GenBank:FE420380.1, GenBank:FE420379.1, GenBank:FE353236.1, GenBank:FE353235.1]
[GenBank:FE303670.1, GenBank:FE303669.1, GenBank:FE394292.1, GenBank:FE394291.1, GenBank:FE400682.1, GenBank:FE400681.1]
[GenBank:FE385611.1, GenBank:FE385610.1, GenBank:FE300572.1, GenBank:FE300571.1, GenBank:FE386869.1, GenBank:FE386868.1]
In the phylogenetic analysis, the Spz2-like sequences that were retrieved by tBLASTN using Spz2 as query form two groups of sequences. Representatives of the first group were named Dappu-Spz7A, 7B etc. while representatives of the second group were named Dappu-Spz8A, 8B etc. to distinguish them from the 6 Spz representatives known to date.
Daphnia Spz7s and Spz8s define new Spz paralogues in crustaceans
Origin of Daphnia pulex Spätzle ESTs.
[GenBank:FE389946] Stress: bacterial infection.
[GenBank:FE334520] Stress: metal, high [c] (20 ug Cd/L;
Stress: 200 ug Zn/L; 1348 ug As/L)
[GenBank:FE376962] Stress: salt (750 mg/L of sodium chloride).
[GenBank:FE399977] Stress: Metal exposure, low [c]
(1 ug Cd/L; 50 ug Zn/L; 500 ug As/L).
[GenBank:FE359418] Stress: nickle (200 ug/Lof Ni as nickle chloride).
[GenBank:FE407285] Stress: monomethylarsenic III
(100 ug/L of As as diiodo-MMAIII).
[GenBank:FE325461] Exposed to Methyl Farnesoate hormone
[GenBank:FE361273] Stress: nickle (200 ug/L of Ni as nickle chloride).
[GenBank:FE413050] Stress: nanoparticles (Titanium Dioxide).
[GenBank:FE363720] Stress: copper (5 ug/L of Cu as copper sulphate).
[GenBank:FE357035] Stress: nickle (200 ug/Lof Ni as nickle chloride).
[GenBank:FE367435] Stress: copper (5 ug/Lof Cu as copper sulphate).
[GenBank:FE380921] Stress: salt (750 mg/L of sodium chloride).
[GenBank:FE309270] Stress: arsenic
(1348 ug/L of As as sodium arsenite)
[GenBank:FE319054] Stress: zinc (200 ug/Lof Zn as zinc chloride).
[GenBank:FE385764] Stress: nanoparticles (Fullerene).
[GenBank:FE299965] Stress: hypoxia.
[GenBank:EG565383] Daphnia magna molting cDNA library
[GenBank:FE328688] Exposed to Methyl Farnesoate hormone.
[GenBank:FE322916] Stress: zinc (200 ug/Lof Zn as zinc chloride).
[GenBank:FE416776] Stress: Microcystis.
[GenBank:FE420380] Stress: calcium starvation (1 mg/Lof Ca).
[GenBank:FE303670] Stress: hypoxia.
[GenBank:FE394292] Stress: mixed metals, high [c]
(20 ug Cd/L; 200 ug Zn/L; 1348 ug As/L).
[GenBank:FE400682] Stress: mixed metals, low [c]
(1 ug Cd/L; 50 ug Zn/L; 500 ug As/L).
[GenBank:FE416560] Stress: Microcystis.
[GenBank:FE373132] Stress: acid (pH 6.0).
[GenBank:FE385611] [GenBank:FE386869] Stress: nanoparticles (Fullerene).
