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The evolution of the metazoan Toll receptor family and its expression during protostome development - PubMed

  • ️Fri Jan 01 2021

The evolution of the metazoan Toll receptor family and its expression during protostome development

Andrea Orús-Alcalde et al. BMC Ecol Evol. 2021.

Abstract

Background: Toll-like receptors (TLRs) play a crucial role in immunity and development. They contain leucine-rich repeat domains, one transmembrane domain, and one Toll/IL-1 receptor domain. TLRs have been classified into V-type/scc and P-type/mcc TLRs, based on differences in the leucine-rich repeat domain region. Although TLRs are widespread in animals, detailed phylogenetic studies of this gene family are lacking. Here we aim to uncover TLR evolution by conducting a survey and a phylogenetic analysis in species across Bilateria. To discriminate between their role in development and immunity we furthermore analyzed stage-specific transcriptomes of the ecdysozoans Priapulus caudatus and Hypsibius exemplaris, and the spiralians Crassostrea gigas and Terebratalia transversa.

Results: We detected a low number of TLRs in ecdysozoan species, and multiple independent radiations within the Spiralia. V-type/scc and P-type/mcc type-receptors are present in cnidarians, protostomes and deuterostomes, and therefore they emerged early in TLR evolution, followed by a loss in xenacoelomorphs. Our phylogenetic analysis shows that TLRs cluster into three major clades: clade α is present in cnidarians, ecdysozoans, and spiralians; clade β in deuterostomes, ecdysozoans, and spiralians; and clade γ is only found in spiralians. Our stage-specific transcriptome and in situ hybridization analyses show that TLRs are expressed during development in all species analyzed, which indicates a broad role of TLRs during animal development.

Conclusions: Our findings suggest that a clade α TLR gene (TLR-Ca) and a clade β/γ TLR gene (TLR-Cβ/γ) were already present in the cnidarian-bilaterian common ancestor. However, although TLR-Ca was conserved in cnidarians, TLR-Cβ/γ was lost during the early evolution of these taxa. Moreover, TLR-Cβ/γ duplicated to generate TLR-Cβ and TLR-Cγ in the lineage to the last common protostome-deuterostome ancestor. TLR-Ca, TLR-Cβ and TLR-Cγ further expanded generating the three major TLR clades. While all three clades radiated in several spiralian lineages, specific TLRs clades have been presumably lost in other lineages. Furthermore, the expression of the majority of these genes during protostome ontogeny suggests a likely role in development.

Keywords: Development; Gene duplication; Innate immunity; Metazoan evolution; Toll receptor; Toll-like receptor.

© 2021. The Author(s).

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1

Structure of TLR and TLR-like receptors. TLRs are constituted by a series of extracellular leucine-rich repeat (LRR) domains, a transmembrane region (TM) and an intracellular Toll/IL-1 receptor (TIR) domain. TLRs are often classified into V-type/scc or P-type/mcc according to the structure of their extracellular region. V-type/scc TLRs have only one LRRCT located next to the TIR domain, while P-type/mcc TLRs have more than one LRRCT and, sometimes, an LRRNT domain. Proteins that lack either the LRR domains or the TIR domain are not considered as TLR receptors. These TLR-like proteins are classified in LRR-only or TIR-only. [Adapted from 7, 13]

Fig. 2
Fig. 2

Review of the number of TLRs across metazoans. Within metazoans, no TLRs have been found outside Cnidaria and Bilateria. Spiralians show a variable number of TLRs, being, for example, 23 TLRs in the annelid C. teleta, but none in the rotifer A. vaga. In ecdysozoans, C. elegans and D. melanogaster have 1 and 9 TLRs, respectively. The number of TLRs in deuterostomes is also variable, being high in S. purpuratus and B. lanceolatum, but reduced in tunicates. References: [, , , , , –66, 69, 70, 72, 75, 82, 84, 88, 92, 94, 96, 97]. Phylogeny according to [98]

