Evolution of immune chemoreceptors into sensors of the outside world

Abstract

Changes in gene expression patterns represent an essential source of evolutionary innovation. A striking case of neofunctionalization is the acquisition of neuronal specificity by immune formyl peptide receptors (Fprs). In mammals, Fprs are expressed by immune cells, where they detect pathogenic and inflammatory chemical cues. In rodents, these receptors are also expressed by sensory neurons of the vomeronasal organ, an olfactory structure mediating innate avoidance behaviors. Here we show that two gene shuffling events led to two independent acquisitions of neuronal specificity by Fprs. The first event targeted the promoter of a V1R receptor gene. This was followed some 30 million years later by a second genomic accident targeting the promoter of a V2R gene. Finally, we show that expression of a vomeronasal Fpr can reverse back to the immune system under inflammatory conditions via the production of an intergenic transcript linking neuronal and immune Fpr genes. Thus, three hijackings of regulatory elements are sufficient to explain all aspects of the complex expression patterns acquired by a receptor family that switched from sensing pathogens inside the organism to sensing the outside world through the nose.

Keywords: gene evolution; neofunctionalization; olfaction; olfactory receptor; vomeronasal organ.

Fig. 1.

The different identities of olfactory and immune Fprs. (A) Phylogenetic relationships between Fprs pertaining to 32 different mammalian species (16 Eumuroida, 12 non-Eumuroida glires, and 4 other mammals). The tree was inferred from a protein alignment with a maximum likelihood approach. The scale bar corresponds, for each branch, to amino acid substitutions per site. Branches with low bootstrap support (<30%) are indicated by a white disk. Black dots indicate species for which Fpr tissue specificity (vomeronasal or immune) was evaluated by qPCR, RNA-seq, or immunohistochemistry. On the bottom left part of the tree, the schematic represents a coronal section through a VNO with its basal and apical neuroepithelium. The dotted area encompasses all FPR-rs3 orthologs. (B) Conserved amino acid residues present in FPR-rs3 of all tested species and never observed in FPR-rs2 or FPR1. Colored residues are conserved at 100% in FPR-rs3 of all tested rodent species (n = 17 species, 18 alleles) (Fig. S3). (C) Pairwise K_a/K_s ratios and K_s rates of immune and vomeronasal Fprs from various rodent species (the row order follows the order in Fig. S3). (D) Intermingling between Fpr and Vr genes is associated with vomeronasal Fpr expression. As landmarks of the synteny, the Has1 and Ppp2r1a nonolfactory genes are shown in green. Genomic coordinates are indicated in Table S6. Genes (colored rectangles) are arranged according to their transcriptional orientation.

Fig. S1.

A fast-evolving Fpr gene family. (A) Synteny between the rat and the mouse Fpr/Vr gene clusters (localized on chromosomes 1 and 17, respectively). Colored shades indicate expansions and contractions of homologous groups of genes between rat and mouse. (B) Phylogenetic relationships between mouse Fpr genes relative to their genomic position. (C) Fpr/Vr gene cluster organization of various species (as presented in Fig. 1D), at scale.

Fig. S2.

Expression of Fprs evaluated by qPCR in tissues from rat, guinea pig, and Chinese hamster. Colored disks indicate the level of gene expression in each tissue. For each condition, expression values were first normalized (Methods and Table S4) and then, for each gene and in each species, displayed relative to the highest expression value.

Fig. S3.

Phylogenetic tree corresponding to the species used to determine the differentially conserved amino acid residues in immune and vomeronasal Fprs. The green disks indicate the Fpr sequences used to determine FPR-rs3–specific amino acids. Overlapping dots show the number of alleles or paralogs used. Yellow disks indicate pseudogenes. A noncolored disk indicates lack of a PCR amplicon with degenerate primers of the corresponding Fpr (Methods), or no sequence identified in the genome assembly. Small red dots under the circles correspond to the sequences used for the K_a/K_s and the K_s matrices shown in Fig. 1C.

Fig. 2.

Fpr promoters driving apical and basal vomeronasal neuron expression were originally V1r and V2r promoters, respectively. (A) Sequence identities between the mouse Fpr-rs3 promoter and first noncoding exon, and the corresponding segments of Fpr, V1r, and V2r genes from various species are shown. Homologies corresponding to the CDS are amino acid identities. The blue color in the vomeronasal schematic indicates the apical layer in which Fpr-rs3, Fpr-rs4, Fpr-rs6, and Fpr-rs7 are transcribed. (B) Blast2 plots showing homologies (the dotted transverse line) between CpV1rx1 and pseudogenic remnants of V1r coding sequences located between exon 1 and the coding sequence of the mouse Fpr-rs7. (C and D) Closest relatives to CpV1rx in the mouse genome (V1rx-ps1 and V1rx-ps2) and in the rat genome (RnV1rx-ps1, RnV1rx-ps2, RnV1rx-ps3, and RnV1rx-ps4). (E) Sequence identities as in A, but with the basally expressed Fpr-rs1.

Fig. S4.

Acquisition of vomeronasal specificity led to the extinction of a V1r family. (A) Blast2 plots showing homologies (the dotted transverse line) between CpV1rx1 and pseudogenic remnants of V1r coding sequences located between exon 1 and the coding sequence of the rat Fpr-rs6. (B) Syntenic analysis of V1rx genes among mammals. (C) Phylogenetic tree corresponding to all V1rs from mouse, rat, guinea pig, horse, cat, and rabbit. (Insert) No intact member of family V1rx remains in the rat or in the mouse genome. The scale bar indicates amino acid substitutions per site.

Fig. 3.

Reacquisition of immune specificity by Fpr-rs1. (A, Upper) Schematic representing the experimental design. (A, Lower) RT-PCR (primer locations indicated in B) showing two Fpr-rs1 transcript variants, one driven by the Fpr-rs1 promoter and the other driven by the immune Fpr-rs2 promoter. The abundance of this latter intergenic splice variant increases in bone marrow cells after LPS treatment. (B) Schematic corresponding to the vomeronasal (1) and immune (2) Fpr-rs1 transcripts.

Fig. S5.

Acquisition of neuronal specificity by Fprs. Schematic indicating the timing of acquisition by Fprs of apical and basal vomeronasal sensory neuron expression during the evolution of rodents.