[GenBank:FE300572] Stress: hypoxia
[GenBank:FE383908] Stress: nanoparticles(Fullerene)
Daphnia neurotrophin gene and homologues in the protostomes
Predicted Daphnia neurotrophin protein
Genomic organization of the putative Daphnia neurotrophin
Other putative protostome neurotrophins
The discovery of a putative neurotrophin gene in Daphnia pulex along side Spz genes prompted to ask whether these genes could be found in other protostomes. Search of genbank sequence data with Dappu-NT as query, did not yield orthologous sequences in flies. ESTs of high similarity to the Dappu-NT were however identified in the ticks Rhipicephalus microplus and Ixodes scapularis (arachnid arthropods). A sequence fragment encoding only part of the Cys knot of the mature neurotrophin was found for Rhipicephalus [Genbank: FG579776.1] while longer and respectively overlapping ESTs were retrieved for Ixodes. Some of the Ixodes sequences were derived from the synganglia, a central nervous system structure [e.g. Genbank: EL516713.1] and the longest EST [Genbank: EL516713.1] encoded a putative signal peptide, prodomain as well as a neurotrophin Cys knot structure in the predicted mature protein. The Ixodes neurotrophin sequence was moreover found along side Ixodes ESTs encoding different Spz paralogues, such as a putative Ixodes Spz1 [Genbank: EW823171.1] and Spz3 [Genbank: EW797276.1]. This suggests that neurotrophins and Spzs are present within several classes of arthropods including crustaceans and arachnids. It is generally accepted that protostomes comprise two groups of animals, the ecdysozoa (comprising nematodes and arthropods such as Daphnia, Ixodes and Rhipicephalus) and the lophotrochozoa (comprising flatworms, annelids and mollusks). A number of genomes representing the Lophotrochozoa have recently been made available, such as those of the annelids Capitella sp. I, Helobdella robusta and the mollusk Lottia gigantea. tBLASTN searches on Helobdella yielded no Spz genes but a sequence encoding a neurotrophin-like Cys knot fragment on Scaffold 16. The same search method on the Capitella genome also resulted in a neurotrophin-like sequence, which was supported by an EST [GenBank: EY519311.1]. The Capitella neurotrophin-like sequence has a signal peptide a prodomain and a putative mature Cys knot. A sequence encoding a neurotrophin like Cys knot was also found on Scaffold 27 in Lottia but with no corresponding EST. No Spz genes were recovered from lophotrochozoans, but the limited amount of representatives with complete genetic data in this group makes it too early to conclude that Spzs are absent.
The Daphnia neurotrophin and its protostome homologues cluster with deuterostome neurotrophins in phylogenetic trees
To verify the relationship of the putative protostome neurotrophin sequences of Daphnia, Ixodes, Rhipicephalus, Capitella, Helobdella and Lottia to deuterostome neurotrophins, the protostome putative neurotrophin sequences were phylogenetically analyzed with both protostome Spzs (including Daphnia and Ixodes Spzs described previously) and deuterostome neurotrophins. In the phylogenetic trees obtained by ML and BI, the Daphnia, Ixodes, Rhipicephalus, Capitella, Helobdella and Lottia neurotrophins cluster with the deuterostome neurotrophins in a "neurotrophin group". Spz, Spz2 (and DNT1), Spz3, Spz4, Spz5 (and DNT2), Spz6, Spz7, and Spz8 paralogues moreover cluster in another large group which is distinct from that formed by the "neurotrophin group". The node at the base of the neurotrophin group is strongly supported both by a ML bootstrap value of 97% and a BI pp value of 1. This implies that neurotrophin and Spz families form paralogous families and in the same time suggests for the first time that protostomes have deuterostome neurotrophin orthologues (Figure 2).
Evolution of neurotrophin/Spz genes in the bilateria
Spz and neurotrophins form two closely related paralogous families in the protostomes
The phylogenetic analyses presented in this work, show that Spzs and neurotrophins form two paralogous families in the protostomes. Figure 3 shows an alignment of the Cys knot of the major Spz paralogues, to neurotrophins from protostome, invertebrate deuterostome, and vertebrate origin. Three sites, which are numbered in reference to the alignment in Figure 3, distinguish the neurotrophin and Spz families. The first is Pro13 (highlighted in bright red, Figure 3) which is conserved in all of the Spz peptide sequences, but not found in the neurotrophins. The second is an aromatic residue in Spz proteins at position 133 (Phe or Tyr (in dark red, Figure 3)), which is replaced by an Ile or Val in neurotrophins. Finally, Trp 130 (blue) is not found in any Spz proteins, yet it is conserved in all neurotrophins (except for the Branchiostoma, "amphioxus NT", that has a Tyr). Despite these three differences, Spzs and neurotrophins share the six Cys forming the Cys knot in neurotrophins as well as Gln44 whose side chain (in neurotrophins), contributes a crucial hydrogen bond to the tertiary structure. Three hydrophobic sites at position 26, 133 and 135 (indicated below the alignment by an H in Figure 3) which locate to the hydrophobic core of the neurotrophins are also common to Spzs and neurotrophins. These similarities and the recent results of Zhu et al. showing that Spzs and neurotrophins have similar functions, argues that the two families are closely related. When the Spz and neurotrophin families diverged, and where they originate in the bilateria, is difficult to pinpoint. Spz genes have not been found in deuterostomes, so if they were already present at the protostome/deuterostome split, they were probably lost in the deuterostome lineage. Alternatively, Spz genes could have evolved from duplications of an ancestral neurotrophin-like gene somewhere in the lineage leading to the protostomes.
