Colonial marine invertebrates often encounter conspecifics when encrusting hard surfaces in the sea. As such animals have limited or no capacity for movement, cell-to-cell contact between individuals inevitably results. Allorecognition at such contacts triggers a sequence of events that typically involves a binary choice between fusion and rejection.
Allorecognition decisions have long been appreciated to be under genetic control based on the observation that fusion is rare except among close kin. A more detailed genetic understanding of fusibility is available for two organisms, both of which have developed into model systems for the study of this phenomenon. The two systems are Botryllus schlosseri
, an invertebrate chordate, and Hydractinia symbiolongicarpus
, a cnidarian and the subject of this study [recently reviewed by Rosengarten and Nicotra (2011)
The genetic analysis of fusibility in Hydractinia
began with the work of Hauenschild (1954)
. In a series of crosses with wild-type animals he provided support for a one-locus system with alleles of different strength. Hauenschild’s scheme was largely successful in accounting for the segregation patterns he observed, but several fusibility results defied explanation based on a one-locus model. Dupasquier (1974)
reanalyzed Hauenschild’s data and noted that, if one assumed the existence of two linked loci and further assumed that one of Hauenschild’s crosses involved a recombinant animal, then several (but not all) of the original inconsistencies were resolved.
No further work was reported for another 22 years until Mokady and Buss (1996)
developed a near-isogenic line. Their study was based on a large number of crosses each with a small segregating population. Fusibility was shown to segregate as a single chromosomal interval within the line. The development of a near-isogenic line was followed by the development of a near-congenic line. Using the congenic line, Cadavid et al. (2004)
mapped the chromosomal interval and showed that it comprised at least distinct two loci. The interval was called ARC (for allorecognition complex), and the allorecognition loci were designated alr1
The allorecognition loci alr1
have recently been identified by positional cloning (Nicotra et al. 2009
; Rosa et al. 2010
). Both genes encode putative transmembrane receptor proteins with extracellular domains resembling Ig-like domains and with intracellular sequence motifs similar to immunoregulatory signaling motifs. Both proteins bear hypervariable domains most similar to Ig-like V-set domains. Comparison of several wild-type alleles of both loci identified multiple V-set residues under positive selection. Notably, alr1
was found to be contained within a large family of structurally similar immunoglobulin superfamily-like genes (Rosa et al. 2010
Knowledge of the alr1
genotype fully predicted fusibility within the inbred and congenic lines (Cadavid et al. 2004
; Powell et al. 2007
). Specifically, if animals shared one ARC haplotype, they fuse, and if they share no ARC haplotype, they reject. Animals that do not share an allele at one alr
locus but do share an allele at the other alr
locus undergo a form of transitory fusion, where colonies initially fuse and thereafter separate from one another. The chronology and other aspects of the phenomenology of the separation vary depending on the locus at which the alleles are shared (Powell et al. 2007
Whether the “fusion rules” elucidated for the congenic lines were sufficient to explain wild-type variation in fusibility remained an open question until the alr
loci were identified. Fusion is rare in the field (Nicotra and Buss 2005
). Two separate tests of fusibility in wild-type colonies have been performed. In the first, wild-type animals were found that fused or underwent transitory fusion with animals from inbred lines, and the sequence of alr
alleles was obtained to determine if they matched. A survey of 535 animals yielded two such animals and both bore matching alleles (Nicotra et al. 2009
; Rosa et al. 2010
). In a related test, wild-type alr1
alleles were sequenced, and fusion tests were subsequently performed on colonies found to bear one or more matching alleles. Five pairs of colonies were identified as bearing matching alleles, and four of the five pairs displayed a fusible phenotype (Rosa et al. 2010
). In both tests, transitory fusion was observed in some colonies, when permanent fusion was expected (or vice versa).
These observations indicate that alr1 and alr2 are major determinants of allorecognition in the wild, but they also indicate that additional allodeterminants are yet to be identified. Two alternative genetic explanations are readily suggested. These additional allodeterminants could be unlinked modifying loci that were heretofore undetected because they were homogenized by the inbreeding program employed to identify the ARC. Alternatively, unlinked modifying loci may play a minimal role, but additional, unidentified loci within different ARC haplotypes may act as allodeterminants. Under this hypothesis, such loci differ between wild-types and inbred haplotypes but not within the congenic lines.
The first of these hypotheses can be easily addressed by classical breeding approaches. The conventional test for dominant modifiers is to introduce alleles from an inbred line into a wild-type genetic background and test for the appearance of the predicted phenotype in homozygotes derived in backcross or F2 incross progeny (Green 1981
). We provide such a test for three different genetic backgrounds and report no effect of genetic background on fusibility. Unexpectedly, these crosses yielded different map distances for the size of the ARC complex from that repeatedly measured for congenic lines.