טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentRinkevich Yuval
SubjectCellular and Molecular Mechanisms of Whole Body
Regeneration in Botrylloides leachi from Blood
Vessel Fragments
DepartmentDepartment of Biology
Supervisor Mr. Reshef Ram
Full Thesis textFull thesis text - English Version


Abstract

Regeneration in adult chordates is confined to a few model cases and terminates in restoration of restricted tissues and organs. Here, we study the unique phenomenon of whole body regeneration (WBR) in the colonial urochordate Botrylloides leachi in which an entire adult zooid is restored from a miniscule blood vessel fragment. We found that the Botrylloides system differs from known regeneration model systems by several fundamental criteria, including epimorphosis without the formation of blastema and the induction of a “multifocal regeneration niche” system. This is also the first documented case of WBR from circulating blood cells that restores not only the soma, but also the germ line. We found that retinoic acid (RA) regulates diverse developmental aspects in WBR. The homologue of the RA receptor and a retinaldehyde dehydrogenase-related gene were expressed specifically in blood cells within regeneration niches and throughout bud development. More importantly, RA inhibitors as well as RNAi knockdown experiments resulted in WBR arrest and bud malformations, while all-trans RA caused   doubly accelerated regeneration and multi-bud formation, leading to restored colonies with multiple zooids, suggesting that RA signaling may have had ancestral roles in body restoration events. We have also assembled a subtracted library of 1326 ESTs from early stages of Botrylloides leachi WBR. Analysis of functional categories revealed a group of 67 transcripts with broad roles in innate immunity signaling. Gene ontology analysis on the immune category, identified “peptidase activity” and “protease inhibitor activity” as entries functioning during WBR. Analyzing the expression of a representative candidate gene from the “peptidase activity” sub-group, Bl-Trsp revealed low transcript levels in naïve vasculature but with upregulated expression during WBR. In situ hybridization revealed staining restricted to a circulating population of macrophage cells. Furthermore, Bl-Trsp was localized in regeneration niches within vasculature, in regenerating buds, and in buds, during blastogenesis. Comparison of Genome-wide transcription of WBR with five other developmental processes in ascidians (including metamorphosis, budding and blastogenesis), revealed conserved immune signaling expressions and suggest co-option of immune-related genes in developmental processes. This, in turn, may have enabled the high diversity of life history traits represented by urochordate ascidians.  Three additional EST library categories were analyzed, including "cell communication and signaling", revealing expression of major signaling pathways including Notch/Delta, Protein Kinases, nuclear hormone receptors and GPCR and TGF beta. Overall, the data presented reveals activation of complex signaling pathways involving multiple signaling   during WBR.