Evolution of neurotrophins in the bilateria
In the phylogenetic trees, protostome neurotrophins fall into several groups. Ecdysozoa arthropod neurotrophins cluster together (Rhipicephalus, Ixodes and Daphnia) while another group is formed by the Lophotrochozoa (Lottia, Capitella and Helobdella) neurotrophins. The invertebrate deuterostome neurotrophin sequences used for the tree include those of a Cephalochordate (Branchiostoma (amphioxus in Figure 2)), a Hemichordate (Saccoglossus (acorn worm, Figure 2)) and an Echinoderm (Strongylocentrotus (sea urchin, Figure 2)). Echinoderms and Hemichordates are closely related and form a clade called Ambulacraria. The two previously described Strongylocentrotus neurotrophin paralogues were included in the phylogenetic analysis. Only one neurotrophin has been described for Saccoglossus but search of the genbank EST database for the purpose of this study revealed two different ESTs that were arbitrarily called "acorn worm Nta" ([GenBank:FF527248.1] undescribed so far) and "acorn worm Ntb" ([GenBank:FF505731.1] & [GenBank:FF504966.1]) [8, 10]. The genome of Saccoglossus is still unavailable, so the full repertoire of neurotrophins in this genome cannot yet be investigated. In the tree, ambulacraria neurotrophins form two groups, in accordance with the two Strongylocentrotus neurotrophins. The Strongylocentrotus neurotrophin Sp-NT2 ("sea urchin NT2" in Figure 2), which does not have the canonical intron-exon organization of vertebrate neurotrophins forms a group with the two Saccoglossus neurotrophin ESTs ("acorn worm NTa" and "acorn worm NTb"). The latter group is more closely related to the protostome neurotrophins, as it diverges between the two groups formed by Ecdysozoa and Lophotrochozoa neurotrophins in the tree (Figure 2). The canonical Strongylocentrotus neurotrophin Sp-NT ("sea urchin NT" in Figure 2), which shares the same intron-exon organization as vertebrates, clusters closer to Branchiostoma ("amphioxus NT", Figure 2) and the vertebrates in the tree, consistent with what would be expected for an invertebrate deuterostome neurotrophin. The genome of Saccoglossus is not released, so it is not possible to know if this genome encodes an additional canonical neurotrophin type, which would in turn cluster with Sp-NT.
The presence of two Strongylocentrotus (Echinoderm) neurotrophins, one more protostome like (Sp-NT2 ("sea urchin NT2", Figure 2)), and the other more vertebrate like (Sp-NT ("sea urchin NT", Figure 2)), may indicate interesting evolutionary transitions occurring at the stem of the deuterostome lineage. The first transitions may be small changes in the protein tertiary and quaternary structure. Sp-NT2 ("sea urchin NT2" in Figure 3) has a Pro at position 136 in the alignment (Figure 3) which is not found in Sp-NT ("sea urchin NT"), yet conserved in some protostome neurotrophins and in all Spz proteins. Pro 136 is at the interface between associated Spz monomers when dimerization occurs and is probably conserved in Spzs for structural reasons. The replacement of Pro136 by other residues in canonical deuterostome neurotrophins may have changed the interface presented by each monomer for dimerization. Sp-NT2 ("sea urchin NT2") also has an additional Cys adjacent to the fifth Cys knot, as does the Saccoglossus "acorn worm NTb", which is reminiscent of some Spz paralogues, and which, together with Pro136 allows tight homodimmer formation by covalent disulfide bonding. Finally, Sp-NT2 ("sea urchin NT2") and "acorn worm NTa" and "b" have an Asn at position 18 in the alignment (Figure 3) which is common to many Spz proteins but strictly replaced with an Asp in all canonical neurotrophins (highlighted in blue, Figure 3). Remarkably, Asp18 (18 = alignment Figure 3 numbering, and equivalent to mammalian neurotrophin Asp30) has been shown to be important for tertiary structure stability, and in particular, stability of a beta-hairpin loop formed in neurotrophins by residues 18-22 (18-22 in the alignment, and equivalent to mammalian neurotrophin residues 30-34).
