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Histocompatibility is the basis by which multicellular organisms of the same species distinguish self from non-self. 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/non-self and determines “graft” outcomes in this organism. This gene is significantly upregulated in colonies poised to undergo fusion 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.
Allorecognition, the capacity to distinguish “self’ from allogeneic “nonself’, is critical for multicellular life. This process also has important implications for humans, as it underlies maternal tolerance of the fetus (1–2) and the outcomes of blood or tissue transplants (3–4). To gain insights into the evolution and molecular characteristics of allorecognition, we are studying Botryllus schlosseri, a member of the urochordates, the closest living sister group of vertebrates (5). B. schlosseri engages in a natural transplantation reaction, whereby colonies undergo self-nonself recognition, which leads to either formation of parabionts with a fused vasculature (i.e., fusion) or an inflammatory rejection response (i.e., rejection) (fig. S1). A polymorphic gene locus governs fusion/rejection outcomes (6–9). This locus, called Fu/HC for fusion/histocompatibility, encodes multiple co-dominant alleles, and progeny from crosses between histocompatible B. schlosseri colonies are known to segregate as a monogenic trait (8, 9). The rules governing fusibility reactions are as follows: AB=AB leads to fusion, AB=CD to rejection, and AB=BC to fusion. Previously, we identified a highly polymorphic candidate allorecognition gene (cFuHC) within the Fu/HC locus (10–12). As the major histocompatibility regions in vertebrates are haplotypes (that is, sets of linked genes), we analyzed the recently completed B. schlosseri genome (13) to determine whether a haplotype or single protein-encoding gene encodes self-nonself recognition.
Using diverse sequencing data, we first attempted to validate the genomic structure of the cFuHC, which previously appeared to correlate with fusion or rejection outcomes (12). The original cFuHC model consists of two dominant isoforms, a secreted form and a membrane-bound form encompassing the entire predicted gene (12). We found that instead of two isoforms, the cFuHC consists of two genes separated by 250 base pairs (bp) (Fig. 1, tables S1–S3). We found no evidence for an mRNA isoform bridging these two genes (table S4). One gene, which we term sFuHC, is identical to the original secreted isoform; the other, termed mFuHC, includes the remaining portion of cFuHC, but has a novel N-terminal exon encoding a signal peptide (14) (table S5). BLAST analysis revealed a homolog of mFuHC, but not sFuHC, in Ciona intestinalis (gi|198429243) [Expectation value (E-value) = 4e−37], which further supported our finding. Both genes are highly polymorphic (fig. S2), as previously reported for cFuHC (12).
Next, we tested whether any genes from the draft assembly encode alleles consistent with a Botryllus histocompatibility factor. We used two complementary strategies, one to assess allelic concordance with known fusibility outcomes and the other to evaluate allelic agreement with Fu/HC genotypes defined by breeding experiments. For the former, we developed a computational pipeline that includes methods to accurately and efficiently phase paired-end RNA sequencing (RNA-Seq) reads into haplotypes, compare phased alleles between colonies, and score each gene based on its ability to stratify known fusibility outcomes (figs. S3 to S7; 15). For the latter, we established lines of distinct Fu/HC genotypes (AA, BB, AB and AX), and used a classical genetics approach (fig. S8). By performing RNA-Seq on colonies with defined Fu/HC genotypes (fig. S8), we could precisely screen for allorecognition factor candidates, because any genes inconsistent with defined genotypes must be incorrect.
In all, 17 colonies encompassing 29 pairs of known fusion-rejection outcomes were analyzed. To increase sensitivity, we included pairs of related rejecting colonies bred in our laboratory and unrelated fusing colonies obtained from the wild (fig S8). Transcriptome sequencing (table S4), followed by haplotype phasing and interallele comparison (fig. S4), revealed that sFuHC and, to some extent, mFuHC, significantly stratify colony pairs by known fusion-rejection outcomes (P=5.6×10−5 and P=0.05, respectively, as determined by 1 million random permutations of known fusion-rejection labels across the genome) (Fig. 2A and tables S5 and S6). Although significant, segregation was not perfect for either gene (Fig. 2A and fig. S9), and neither sFuHC nor mFuHC are concordant in primary sequence among all AA colonies (fig. S9), and so, they fail the classical genetics test. These results indicate that the allorecognition factor in B. schlosseri is encoded by another gene, consistent with a recent report (16).
