Transcriptome profiling of Trichoplax adhaerens highlights its digestive epithelium and a rich set of genes for fast electrogenic and slow neuromodulatory cellular signaling

Abstract

Background Trichoplax adhaerens is a fascinating early-diverging animal that lacks a nervous system and synapses, and yet is capable of directed motile feeding behavior culminating in the external digestion of microorganisms by secreted hydrolytic enzymes. The mechanisms by which Trichoplax cells communicate with each other to coordinate their activity and behavior is unclear, though recent studies have suggested that secreted regulatory peptides might be involved.Results Here, we generated a high quality mRNA transcriptome of Trichoplax adhaerens , and predicted secreted proteins to identify gene homologues for digestion, development, immunity, cell adhesion, and peptide signaling. Detailed annotation of the expressed Trichoplax gene set also identified a nearly complete set of electrogenic genes involved in fast neural signalling, plus a set of 665 G-protein coupled receptors that in the nervous system integrate with fast signalling machinery to modulate cellular excitability. Furthermore, Trichoplax expresses an array of genes involved in intracellular signaling, including the key effector enzymes protein kinases A and C that functionally link fast and slow cellular signaling. Also identified were nearly complete sets of pre- and post-synaptic scaffolding genes, most encoding appropriate protein domain architectures. Notably, the Trichoplax proteome was found to bear slightly reduced counts of synaptic protein interaction domains such as PDZ, SH3 and C2 compared to other animals, but abundance of these domains did not appear to predict the presence of synapses in early-diverging groups.Conclusions Despite its apparent cellular and morphological simplicity, Trichoplax expresses a rich set of genes involved in complex animal traits. The transcriptome presented here adds a valuable additional resource for molecular studies on Trichoplax genes, exemplified by our ability to clone cDNAs for nine full-length acid sensing ion channel proteins with almost perfect matches with their corresponding transcriptome sequences.

Early metazoan origin and multiple losses of a novel clade of RIM pre-synaptic calcium channel scaffolding protein homologues

“…Work in our laboratory identified two RIM homologues in the transcriptome of Trichoplax adhaerens , a small sea water invertebrate that diverged from other animals roughly 600 million years ago (Dos Reis, et al 2015), and that lacks a nervous system and synapses (Smith, et al 2014). One paralogue was found to be considerably longer in protein sequence (2,487 residues) and to lack a predicted PDZ domain for interactions with the C-termini of Ca V 2 channels (Figure 1A) (Wong, et al 2019). We decided to name this particular homologue type I RIM (I-RIM), based on similarities with canonical RIM genes described in other animals.…”