The other transition that may have occurred at the stem of the deuterostome lineage is the occurrence of gene duplications and the adoption by one of the duplicates, of a more compact genomic structure. The precursor sequence of Sp-NT2 is split into several exons, as is the precursor of Daphnia neurotrophin and Spzs. The canonical neurotrophin paralogue Sp-NT in Strongylocentrotus, however, has a precursor encoded by one exon like vertebrate neurotrophins. The two Strongylocentrotus neurotrophins could have arisen from the duplication of an ancestral gene at the base of the deuterostome lineage whereby one or several duplicates retained a more ancestral character leading to Sp-NT2 ("sea urchin NT2") and Saccoglosssus "acorn worm NTa" and "b", while another type of duplicate adopted a more compact genomic organization. (The genetic organization of the Saccoglossus genes will be interesting to examine in this respect). The gene(s) with ancestral character could have been lost before the divergence of the Cephalochordates on the lineage leading to the vertebrates, leaving a canonical neurotrophin to duplicate via genome duplications along the lineage leading to the vertebrates. It is noteworthy in this regard that neither protostome neurotrophins nor deuterostome invertebrate neurotrophins share a closer relationship to any of the vertebrate neurotrophin paralogues (NGF, BDNF, NT3 and NT4/5). In the phylogenetic tree (Figure 2) the vertebrate genes form a tight cluster. In the alignment, all vertebrate neurotrophins share Thr116 (highlighted in green, Figure 3). Thr116 which is known to form hydrogen bonds across a beta hairpin is not found in any of the invertebrate homologues, thus defining a vertebrate neurotrophin-like characteristic.
Trk and Trk-related families in the protostomes and evolution in bilateria
The Daphnia and protostome Trks
Daphnia Trk receptor predicted peptide structure
On the cytoplasmic side, Dappu-Trk includes a tyrosine kinase (TK) domain that has all the key residues necessary to carry out its function as a catalytic receptor. Like its vertebrate counterparts, it contains the signature pattern of class II tyrosine kinase receptors DIYSSDYYK (Figure 7, grey background). Within this short amino acid sequence, three tyrosine residues (Y755, Y759 and Y760; Figure 7, black background) constitute the putative auto-phosphorylation activation loop. A presumptive ATP-binding region is located at the N-terminal pole of the TK domain and contains a conserved lysine (K606, Figure 7, black background) responsible for binding ATP. In addition, two potential phosphorylation sites for cAMP/cGMP-dependent kinase proteins lie in positions comparable to those in vertebrates: RYS at the juxtamembrane intracytoplasmic part preceding the TK domain and RKFT following the activation loop within the TK domain (Figure 7, black background). In vertebrates, the former motif is a binding site for SNT, a protein involved in neuronal differentiation and neurite outgrowth pathways. Also present in Daphnia Trk is the docking site for Shc, an adaptor protein which in vertebrates activates the Ras-Raf-Erk and PI3kinase-AKT signaling pathways involved in neuronal survival and differentiation events. It is identically placed, preceding the tyrosine kinase domain (Y531, Figure 7, black background). Remarkably, at the furthest C terminus of the protein, outside the TK domain, Daphnia Trk has a tyrosine (Y874), which in mammals serves as the docking site for PLC, whose transduction pathway leads to initiation and maintenance of long-term potentiation events. Contrarily to vertebrates, this tyrosine is absent in invertebrate deuterostomes (Branchiostoma floridae and Strongylocentrotus purpuratus). In this respect, it is noteworthy that out of the two mollusk Trk-like proteins (LTrk and ApTrkl), which share with Trks some of the intracellular region comprising the tyrosine kinase domain, LTrk but not ApTrkl has the vertebrate equivalent tyrosine for PLC gamma activation. This suggests that the tyrosine could either be ancient and lost in specific lineages, or that it occurred independently in several lineages. Two questions are important in this respect, and these are whether LTrk or DaphniaTrk can in fact activate the PLC gamma pathway and if the tyrosine residue which has been shown to be absolutely necessary for PLC gamma activation can do this alone outside a consensus vertebrate sequence [P(VIS)YLD(IV)L(GE)] in this region of the protein.