Our unbiased genome-wide scan revealed three candidate genes with perfect classification performance (Fig. 2A). Among them, only one gene is also fully consistent with genetically defined lines (Fig. 2B; Database S2 and table S6). This gene is free of any amino acid differences between histocompatible pairs (Fig. 2B); is highly polymorphic (Fig. 2C); and, on the basis of RNA-Seq, is expressed more highly than either sFuHC or mFuHC (fig. S10). It is striking that analysis of the fosmid sequence used to identify cFuHC revealed that this gene is located ~62kb away from sFuHC and mFuHC (Fig. 2C and table S5). Analysis of the draft genome confirmed physical linkage for these three genes (13).
We termed this candidate Fu/HC gene, “Botryllus Histocompatibility Factor” (BHF), and further analyzed its sequence, relationship to fusibility outcomes, and expression patterns. BHF is composed of three exons, encoding a highly charged and partially unstructured 252- amino acid protein (Fig. 2C, fig. S11, and tables S5 and S7), with no detectable domains or signal peptide (14). BHF has three remote homologs in the National Center for Biotechnology Information (NCBI) database, all of which encode uncharacterized proteins from solitary tunicates (fig. S12). Because colonial but not solitary tunicates participate in fusibility reactions, we attempted to amplify BHF from two other colonial tunicate species (Botrylloides sp. and Diplosoma sp.). We succeeded in recovering highly similar sequences from both species (fig. S12), which indicated that BHF may represent a general colonial tunicate allorecognition actor. To validate BHF, we sequenced four additional B. schlosseri colonies by RNA-Seq (Fig. 3A and table S4), and performed BHF Sanger-sequencing on two additional AA colonies (fig. S13). We found that BHF absolutely aligns with fusibility outcomes in the validation cohort (Fig. 3A), and is homozygous and identical in sequence among all AA colonies (fig. S14A). Moreover, polymorphisms within the first 100 amino acids could predict the outcomes of all histocompatibility reactions (fig. S14B), and at the nucleotide level, BHF remains absolutely predictive (fig. S15).
Among the 23 colonies examined, we determined 10 unique BHF alleles that not only agree with all known fusibility outcomes (Fig. 3A and fig. S14) and known pedigree relations (fig. S13), but also allow for the confirmation of precise predictions of B. schlosseri self-non-self recognition events. As an example, we predicted that colony 31 (genotype AD) would fuse with colony 944 (genotype AD) and reject colony 4 (genotype BI), and that colony Sc109e would fuse with colony 31. Indeed, we confirmed our predictions for these pairs, along with all other pairs tested (n=6 of 6) (Fig. 3A and, e.g., fig. S16).
We next asked whether BHF is up-regulated under the conditions preceding fusion or rejection, a potential outcome of a bona fide fusibility factor. In tissues that participate in allorecognition (vasculature/tunic), levels of BHF, sFuHC, and mFuHC were assessed by real-time polymerase chain reaction (PCR) in both apposing colonies (“challenged”) and physically unpaired colonies (“naïve”). We found a significant upregulation of BHF but not sFuHC or mFuHC in challenged colonies (two-tailed t test, P=0.009) (Fig. 3B). Moreover, among these three genes, only BHF was found among transcripts associated with the B. schlosseri rejection response (17) (table S6).
We next investigated BHF localization and expression. Using whole-mount in situ hybridization, we found high expression levels of BHF in blood vessels, including cells in the ampullae (Fig. 3C, top). Increased BHF expression was also observed on cells lining the periphery of blood vessels, consistent with epithelium (Fig. 3C, bottom). By RNA-Seq, we found enriched expression of BHF in the vasculature compared to endostyle (fig. S17), and by semi-quantitative PCR and Sanger-sequencing, we found broad expression of BHF in blood, ampullae, bud, endostyle region, tadpole, and sperm (fig. S18). These data are consistent with a histocompatibility-related function for BHF.
