טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentHaber Omer
SubjectWound-Healing Cascades Initiate Urochordate Whole Body
Regeneration
DepartmentDepartment of Biology
Supervisor Mr. Reshef Ram
Full Thesis textFull thesis text - English Version


Abstract

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 few and 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 a previous study in our lab that investigated the unique phenomenon of WBR in the colonial B.leachi.. This study also has shown that retinoic acid regulates diverse developmental aspects in WBR. However, the initial signaling events that trigger this process remain unknown.

In the present study we tested the hypothesis that early WBR events resemble wound-healing. Therefore, we decided to investigate signaling cascades that occur rapidly after injury of blood vessels and ampullae in the B.leachi colony, leading to WBR.

It is known from literature that a physical injury causes a release of ATP from injured cells. These extracellular nucleotides stimulate cells by activating the P2 purinergic receptors present on the cell surface and by activation of the mitogen-activated protein kinases cascade.

We found that wounding the blood vessels and ampullae, in the B.leachi colony, result in ATP release into the medium. We demonstrate that the inhibition of the purinergic receptors, P2Y, abrogate the migration of ampullae and arrest WBR. Moreover, we reveal that wounding induce rapid activation of extracellular signal-regulated kinase (ERK) pathway. This activation was attenuated by the administration of a MEK inhibitor that is upstream to ERK.  Furthermore, this inhibition results in a complete WBR arrest.

Searching for the cellular source of WBR, we found that Oct4, a stem cell marker, is expressed in ampulae but not in the zooids. We also found that WBR accompanied by a reduction in the protein level of Oct4, suggesting differentiation of stem cells during WBR. Furthermore, we demonstrated that inhibition of RA synthesis, maintains, 24 hours after dissection, Oct4 protein expression. These results suggest that the early events of the WBR phenomenon resemble those occur during wound-healing. Moreover, these initial events are critical for appropriate continuance of WBR. Since wound-healing but not regeneration is an obvious event in higher chordates, it is therefore, plausible that some regeneration abilities that are expressed in early chordates as natural continuity of wound-healing potentially exist in higher vertebrates but during the course of evolution were altered or repressed.