Daphnia Trk receptor genomic structure
The second IgC2 domain in Daphnia Trk is also encoded by 2 exons (Daphnia Exon VIII and IX) unlike vertebrate and amphioxus Trks. Exon IX also spans the extra-juxta-membrane sequence of the receptor and covers what is part of vertebrate exon 8, 9 and 10. The extracellular juxta-membrane region in amphioxus and vertebrate Trks is encoded by distinct exons to those bearing the second Ig. In vertebrates, exons 9 and 10 encode the extracellular juxta-membrane region, while in amphioxus Trk a single compact exon encompasses vertebrate exons 9 and 10. Interestingly, human Trk exon 9 encodes a short amino acid sequence alternatively spliced in several of the Trk isoforms described in vertebrate species. The presence or absence of this short amino acid sequence is tissue specific and affects ligand preference. Human and rat TrkAI isoforms, which lack six amino acids in this region (encoded by exon 9), are expressed in non-neuronal tissues and bind only NGF, while TrkA-II isoforms, which contain the six amino acid insertion, are localized in neuronal tissues and can interact with both NGF and NT3 . The absence of this mini-exon in amphioxus and Daphnia Trks suggests that Trks may have acquired more ligand flexibility in the lineage leading to the vertebrates. The transmembrane region of the Daphnia Trk protein and the first phosphorylation site for cAMP/cGMP-dependent kinase proteins, RYS, are encoded by Daphnia exon X. Daphnia exon X aligns to vertebrate exon 11, with similar intron-exon boundaries. Vertebrate exons 12 and 13 align to Daphnia exon XI which is more compact and spans not only the tyrosine responsible for Shc recruitment Y533 (covered by exon 12 in vertebrates) but also part of the tyrosine kinase domain (encoded by vertebrate exon 13). The breakpoint between exon XI and XII in Daphnia corresponds to the breakpoint between human Trk exon 13 and 14 centrally located in the ATP binding residue K606. The tyrosine kinase domain and the intracytoplasmic C-terminus are distributed over five exons (Daphnia exons XII, XIII, XIV, XV and XVI). Daphnia exon XII corresponds to vertebrate exon 14, while Daphnia exon XIII does not span as much peptide sequence as vertebrate exon 15 and does not include the autophosphorylation sequence. This sequence is present instead in the next Daphnia exon XIV. The breakpoint between exon XIV and XV corresponds roughly to the breakpoint between vertebrate exon 16 and 17. In vertebrates, the C-terminal of the tyrosine kinase domain and the tyrosine responsible for PLC gamma docking are encoded by one exon (exon17). In Daphnia, these entities are encoded by two separate exons (exon XV and XVI). The intron-exon organization of Daphnia Trk, with more exons than amphioxus Trk, and several intron-exon boundaries that are conserved with vertebrate Trks, reopens the question on how the ancestral organization of the Trk gene looked like.
Daphnia Trk is a deuterostome Trk orthologue
Search for other protostomian Trks
The presence of a putative Trk orthologue in Daphnia suggested that Trks could perhaps be found in other protostomes. tBLASTN of genbank, with the Daphnia sequence did not yield any Trks in flies, but a search on the Capitella and Lottia genomes gave promising results. The Capitella and Lottia genes retrieved from the respective genome sequences are incomplete and may have errors particularly in the first half of the sequences. This could be due to missing sequence data and/or assembly problems. For both Capitella and Lottia, the sequence encoding the signal peptide and the first Cys cluster expected for Trks is lacking. A rather complete LRR is present at the N terminal of the Capitella amino acid sequence, while the Lottia sequence begins at what could be the end of the LRR. Both Lottia and Capitella sequences present a Cys Cluster after the LRR followed by a sequence encoding an IgC2 domain, similar to deuterostomes and Daphnia in this respect, but different from Lymnaea Trk (which is a Mollusk representative like Lottia). Indeed, Lymnaea Trk does not have an immunoglobulin domain in this region. What could be remnants of a second IgC2 domain (or an incorrect IgC2 sequence due to errors) follows the first IgC2 in Lottia and Capitella. Deletions occur, however, relative to the Daphnia sequence in this region, particularly in Capitella. The deletions are most important in the region after the two Asn residues which are known to interact with neurotrophin ligands in vertebrates. Lymnaea has an immuglobulin of the C1 type in this equivalent region. Presently, it is difficult to judge whether the deletions are "true" in evolutionary terms, or if they represent sequence errors, because in Capitella, the transmembrane domain which should follow the second Ig is absent and there are deletions relative to Daphnia in what should be the extracellular and intracellular regions immediately surrounding the transmembrane domain. The Lottia Trk sequence has a recognizable transmembrane domain. Despite possible errors in the first halves of the Lottia and Capitella sequences, the sequences encoding the intracellular part of the receptor prior to and within the tyrosine kinase domains meet the expectation for Trk sequences. Prior to the tyrosine kinase domain, the the NPxY motif is present in both Lottia and Capitella sequences. Also, the tyrosine kinase domains of both Lottia and Capitella have the typical autophosphorylation sequence [D(V/I)(S/T)(S/T)DYYR]. At the furthest C-terminus of the proteins, outside the TK domain, Lottia and Capitella Trks have a tyrosine equivalent to the mammalian Trk PLC docking site.