Fig. 3
Fig. 3

Number of TLRs in species included in the genome/transcriptome analyses. In general, the number of TLRs in spiralians (purple) is higher and more variable between species when compared to ecdysozoans (magenta). Species for which TLRs were not detected are excluded from the graph

Fig. 4
Fig. 4

TLR phylogenetic analysis and distribution of P-type/mcc or V-type/scc. A Phylogenetic analysis of TLRs based on maximum likelihood. Bootstrap values are indicated next to the main nodes, being all nodes with bootstrap values > 60 marked with full dots and colored differently according to the support values. Tip labels contain an abbreviation of the species name and the gene name given in this study (for sequences searched de novo here) or in the original study (for sequences obtained from the literature). Numbers in the gene name do not imply gene orthology. Species abbreviations: Ael: A. elegans; Ad: A. digitifera; Am: A. millepora; Bgl: B. glabrata; Ce: C. elegans; Cgi: C. gigas; Ci: C. intestinalis; Cs: C. sinensis; Dm: D. melanogaster; Dpu: D. pulex; Efe: E. fetida; Ese: E. senta; Goc: G. oculata; Hex: H. exemplaris; Hps: H. psittacea; Hro: H. robusta; Hsa: H. sapiens; Hsp: H. spinulosus; Isc: I. scapularis; Mme: M. membranacea; Nge: N. geniculatus; Nv: N. vectensis; Lan: L. anatina; Lloa: L. loa; Llon: L. longissimus; Lrub: L. ruber; Lrug: L. rugatus; Obi: O. bimaculoides; Od: O. dioica; Of: O. faveolata; Ovo: O. volvulus; Pau: P. australis; Pcap: P. capensis; Pcau: P. caudatus; Phe: P. hermeri; Ppe: P. peregrina; Ppr: P. prolifca; Pps: P. psammophila; Pva: P.vancouverensis; Rva: R. varieornatus; Sp: S. purpuratus; Ttr: T. transversa. B Presence/absence of the TLR clades in the metazoan groups included in our study

Fig. 5
Fig. 5

TLR expression in developmental stage-specific transcriptomes of (A) H. exemplaris, (B) P. caudatus, C C. gigas and (D) T. transversa. Heatmaps corresponding to the average of the RSEM analyses are shown. For heatmaps corresponding to Kallisto analyses see Additional files 6, 7, 8 and 9: Tables S3–S6. Bold indicates stages and genes for which in situ hybridization was performed. TMM: Trimmed means of M values

Fig. 6
Fig. 6

Expression of TLRs during the development of the brachiopod T. transversa. Whole-mount in situ hybridization (WMISH) of TLRs in T. transversa embryos and larvae. Above the WMISH plates, there are schematic representations of each developmental stage analyzed. These representations are not to scale. The name of each gene is indicated in the rectangles on the left. All panels show dorso-ventral views and anterior to the top. Squares in the top-right of each plate indicate whether the expression was detected (yellow) or not (blue) in the stage-specific transcriptome analysis. Ectoderm, mesoderm and endoderm is indicated with blue, red and yellow arrowheads, respectively. The red and yellow arrowhead indicates endomesoderm. The ring-shape (indicated with white asterisks) staining present in the late larvae is background staining (Additional file 10: Fig. S4), probably related with the spicule formation described by Stricker and Reed [–128]. Scale bar indicates 50 μm. al: apical lobe; bp: blastopore; cs: chaetal sacs; em: endomesoderm; me: mesoderm; ml: mantle lobe; pl: pedicle lobe

Fig. 7
Fig. 7

Comparison between Davidson et al. [65] and this study. The main conclusions and the number of TLRs and species included in the two studies are compared. Cnidaria (C), Spiralia (S), Edysozoa (E) and Deuterostomia (D)

Fig. 8
Fig. 8

Origin and evolution of TLRs. Gene lineages are depicted in different colors (TLR-Cα: light brown; TLR-Cβ/γ and TLR-Cβ: light grey; and TLR-Cγ: dark grey) within the metazoan tree. Gene losses are indicated with a cross. Phylogeny according to: [98]. For other hypotheses see Additional file 11: Fig. S5

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