Isolation and characterization of uncle fester, an allorecognition molecule in the primitive chordate, Botryllus schlosseri [electronic resource]

The ability to molecularly distinguish self from non-self is a common feature throughout the metazoa and is the fundamental basis of immune function. Although this phenomenon is pervasive throughout the animal kingdom, we often find examples of highly polymorphic, naturally occurring allorecognition systems within the colonial marine invertebrates. These types of animals, specifically the sponges, hydroids, anemones, bryozoans and ascidians, are permanently attached to the substratum and typically propagate continuously via asexual reproduction, which often results in physical contact between adjacent colonies. If the colonies are compatible they will often blend together, and depending on the species will form a single chimeric individual or they will reject, during which the interacting tissues are destroyed. In the vertebrates, histocompatibility is ultimately an artifact of medical intervention, considering that vertebrates are only naturally challenged to immunologically tolerate individuals of their own species during pregnancy. Ultimately, allorecognition in the vertebrates is controlled by polymorphisms at the MHC, which are recognized by effector cells in both the adaptive (T-cells) and innate (Natural Killer cells) branches of the immune system. Interestingly, the molecular components responsible for driving adaptive immunity within the vertebrates are entirely absent within the lower taxa. Thus, while it is clear that highly polymorphic allorecognition systems have an early phylogenetic origin, the molecular and cellular basis driving their specificity, and their relationship to the more sophisticated vertebrate immune system has remained elusive. The primary focus of my dissertation has been to characterize a candidate allorecognition protein, called uncle fester, and determine if it plays a role in a naturally occurring transplantation reaction that occurs in the primitive chordate Botryllus schlosseri. As the closest living invertebrate relative to the vertebrates, B. schlosseri occupies a key position within chordate lineage and is thus phylogenetically poised to address questions regarding the origins of vertebrate innate and adaptive immunity. In chapter 2, I provide an in-depth review of the allorecognition phenotype found within Botryllids, as well as a summary of the previously characterized molecular components implicated in the response. Briefly, as colonies of Botryllus schlosseri grow within their natural habitat, they often come into contact with one another at extracorporeal vascular structures called ampullae. After this initial interaction, colonies will either fuse, forming a parabiotic pair and hematopoietic chimera, or they will reject, initiating a blood-based inflammatory reaction that causes the two colonies to retreat. Fusion or rejection is controlled by the polymorphisms of a single locus, called the fuhc, and in order for two colonies to fuse they must share one or both alleles at this gene. In addition to the self-ligand, a putative receptor called fester has also been identified. Functional assays indicate that fester is playing dual roles in the allorecognition response, both as an inhibitory receptor involved in discriminating between different fuhc alleles and as an activating receptor, responsible for the initiation of both fusion and rejection. Encoded between the fuhc and fester, is a distantly related member of the fester family, a gene which we called uncle fester and its description is found within chapter 3. To characterize this locus, I posed the following questions: 1) Is uncle fester polymorphic at the nucleotide or amino acid level? 2) Are there alternative splice forms? 3) Where are uncle fester mRNA and protein expressed in juveniles and adults? and 4) What is its function in vivo? What I discovered is that the uncle fester gene consists of 9 exons spanning a genomic region of ca. 46 Kb., contains a signal sequence, an extracellular SCR domain, three contiguous transmembrane helices and a short intracellular tail. Sequences from over 70 individuals collected from populations along the West Coast of North America and two populations from the North East coast revealed that uncle fester is remarkably non-polymorphic, with only two single nucleotide polymorphisms and one amino acid deletion found to be segregating within these populations. I first determined uncle fester expression by RT-PCR, and found that expression begins in the early stages of embryonic development, and continues throughout the life cycle of the individual. These studies also revealed that uncle fester is alternatively spliced in the embryo, tadpole and adult stages of development. Each adult examined thus far expresses three alternative splice variants; however, embryos and tadpole larvae express an additional set of splice variants. The roles of these splice variants remain unknown. I then performed in situ hybridization on tadpole larvae, juveniles and adult colonies using an uncle fester specific riboprobe, to determine where the uncle fester mRNA is localized. The results indicate that uncle fester mRNA is found on the larval adhesive papillae, the epithelia of the ampullae and on a subset of blood cells, all tissues known to be involved in the allorecognition response. I also created two monoclonal antibodies to the uncle fester protein, and results from whole-mount immunohistochemistry are concordant with the mRNA in situ hybridization data. The methods and characterization of the uncle fester antibodies can be found in Chapter 4. Previous studies have shown that fester is also expressed along the epithelia of the ampullae. To determine if both proteins are co-expressed on the same populations of cells, I performed double-labeled FACS and IF using directly conjugated fester and uncle fester mAbs. Results indicated that all cells that express uncle fester also express fester, and in addition there is a population of fester+/uncle fester- cells found migrating within the tunic. To determine if uncle fester was functionally involved in the allorecognition response, I used two distinct approaches. First, I delivered custom siRNA to individuals while surgically removing the ampullar tissue, essentially forcing the animals to regenerate new ampullae under the effects of the siRNA and eliminating the uncle fester protein. When incompatible colonies were paired while under the effects of siRNA, the allorecognition response was never initiated and the two colonies eventually grew over the top of each other. However, the application of uncle fester siRNA had no effect on compatible colonies, suggesting that the activation of a rejection reaction is independent of a fusion response. The second approach was to stimulate function using the uncle fester specific monoclonal antibodies. By conjugating the mAb to magnetic beads, I was able to localize the antibody to the epithelia of ampullae, which resulted in the formation of strong points of rejection on a single colony. In summary, I found that uncle fester is a non-polymorphic type I transmembrane protein that expresses a limited repertoire of alternative splice variants. It is co-localized with both the fuhc and fester on all tissues important to the allorecognition response. Functionally, uncle fester is required to activate a rejection response, but plays no role in compatible interactions. These results have transformed our working hypothesis from allorecognition consisting of a single activating pathway, to one consisting of two independent activating and inhibitory pathways that control histocompatibility outcomes.

Keywords: Biology.
Advisor: De Tomaso Anthony (Anthony William)
Somero George N. primary advisor. Thesis advisor
Mudgett Mary Beth
Parham Peter