Phylogenetic analysis of Trk receptors in the bilateria
The sequence of the mollusk Lottia Trk appeared to be more similar to Daphnia and deuterostome Trks in the encoded extracellular domain, than to Lymnaea Trk, another molluskan Trk. The Lottia genome was therefore searched with the Lymnaea Trk as query, to check for the presence or absence of a Lymnaea Trk type (with an IgC1 in the extracellular domain) in Lottia. The Lymnaea Trk type could not be found in Lottia. Because of some uncertainties remaining regarding the first respective halves (encoding N terminal) of the Capitella and Lottia Trk sequences, phylogenetic trees were derived from tyrosine kinase domains only (Figure 9B). The phylogenetic analysis also included closely related RTK tyrosine kinase sequences (RORs, NRKs and Trkl) as well as the tyrosine kinase domains of Daphnia and deuterostome (Branchiostoma (amphioxus in Figure 9B), Strongylocentrotus (sea urchin), Homo (human), Gallus (chicken) and Danio (zebrafish)) Trks "see additional file 3" (Figure 9B)[12, 39, 40]. The orthology of Daphnia, Capitella, Lymnaea and Lottia tyrosine kinase domain to deuterostome Trks is supported by BI and ML. The node supporting the Trk family within the RTKs has a ML bootstrap value of 90% and a BI pp value of 1. Within the Trk tree, vertebrate Trks cluster together. Invertebrate Trks do not bear any closer relationship to any vertebrate Trk A, B or C paralogue. Despite the fact that Lottia and Lymnaea have different extracellular domains, the molluskan Trk tyrosine kinases cluster together with high support and are closely related to the Capitella and Daphnia Trks (Figure 9B). The finding of Trks in some protostomes but not in Drosophila and Caenorhabditis suggests that Trks are ancient but have been lost in some lineages. The finding of two IgC2 domains on the extracellular side of Daphnia Trk (ecdyzosoan) and at least one IgC2 domain (and possibly traces of another) in Capitella and Lottia (lophotrochozoa) suggests that a Trk bearing all the characteristics of deuterostome Trks was probably present before the protostome/deuterostome split.
Daphnia Trkl receptor and a Trkl-related lineage in the protostomes
Before the discovery of the Dappu-Trk, the closest protostomian related Trks were the Lymnaea Trk (which now appears to be a Trk orthologue) and the Aplysia Trkl. The search of the Daphnia genome with vertebrate Trks and Lymnaea Trk yielded the previously described Dappu-Trk gene. Investigating the Daphnia genome for Aplysia Trkl sequences however, uncovered a Trkl homologue in Daphnia (Dappu-235827). When Aplysia Trkl was first reported, it was described as having the tyrosine kinase domain most similar to vertebrate Trks but less so than Lymnaea Trk. The precise phylogenetic relationship between Lymnaea Trk and Aplysia Trkl is so far not known, but the existence of a putative Trkl protein in Daphnia led to hypothesize that Trks and Trkls could form two paralogous families in the protostomes. Despite a strong similarity to Trks in the intracellular region, Trkl bears little similarity to Trks in the extracellular domain of the receptor. In Aplysia Trkl, the initiating Met is followed by a relatively small ectodomain including the fragment of an EGF domain. The ectodomain is followed by a single transmembrane region, a tyrosine kinase domain and a long C-terminal extension.
The Aplysia Trkl also differs from Trks in that it does not have a precursor sequence with a signal peptide.
The Daphnia ROR and NRK receptors and their protostome homologues
Daphnia and protostome RORs
Daphnia and protostome NRKs
Evolution of Trk and Trk-related RTKs in the bilateria
The finding of Trk orthologues in the protostomes suggests that a Trk protein was already present at the protostome/deuterostome split. The absence of a Trk in Drosophila has been attributed to a lesser need for plasticity of the insect nervous system. This may not be the case for all arthropods as reflected by the Daphnia Trk. The additional finding of a Trkl family of protein in the protostomes, which is very closely related to Trks, suggest that protostomes have a complex array of Trk related proteins, including NRKs, which are not found in deuterostomes.
P75NTR genes in the protostomes and evolution in the bilateria
p75NTR in Daphnia and other protostomes
Evolution of p75NTR in the bilateria
The finding in several protostome representatives of p75NTR, a TNFRSF member endowed with a death domain suggests that death domain TNFRSF could have appeared early in evolution.
