Vascular regeneration in a basal chordate is due to the presence of immobile, bi-functional cells.

The source of tissue turnover during homeostasis or following injury is usually due to proliferation of a small number of resident, lineage-restricted stem cells that have the ability to amplify and differentiate into mature cell types. We are studying vascular regeneration in a chordate model organism, Botryllus schlosseri, and have previously found that following surgical ablation of the extracorporeal vasculature, new tissue will regenerate in a VEGF-dependent process within 48 hrs. Here we use a novel vascular cell lineage tracing methodology to assess regeneration in parabiosed individuals and demonstrate that the source of regenerated vasculature is due to the proliferation of pre-existing vascular resident cells and not a mobile progenitor. We also show that these cells are bi-potential, and can reversibly adopt two fates, that of the newly forming vessels or the differentiated vascular tissue at the terminus of the vasculature, known as ampullae. In addition, we show that pre-existing vascular resident cells differentially express progenitor and differentiated cell markers including the Botryllus homologs of CD133, VEGFR-2, and Cadherin during the regenerative process.

Discovery of amino acid motifs for thrombin cleavage and validation using a model substrate.

Understanding the active site preferences of an enzyme is critical to the design of effective inhibitors and to gaining insights into its mechanisms of action on substrates. While the subsite specificity of thrombin is understood, it is not clear whether the enzyme prefers individual amino acids at each subsite in isolation or prefers to cleave combinations of amino acids as a motif. To investigate whether preferred peptide motifs for cleavage could be identified for thrombin, we exposed a phage-displayed peptide library to thrombin. The resulting preferentially cleaved substrates were analyzed using the technique of association rule discovery. The results revealed that thrombin selected for amino acid motifs in cleavage sites. The contribution of these hypothetical motifs to substrate cleavage efficiency was further investigated using the B1 IgG-binding domain of streptococcal protein G as a model substrate. Introduction of a P(2)-P(1)' LRS thrombin cleavage sequence within a major loop of the protein led to cleavage of the protein by thrombin, with the cleavage efficiency increasing with the length of the loop. Introduction of further P(3)-P(1) and P(1)-P(1)'-P(3)' amino acid motifs into the loop region yielded greater cleavage efficiencies, suggesting that the susceptibility of a protein substrate to cleavage by thrombin is influenced by these motifs, perhaps because of cooperative effects between subsites closest to the scissile peptide bond.

Allorecognition in a basal chordate consists of independent activating and inhibitory pathways.

Histocompatibility in the basal chordate Botryllus schlosseri is controlled by the polymorphisms of a single gene: the fuhc. A polymorphic candidate receptor (fester) appeared to play roles in both initiating the reaction and discriminating between fuhc alleles. Here we report the characterization of a related protein, uncle fester. uncle fester is not polymorphic, and although coexpressed with fester, has different functional properties. Loss-of-function studies demonstrate that uncle fester was required for incompatible reactions but has no role in interactions between compatible individuals. Furthermore, stimulation with monoclonal antibodies could initiate a rejection phenotype on a single colony, and in both assays the severity of the rejection could be manipulated. These findings suggest that allorecognition in Botryllus consists of independent pathways that control compatible and incompatible outcomes that are integrated within the interacting cells, and may provide insight into basal processes conserved in allorecognition responses throughout the metazoa.