Finally, to assess BHF function, we performed morpholino-mediated knockdown experiments (15). In colony allorecognition assays, three of four isogenic pairs receiving control morpholinos fused within 24 hours of ampullae contact. By contrast, no reactions were observed in isogenic pairs receiving BHF translation-blocking morpholinos (n=6), despite constant physical contact over observational periods ranging from 2 to 7 days (Fig. 3D, fig. S19, and table S8). To exclude nonspecific effects, we also tested BHF splice-inhibiting morpholinos, using the progeny of wild-type colonies (15). Within 2 days of ampullae contact, all control pairs had fused (n=2) or rejected (n=1), whereas colony pairs receiving splice-inhibiting morpholinos did not react (n=5) (figs. S20 and S21, table S9, and movies S1 and S2). These data support our genomic analysis and indicate that BHF participates in fusion and rejection initiation.
In the jawed vertebrates the MHC is a haplotype, each sublocus of which specifies a different recognition process, usually by unique subsets of cells (18–20). By contrast, the B. schlosseri Fu/HC locus is a single gene (BHF) embedded in a haplotype of several genes with high polymorphism. Unlike the secreted (sFuHC) and membrane-bound (mFuHC) genes, BHF has none of the domains expected for a cell surface recognition protein or, in fact, domains that are conserved throughout protein evolution. Because BHF does not follow biological precedence by either sequence or domains, future investigations of this gene will likely reveal new mechanisms of recognition.
The ability to reliably predict histocompatibility outcomes on the basis of a single gene has broad implications for the study of allorecognition. For example, after vasculature fusion, stem cells from each B. schlosseri colony compete to overtake germline and/or somatic lineages (21–24). Stem cell competition may lead to elimination the other colony’s genome, or may produce a chimeric colony with mixed genotypes. To date, induction of chimerism using hematopoietic stem-cell transplantation is the only way to achieve long-term donor-specific tolerance to human organ allografts (25). Chimerism can be short-lived, and if lost, the threat of allograft rejection emerges. B. schlosseri is a unique species for studying stem cell-mediated chimerism, and such research will be facilitated by BHF.
We thank B. Rinkevich for pointing out the difficulty with the original cFuHC assignments, T. Snyder, J. Okamoto, L. Me, L. Ooi, A. Dominguez, C. Lowe, K. Uhlinger, L. Crowder, S. Karten, C. Patton, L. Jerabek, and T. Storm for invaluable technical advice and help. A. De Tomaso provided the fosmid sequence used to characterize cFuHC (12) (table s5). D.P., A.V., and S.R.Q. have filed U.S. and international patent applications (61/532,882 and 13/608,778, respectively) entitled “Methods for obtaining a sequence.” This invention allows for the sequencing of long continuous (kilobase scale) nucleic acid fragments using conventional short read–sequencing technologies, useful for consensus sequencing and haplotype determination. This study was supported by National Institutes of Health (NIH) grants 1R56AI089968, RO1GM100315 and 1R01AG037968 awarded to I.L.W, A.V., and S.R.Q., respectively, and the Virginia and D.K. Ludwig Fund for Cancer Research awarded to I.L.W. D.S. was supported by NIH Grant K99CA151673-01A1 and Department of Defense Grant W81XWH-10-1-0500, and A.M.N., D.M.C., D.S., and I.K.D. were supported by a grant from the Siebel Stem Cell Institute and the Thomas and Stacey Siebel Foundation. The data in this paper are tabulated in the main manuscript and in the supplementary materials. BHF, sFuHC, and mFuHC sequences are available in GenBank under accession numbers KF017887-KF017889, and the RNA-Seq data are available on the Sequence Read Archive (SRA) database: BioProject SRP022042.