While p75NTR was the first TNFRSF member to be discovered, and its sequence characterized the TNFRSF CRDs, the phylogenetic relationship of p75NTR to other TNFRSF is not well understood. p75NTR has several features that distinguish it from other members of the TNFRSF. While TNFRSF proteins bind to tumor necrosis factor superfamily (TNFSF) ligands, p75NTR binds to neurotrophins which are structurally unrelated to TNFSF proteins. In vertebrates, over 20 TNFRSF members have been identified, most of which function in the immune system. The multiplicity of TNFRSF representatives has been suggested to arise from multiple rounds of duplications in concert with the appearance of adaptive immunity in evolution. Aside from functioning in the immune system, p75NTR has a role in ectodermal development and this function is shared by few TNFRSF members such as Troy, and the EDA receptors (EDAR and XEDAR). Sequence alignment indicates that Troy, EDAR and XEDAR fall within a subfamily of structurally similar TNFSF proteins. Although p75NTR does not have greater overall sequence similarity to Troy than to other TNFRSF proteins, p75NTR and Troy have been shown to share a very similar transmembrane domain. Compared to vertebrates, a small fraction of TNFRSF members were identified in invertebrate deuterostomes, such as Strongylocentrotus and Saccoglossus. Among these, p75NTR, p75NTR-like and Troy sequences were present, suggesting that the duplication separating the p75NTR gene lineage from the Troy/XEDAR/EDAR lineage had already taken place at the root of the deuterostomes. Although the ligand for Troy is unknown, XEDAR and EDAR are known in vertebrates to bind to two different isoforms of EDA, a protein with typical features of the TNFSFs. This may suggest that within the TNFRSF, neurotrophin binding receptors (p75NTR) diverged from portential TNFSF binding receptors at least at the stem of the deuterostome lineage, if not earlier. Prior to the current finding of p75NTRs in protostomes, the only TNFR representative known in protostomes was Drosophila Wengen. Wengen has some degree of similarity to p75NTR but also similarity to TNFR1 and XEDAR, which are receptors for TNFSF members. A ligand for Wengen, called Eiger has been reported, and Eiger is most similar to EDA, which is the XEDAR ligand. Eiger is clearly a TNFSF member and is not related to neurotrophins. tBLASTN searches against gene predictions on the Daphnia genome sequence with Drosophila Wengen as query, yields a hit with a relatively high e-value (0.044) to a gene prediction on scaffold 4 (Dappu-305473). The genomic sequence is supported by several ESTs ([Genbank: FE377316.1], [Genbank: FE348619.1]) and it encodes a TNFR domain protein which like Wengen lacks a death domain. In addition to this gene, partial ESTs in Daphnia present high similarity to the Eiger/EDA gene ([Genbank: FE337796.1], [Genbank: FE355401.1]) and encode a TNF domain. The ESTs are represented in the genomic sequence on scaffold 9. Although the evolution of the TNFRSF is beyond the scope of the present study, these preliminary results may suggest that Wengen and p75NTR are paralogues. Since Wengen binds to a TNFSF member, future research will be needed to investigate whether protostomian p75NTRs can indeed bind to protostome neurotrophins. If so, this may indicate that the prerequisites for separate evolution of TNFRSF binding to neurotrophins and TNFRSF binding to TNFSF members were already established before the protostome/deuterostome split.
In this work, the genome sequence of Daphnia pulex provides the first evidence for components of the neurotrophin/Trk/p75NTR signaling system in a protostome. Also, for the first time, Daphnia represents an organism where the genome encodes both Spz proteins and a neurotrophin, thus allowing a clarification of the relationship between neurotrophin and Spz proteins as paralogous families. Two new Spz protein families (Spz7 and Spz8) are moreover described, and representatives of these families are found in other crustaceans. Spz7 and Spz8 proteins are represented in Daphnia by 14 or more representatives, suggesting that the genes encoding these proteins could be under selective pressure. The Daphnia genome also reveals the most conserved Trk relative to the deuterostomes found so far in a protostome. The Daphnia Trk is found along side a Daphnia Trkl which is orthologous to Aplysia Trkl. The search of other genomes and ESTs derived from several protostome species reveals moreover that Trk and Trkl form closely related paralogous families in the protostomes within a "Trk/Trkl family" that is paralogous to NRKs. This highlights a previously unsuspected complexity in Trk and Trk related receptors in protostomes. For the first time, the Daphnia genome presents a protostome p75NTR receptor and the p75NTR is subsequently shown to exist in other protostomes. Thus the neurotrophin signaling ligand and receptors were probably already present before the protostome/deuterostome split. The finding in protostomes of p75NTR, which is a TNFRSF member endowed with a death domain, reopens the question as to whether the TNFRSF ancestral configuration was "death domain less" as previously suggested. It will be interesting to see whether a protostomian p75NTR functions in the immune system, the nervous system or both. Moreover, because Wengen, the other known protostomian TNFRSF member binds to a TNFSF ligand, it will be interesting to find out if and how protostomian p75NTRs bind to neurotrophins. Also, in contrast to Trks which bind neurotrophins with high selectivity, p75NTR binds indiscriminately to neurotrophins, so future work may reveal whether p75NTR binds to Spzs. The presence of the neurotrophin/Trk/p75NTR signaling system in Daphnia highlights the importance of Daphnia for phylogenetic studies, but also as a medical model, since neurotrophins, Trk receptors and p75NTR are involved in the nervous system, the immune system, and in various diseases ranging from Alzheimers to cancer[46, 47], including parasite infection.
