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. 2013 Jul 26;341(6144):384-7.
doi: 10.1126/science.1238036.

Identification of a Colonial Chordate Histocompatibility Gene

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Free PMC article

Identification of a Colonial Chordate Histocompatibility Gene

Ayelet Voskoboynik et al. Science. .
Free PMC article

Abstract

Histocompatibility is the basis by which multicellular organisms of the same species distinguish self from nonself. Relatively little is known about the mechanisms underlying histocompatibility reactions in lower organisms. Botryllus schlosseri is a colonial urochordate, a sister group of vertebrates, that exhibits a genetically determined natural transplantation reaction, whereby self-recognition between colonies leads to formation of parabionts with a common vasculature, whereas rejection occurs between incompatible colonies. Using genetically defined lines, whole-transcriptome sequencing, and genomics, we identified a single gene that encodes self-nonself and determines "graft" outcomes in this organism. This gene is significantly up-regulated in colonies poised to undergo fusion and/or rejection, is highly expressed in the vasculature, and is functionally linked to histocompatibility outcomes. These findings establish a platform for advancing the science of allorecognition.

Figures

Fig. 1
Fig. 1. Genomic characterization of the cFuHC locus in B. schlosseri reveals two tightly linked genes
The cFuHC locus encodes two gene products, sFuHC (a secreted form) and mFuHC (a membrane-bound form). Sequences aligned, from bottom to top: (i) Genomic contigs from B. schlosseri draft assembly; (ii) Fosmid clone used to characterize cFuHC (12) (table S5); (iii) predicted exon structures, with genomic coordinates indicated below in red (contigs with identical interexon distances to the fosmid are colored gray); (iv) B. schlosseri expressed sequence tags (ESTs) obtained from NCBI; (v) Sanger-sequenced PCR products resulting from selected cFuHC amplicons (table S1); (vi) representative RNA-Seq reads (100 bp × 2) from 17 colonies (table S4); (vii) translated primary sequences with predicted functional domains (14). All alignments were performed with megablast (mismatch penalty = −2, ≥90% identity, no query filtering, and otherwise default parameters). EGF, epidermal growth factor; IG, immunoglobulin domain; SP, signal peptide; TM, transmembrane domain.
Fig. 2
Fig. 2. Genome-wide analysis for candidate Fu/HCs reveals a single gene that exhibits perfect alignment with fusibility outcomes and defined Fu/HC genotypes
The ability to stratify known fusion or rejection outcomes was tested for all predicted genes from the draft assembly having transcriptome data covering ≥6 fusion and ≥6 rejection pairs, ≥20 common sites sequenced per pair, and at least 1 amino acid polymorphism (after filtering, n = 7,523 genes) (table S5). A, Classification errors across the genome are depicted as a boxplot showing the median (horizontal line), 25th to 75th percentiles (within the box), and 1st to 99th percentiles (whiskers). Although sFuHC is in the top 1% of best-performing genes, novel Fu/HC candidates with equal or better performance were also identified and are indicated in pink beneath the boxplot. Classification errors <0.2 (dotted line) have a p-value < 0.001, as determined by 1 million random permutations of known fusibility outcomes for each gene analyzed in the assembly (table S6). B, A B. schlosseri gene that exhibits perfect sequence concordance with fusion or rejection outcomes and defined genotypes, termed BHF. C, BHF genomic and message sequence architecture (table S7), representative RNA-Seq coverage and amino acid polymorphisms across all 17 colonies from the exploratory cohort (tables S4).
Fig. 3
Fig. 3. BHF accurately predicts new fusibility outcomes and has expression patterns and function consistent with a Botryllus allorecognition determinant
A, Known and predicted fusion or rejection outcomes among all 23 B. schlosseri colonies analyzed (table S4), including exploratory (n = 17) and validation cohorts (n = 6). All “blind” predictions were confirmed (6 of 6). B, Expression analysis of BHF, sFuHC, and mFuHC under the conditions preceding fusion or rejection (“challenged”; n = 6) compared to unchallenged control colonies (“naïve”; n = 4) (*P = 0.009, two-tailed unequal variance t test; NS, not significant). Values are presented as mean +/−SEM. C, BHF expression patterns assessed by whole-mount in situ hybridization, compared with control (sense probe). amp, ampullae. Scale bars, 50 μm. D, Analysis of morpholino-induced knockdown of BHF. Top: Unreactive ampullae from apposing colonies under BHF-knockdown conditions (left) and at lower magnification (right). (Bottom) Fused blood vessels between colonies injected with morpholino control (left) and at lower magnification (right). amp, ampullae. Scale bars, 1mm.

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