“…Phylogenetic trees were visualized with MEGA X (Kumar, et al 2018) and FigTree 1.4.3 (Rambaut 2007), and shared nodes with respectively strong bootstrap support and high posterior probability values were annotated on the maximum likelihood tree shown in Figure 1. NCBI protein sequence accession numbers (unless otherwise indicated) are as follows: H.sapiens I-RIM1α: NP_055804.2; M.musculus I-RIM1α: NP_444500.1; R.norvegicus I-RIM1α: Q9JIR4.1; G.gallus I-RIM1α: XP_025004772.1; H.sapiens I-RIM2α: Q9UQ26.2; M.musculus I-RIM2α: NP_444501.1; R.norvegicus I-RIM2α: NP_446397.1; G.gallus I-RIM2α: XP_015138471.1; S. kowalevskii I-RIM: sakowv30037565m (OIST Marine Genomics Unit); P.flava I-RIM: pfl_40v0_9_20150316_1g5731.t1 (OIST Marine Genomics Unit); S.purpuratus I-RIM: XP_011664650.1; A.planci I-RIM: XP_022091188.1; A.californica I-RIM: XP_005106594.2; C.gigas I-RIM: XP_019921964.1; M.yessoensis I-RIM: XP_021377513.1; O.bimaculoides I-RIM: XP_014789722.1; E.granulosus I-RIM: XP_024347539.1; S.haematobium I-RIM: XP_012796083.1; L.anatina I-RIM: XP_013421607.1; L.polyphemus I-RIM (1065aa): XP_022257304.1; L.polyphemus I-RIM (1630aa): XP_022243248.1; C.sculpturatus I-RIM: XP_023224599.1; H.azteca I-RIM: XP_018007323.1; D.melanogaster I-RIM: NP_001247161.2; H.dujardini I-RIM: OQV23161.1; R.varieornatus I-RIM: GAV01719.1; C.elegans I-RIM: NP_741831.1; N.vectensis I-RIM: evg1261940 (from in house transcriptome assembly (Wong, et al 2019)); E.pallida I-RIM: XP_020898842.1; H.vulgaris I-RIM: XP_012564401.1; T.adhaerens I-RIM: evg1642237 (from in house transcriptome assembly (Wong, et al 2019)); O.carmela I-RIM (739aa): m.21147 (Compangen); O.carmela I-RIM (1164aa): m.26069 (Compangen); S. kowalevskii II-RIM: sakowv30000298m (OIST Marine Genomics Unit); A.californica II-RIM: XP_012944640.1; C.gigas II-RIM: XP_011422085.1; M.yessoensis II-RIM: XP_021345230.1; O.bimaculoides II-RIM: XP_014774175.1; L.anatina II-RIM: XP_013397099.2; C.sculpturatus II-RIM: XP_023227959.1; L.polyphemus II-RIM (1313aa): XP_022258855.1; L.polyphemus II-RIM (1323aa): XP_022240908.1; M.leidyi II-RIM: evg198193 (Wong, et al 2019); B.ovata II-RIM: combined overlapping fragmented transcripts TR51711|c1_g1_i2 and TR51711|c1_g3_i7 from unpublished transcriptome generated by Mark Martindale and Joseph Ryan, Whitney Lab, Florida; H.californiensis II-RIM: evg158061 (Wong, et al 2019); E.pallida II-RIM: KXJ21887.1; T.adhaerens II-RIM: evg1176111 (Wong, et al 2019); H.sapiens rabphilin: NP_001137326.1; M.musculus rabphilin: NP_001289273.1; R.norvegicus rabphilin: NP_598202.1; G.gallus rabphilin: XP_015131024.1; S. kowalevskii rabphilin: XP_006817123.1; S.purpuratus rabphilin: XP_030848070.1; A.planci rabphilin: XP_022085716.1; A.californica rabphilin: XP_012945559.1; C.gigas rabphilin: XP_011424844.1; M.yessoensis rabphilin: XP_021345406.1; O.bimaculoides rabphilin: XP_014779575.1; L.anatina rabphilin: XP_013406958.1; L.polyphemus rabphilin: XP_013782034.1; R.varieornatus rabphilin: GAV03979.1; H.dujardini rabphilin: OQV25295.1; D.melanogaster rabphilin: NP_572651.1; H.azteca rabphilin: XP_018018219.1; C.elegans rabphilin: NP_001022566.1; S.haematobium rabphilin: XP_012797599.1; E.granulosus rabphilin: CDS16155.1; A.digitifera rabphilin: XP_015773295.1; E.pallida rabphilin: XP_020895735.1; H.vulgaris rabphilin: XP_012561655.1; M.leidyi rabphilin: evg19024 (Wong, et al 2019); B.ovata rabphilin: TR42475|c0_g1_i1 (Mark Martindale and Joseph Ryan); H.californiensis rabphilin: evg140073 (Wong, et al 2019); T.adhaerens rabphilin: evg1107189.2 (Wong, et al 2019); O.carmela rabphilin: m.15463 (Compangen).…”

