טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentBen Eliezer Eran
SubjectPluripotency Derived Regeneration in Urochordates
DepartmentDepartment of Biology
Supervisor Mr. Reshef Ram
Full Thesis textFull thesis text - English Version


Abstract

Regeneration in adult chordates is investigated in a few model organisms. The degree of regeneration varies considerably, from simple cellular replacement patterns in organs to elaborate types of whole organ regeneration. Yet, massive regeneration events, such as whole body regeneration (WBR), are mostly restricted to low complexity multicellular organisms. However, in the urochordate Botrylloides leachi, a close vertebrate relative, a fully functional adult regenerates from an isolated minute blood vessel fragment. Here, we extend at the molecular level a previous study in our lab that investigated the unique phenomenon of WBR in the colonial ascdian Botrylloides leachi and described it in detail at the morphological and histological level. However, the complete regulatory mechanisms and the cellular source that underlie the WBR process remain unknown.

We tracked two stem cell markers, Sox2 and Oct4, which are known for their regulatory function in pluripotency maintenance, in B. leachi native colonies and during regeneration. We show that Bl-Sox2 and Oct4 are expressed in specific regions of the endostyle, a characterized stem cells niche, as well as in blood vessels epithelial sheet and circulating cells. Oct4 staining is also observed in undifferentiated cells in organs.

During regeneration Oct4 and Sox2 are induced in the first few hours post incision in specific cells, which have been characterized as B. leachi circulating stem cells. In later stages of WBR both genes are expressed in the formation of the blastula-like structure (BLS), an undifferentiated cellular structure that eventually will form the zooid. Moreover, using siRNA techniques we show that Bl-Sox2 is essential for the bud formation in the WBR process. Finally, we revealed that the BLS formation during regeneration occurs through vascular budding, a similar asexual reproduction process adopted by a close related colonial tunicate Botryllus primigenus.

These results demonstrate that stem cells participate and contribute to B. leachi native colony maintenance and WBR. Moreover, Oct4 and Sox2 co-expression in native colonies and regenerating fragments indicates that stem cells regulatory pathways are evolutionary conserved. In addition, the inhibition of Bl-Sox2 translation demonstrates that stem cells are crucial for WBR in B. leachi. Yet, the cellular source of the WBR process and the molecular mechanisms regulating these cells remain to be determined.