Search for new genes
tBLASTN searches were done against NCBI database ESTs http://www.ncbi.nlm.nih.gov/ and also against the Daphnia pulex, Lottia gigantea, Helobdella robusta and Capitella Sp. I genome data made available from US Department of Energy Joint Genome Institute http://www.jgi.doe.gov/Daphnia. Information available on wFleaBase http://wFleaBase.org was also used. Searches on the Joint Genome Institute (JGI) website were done against all gene prediction models, and gene predictions from different software were aligned to each other as well as to EST sequences when available, to select the best prediction. When the models appeared incorrect based on knowledge of related sequences from the same gene family or superfamily, tBLASTN searches were done directly on the scaffold sequences. The genome browser from the JGI website was used to view the position of identical sequence duplicates on a given scaffold or different scaffolds. Additional analyses of the genes predicted, such as the presence of signature sequences in the translated polypeptide, were sought with prosite http://www.expasy.org/tools/scanprosite/. Domain architecture comparisons were generated by SMART http://smart.embl-heidelberg.de/. Potential signal peptidase cleavage sites on translated gene sequences were mapped with SignalP 3.0 http://www.cbs.dtu.dk/services/SignalP/ and prediction of arginine and lysine propeptide cleavage sites was performed using ProP 1.0 http://www.cbs.dtu.dk/services/ProP/.
Alignment and Phylogenetic analysis
Nucleotide sequences to be used in phylogenetic analysis were translated to amino acid sequences and the amino acid sequences were aligned with MUSCLE. The nucleotide sequences were subsequently aligned by RevTrans, using the MUSCLE amino acid alignment as a template. Phylogenetic analyses were performed with two different methods, maximum likelihood (ML) and Bayesian inference (BI). ML tree estimation was done using RaxML-VI v.7.0.0[55, 56]. BI analyses were performed using MrBayes 3.1.2 [57, 58]. Gaps and N's were treated as missing data.
The ML analysis consisted of 100 independent runs on the original alignment using RAxML under the GTRMIX substitution model (re-estimated all free model parameters) with estimated rearrangement settings, a rate category value of 25 and randomized MP (maximum parsimony) starting trees. This model implies an initial tree inference under the GTRCAT model and thereafter evaluates the final tree topology under the GTRGAMMA model until the likelihood values are stable. Branch support was assessed with 1000 non-parametric bootstrap replicates under the GTRMIX model (random number seed = 180874) and plotted on the ML tree with the best likelihood value. For the BI analyses, MrModeltest v.2  was used for search of best fit models for each codon position; In the RTK tree based on full length sequences (Figure 9A) positions 1 and 2 followed the (GTR+I+G) while position 3 followed the (HKY+I+G) of the Akaike information criterion (AIC). In the RTK tree based on sequences encoding the tyrosine kinase domains only (Figure 9B), positions 1 to 3 followed the (GTR+I+G) model. In the Neurotrophin/Spz tree (with Spz6 included), position 1 and 2 followed the (GTR+I+G) model while position 3 followed (GTR+G) (Figure 1 and Figure 2). The BI analyses consisted of four million generations in two parallel chains executed in three separate runs. The seed number was 180874. The first 2500 trees were discarded as "burn in".
The sequencing and portions of the analyses were performed at the DOE Joint Genome Institute under the auspices of the U.S. Department of Energy's Office of Science, Biological and Environmental Research Program, and by the University of California, Lawrence Livermore National Laboratory under Contract No. W-7405-Eng-48, Lawrence Berkeley National Laboratory under Contract No. DE-AC02-05CH11231, Los Alamos National Laboratory under Contract No. W-7405-ENG-36 and in collaboration with the Daphnia Genomics Consortium (DGC) http://Daphnia.cgb.indiana.edu. Additional analyses were performed by wFleaBase, developed at the Genome Informatics Lab of Indiana University with support to Don Gilbert from the National Science Foundation and the National Institutes of Health. Coordination infrastructure for the DGC is provided by The Center for Genomics and Bioinformatics at Indiana University, which is supported in part by the METACyt Initiative of Indiana University, funded in part through a major grant from the Lilly Endowment, Inc. Our work benefits from, and contributes to the Daphnia Genomics Consortium.
I wish to acknowledge the DOE Joint Genome Institute for permitting me to search and use information from the Helobdella robusta, Lottia gigantea and Capitella Sp. I genome sequences.
I wish to thank Dr. Matthias Obst for his help and support with phylogenetic programs and Prof. Michael Thorndyke for his strong support and for proofreading this manuscript.
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