טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentZohar Wolchinsky
SubjectFunction and Regulation of p63 during Early Mammalian
Cardiogenesis
DepartmentDepartment of Medicine
Supervisors Full Professor Aberdam Daniel
Professor Emeritus Itskovitz Joseph
Full Thesis textFull thesis text - English Version


Abstract

p63, member of the p53 gene family, encodes two main isoforms, TAp63 and ΔNp63. Each of these isoforms exhibits a distinct gene expression profile and opposite functions on epithelial homeostasis and cancer. While TAp63 controls cell apoptosis and ageing, ΔNp63 is implicated in cell proliferation, cell adhesion and is mandatory for epidermal commitment of the embryonic ectoderm during the embryonic skin formation. Previous research in our laboratory demonstrated using ES cells that TAp63 is expressed by the embryonic endoderm and acts in a non-cell autonomous manner on cardiac progenitor cells. In addition in-vivo study showed that p63 KO mice suffer from severe congenital cardiomyopathy. It is well known that endoderm-emitting inductive signals during gastrulation are required for the genesis of functional cardiomyocytes. Thus, our hypothesis was that TAp63 expressed in endodermal cells regulates secreted molecules that are required for proper differentiation of cardiac progenitor cells to mature cardiomyocytes.  To address this question, we decided to take advantage of the next-generation sequencing and its application to chromatin immunoprecipitation (ChIP-seq) which has enabled us to measure TAp63 direct interactions on a genome-wide basis.

In the present study we identified several new molecules involved in TAp63 signaling in endodermal cells, among them we have revealed three novel TAp63 target genes involved in cardiomyocyte differentiation using combined genome wide approach. We demonstrated that TAp63 binds to Activin A, AGM (Angiomodulin also named IGFBP7) and Maspin enhancers and transcriptionally regulates their expression, and that specific inhibitions of each of these molecules interfere with proper cardiogenesis. We also showed that ΔNp63 modulates ESC cardiogenesis by competing with TAp63 on consensus p63 binding sites located within the vicinity of AGM and Activin-A genes. In addition we found that AGM and Activin-A can regulate TAp63 and ΔNp63 by a feedback regulation mechanism which may be necessary to modulate cardiogenesis activation by endodermal TAp63, demonstrating the need for a balanced expression between TAp63 and ΔNp63.

Understanding heart development on the molecular level is essential for uncovering the causes of congenital heart diseases. Therapeutic approaches that try to enhance cardiac regeneration or that involve the differentiation of patient-specific induced pluripotent stem cells will also benefit from this knowledge. Our study identified AGM, Activin A and Maspin as novel molecules regulated directly by endodermal TAp63 with crucial role during cardiogenesis. Our findings provide better understanding of the significance of fine tuning of p63 isoforms for proper cardiogenesis, which might have implication for the future design of therapeutic approaches.