Genotype-specific expression of uncle fester suggests a role in allorecognition education in a basal chordate

Abstract

Histocompatibility is the ability to discriminate between self and non-self tissues, and has been described in species throughout the metazoa. Despite its universal presence, histocompatibility genes utilized by different phyla are unique- those found in sponges, cnidarians, ascidians and vertebrates are not orthologous. Thus, the origins of these sophisticated recognition systems, and any potential functional commonalities between them are not understood. A well-studied histocompatibility system exists in the botryllid ascidians, members of the chordate subphylum, Tunicata, and provides an opportunity to do so. Histocompatibility in the botryllids occurs at the tips of an extracorporeal vasculature that come into contact when two individuals grow into proximity. If compatible, the vessels will fuse, forming a parabiosis between the two individuals. If incompatible, the two vessels will reject- an inflammatory reaction that results in melanin scar formation at the point of contact, blocking anastomosis. Compatibility is determined by a single, highly polymorphic locus called the fuhc with the following rules: individuals that share one or both fuhc alleles will fuse, while those who share neither will reject. The fuhc locus encodes multiple proteins with roles in allorecognition, including one called uncle fester, which is necessary and sufficient to initiate the rejection response. Here we report the existence of genotype-specific expression levels of uncle fester, differing by up to 8-fold at the mRNA-level, and that these expression levels are constant and maintained for the lifetime of an individual. We also found that these differences had functional consequences: the expression level of uncle fester correlated with the speed and severity of the rejection response. These findings support previous conclusions that uncle fester levels modulate the rejection response, and may be responsible for controlling the variation observed in the timing and intensity of the reaction. The maintenance of genotype specific expression of uncle fester is also evidence of an education process reminiscent of that which occurs in mammalian Natural Killer (NK) cells. In turn, this suggests that while histocompatibility receptors and ligands evolve via convergent evolution, they may utilize conserved intracellular machinery to interpret binding events at the cell surface.

Keywords: Botryllus schlosseri; Natural Killer Cell; allorecognition; genotype-specific expression; uncle fester.

Figure 1.

DE-Seq analysis of uncle fester gene expression levels in nine B. schlosseri genotype. Each DE-Seq analysis was compared against SB825FEA with the normalized ratio plotted. Asterisks represent a statistical difference (padj < 0.05)

Figure 2.

(a) Graphical representation of histocompatibility outcome amongst different lines used in this study. fuhc alleles are denoted below the genetic line. (b) Relative qPCR analysis across several genotypes. Each sample was normalized ratio against SB825FEA was plotted with standard deviation as the error bars. Plot represents a minimum of 3 qPCR runs. (c) Graphical representation of the predicted promoter region of uncle fester with identified polymorphisms from each genotype listed below. Annotated positions are relative to the TSS (+1). TRR = Tandem Repeat Region; TATA = TATA box; TSS = Transcriptional Start Site; N.D. = Not Determined

Figure 3.

Examples of equal and preferential rejection. Interface of two incompatible individuals during a rejection response. Ampullae from top colony are clear, ampullae from bottom colony are dark. Equivalent points of rejection (POR) are highlighted with black arrows, where the two ampullae are in contact and POR forming between them. Preferential POR are highlighted with white arrows, where POR are forming from ampullae of only one of the colonies, here from either the top (*), or bottom colony (**).