“…To infer the phylogenetic relationships of Ca V channels, a similar approach was used as described for the RIM/rabphillin maximum likelihood tree, with manual trimming of the protein alignment, and a substitution model of LG+I+G4 selected by IQ-TREE (sequences and trimmed alignment provided in respective supplementary files 2 and 3). NCBI protein sequence accession numbers (unless otherwise indicated) are as follows: C.porosus Ca V 1.1: XP_019401893.1; G.gallus Ca V 1.1: NP_001292076.1; P.vitticeps Ca V 1.1: XP_020660962.1; G.japonicus Ca V 1.1: XP_015267320.1; H.sapiens Ca V 1.1: NP_000060.2; D.rerio Ca V 1.1: NP_001139622.1; C.porosus Ca V 1.2: XP_019400097.1; G.gallus Ca V 1.2: XP_015142138.1; H.sapiens Ca V 1.2: Q13936.4; P.vitticeps Ca V 1.2: XP_020644644.1; G.japonicus Ca V 1.2: XP_015278355.1; C.picta bellii Ca V 1.2: XP_008162493.1; D.rerio Ca V 1.2: XP_009298610.1; P.vitticeps Ca V 1.3: XP_020659553.1; G.japonicus Ca V 1.3: XP_015274327.1; C.picta bellii Ca V 1.3: XP_023956012.1; G.gallus Ca V 1.3: XP_015148473.1; H.sapiens Ca V 1.3: NP_001122312.1; C.porosus Ca V 1.3: XP_019392560.1; D.rerio Ca V 1.3: XP_021335256.1; P.vitticeps Ca V 1.4: XP_020648615.1; A.carolinensis Ca V 1.4: XP_016846283.1; P.humilis Ca V 1.4: XP_005533405.1; H.sapiens Ca V 1.4: NP_005174.2; C.intestinalis Ca V 1: XP_018667169.1; H.roretzi Ca V 1: BAA34927.2; A.melifera Ca V 1: XP_016766333.1; A.aegypti Ca V 1: XP_021699870.1; D.melanogaster Ca V 1: AAF53504.1; D.pulex Ca V 1: EFX89598.1; D.magna Ca V 1: KZS09199.1; C.elegans Ca V 1: NP_001023079.1; C.nigoni Ca V 1: PIC34019.1; D.pachys Ca V 1: PAV74346.1; P.pacificus Ca V 1: PDM72512.1; S.ratti Ca V 1: XP_024499702.1; T.britovi Ca V 1: KRY55670.1; T.pseudospiralis Ca V 1: KRY90155.1; L.stagnalis Ca V 1: AAO83839.1; C.gigas Ca V 1: XP_011452714.1; O.bimaculoides Ca V 1: XP_014774811.1; L.anatina Ca V 1: XP_023932094.1; A.planci Ca V 1: XP_022081221.1; S.purpuratus Ca V 1: XP_011670134.1; N.vectensis Ca V 1: combined overlapping fragmented transcripts evg1131374 and TRINITY_DN40077_c0_g1_i4 (Wong, et al 2019); E.pallida Ca V 1: XP_020903719.1; A.digitifera Ca V 1: XP_015778662.1; C.capillata Ca V 1: AAC63050.1; T.adhaerens Ca V 1: evg1032956 (Wong, et al 2019); O.carmela Ca V 1: combined overlapping fragmented transcripts m.84361 and m.186460 and g4118.t1 (Compagen); A.mississippiensis Ca V 2.1: XP_019354952.1; S.vulgaris Ca V 2.1: XP_014748708.1; G.gallus Ca V 2.1: ATE62979.1; C.picta bellii Ca V 2.1: XP_008161515.1; H.sapiens Ca V 2.1: O00555.3; P.vitticeps Ca V 2.1: XP_020656028.1; G.japonicus Ca V 2.1: XP_015274358.1; D.rerio Ca V 2.1: XP_021330116.1; C.picta bellii Ca V 2.2: XP_008172570.1; C.porosus Ca V 2.2: XP_019396155.1; G.gallus Ca V 2.2: XP_015134766.1; P.vitticeps Ca V 2.2: XP_020668759.1; G.japonicus Ca V 2.2: XP_015276634.1; H.sapiens Ca V 2.2: NP_000709.1; D.rerio Ca V 2.2: XP_021331856.1; C.porosus Ca V 2.3: XP_019386495.1; G.gallus Ca V 2.3: XP_015145962.1; C.picta bellii Ca V 2.3: XP_008164802.1; P.vitticeps Ca V 2.3: XP_020645405.1; A.carolinensis Ca V 2.3: XP_008107103.1; H.sapiens Ca V 2.3: NP_001192222.1; D.rerio Ca V 2.3: XP_017206695.2; C.intestinalis Ca V 2: XP_018670105.1; A.melifera Ca V 2: XP_016766516.1; A.aegypti Ca V 2: XP_021710362.1; D.melanogaster Ca V 2: AFH07350.1; H.azteca Ca V 2: XP_018019172.1; C.elegans Ca V 2: NP_001123176.1; C.nigoni Ca V 2: PIC16379.1; H.contortus Ca V 2: CDJ96819.1; S.ratti Ca V 2: XP_024503488.1; T.britovi Ca V 2: KRY49212.1; T.pseudospiralis Ca V 2: KRY73966.1; L.stagnalis Ca V 2: AAO83841.1; A.californica Ca V 2: AVD53847.1; C.gigas Ca V 2: XP_019920407.1; H.bleekeri Ca V 2: BAA13136.2; A.planci Ca V 2: XP_022085274.1; S.purpuratus Ca V 2: XP_011662956.1; N.vectensis Ca V 2a: combined overlapping fragmented transcripts evg194605 (Wong, et al 2019) and NVE4667 (David, et al 2013); E.pallida Ca V 2a: XP_020910418.1; O.faveolata Ca V 2a: XP_020626975.1; A.digitifera Ca V 2a: XP_...…”

“…The dashed line denotes the absence of a predicted PDZ domain for the Trichoplax I-RIM homologue. B) Average transcripts per million (TPM) expression level analysis of the Trichoplax whole animal transcriptome (Wong, et al 2019) reveals expression of a rich set of active zone proteins, plus all three voltage-gated calcium channel paralogues (the color scheme follows that of the Ca V -interactome depicted in panel A. The housekeeping genes eukaryotic translation elongation factor-1α (EF-1αa) and EF-1αb, hypoxanthine phosphoribosyltransferase 1 (HPRT1), and succinate dehydrogenase (SDHA) were used as standards for expression level.…”

“… A) Schematic of pre-synaptic active zone proteins identified in the transcriptome of Trichoplax adhaerens (Wong, et al 2019). Colored proteins represent key pre-synaptic scaffolding proteins that interact with N-, P/Q-, and R-type Ca V 2 channels.…”