Christian Alexandro Clement:
Primary Cilia and Coordination of Signaling Pathways in Heart Development and Tissue Homeostasis

Date: 15-11-2009    Supervisor: Søren Tvorup Christensen

This thesis focuses on cilia and their sensory function in the mammalian organism. In particular, the Hedgehog (Hh) signaling pathway functions via the primary cilium and plays a unique role in development, differentiation, cancer and possibly in stem cell fate. Defects in primary cilia assembly or function are tightly coupled to developmental disorders and diseases in mammals termed “ciliopathies”.

The primary objective of this thesis was to investigate the role the primary cilium in coordinating Hh signaling in stem cell differentiation and heart development in the mouse. We show that human embryonic stem cells (hESC) and mouse embryonal carcinoma stem cells (P19.CL6 EC cells) have primary cilia that display ciliary localization of the essential Hh proteins; Gli2, Ptc1 and Smo. Inhibition of the Hh pathway by KAAD-cyclopamine in P19.CL6 cells hinder formation of synchronously beating clusters of cardiomyocytes. Knockdown of the primary cilium in P19.CL6 EC cells by nucleofection with plasmids expressing Ift20 and Ift88 siRNA significantly reduced the appearance of beating cardiomyocyte clusters thereby mimicking the effect of cyclopamine treatment. In vivo experiments revealed that mouse E11.5 Ift88-/- null mutants (which have no primary cilia) have severe endocardial cushion defects, decreased trabeculation and increased pericardial space along with shortened and malformed cardiac outflow tract. These observations suggest that primary cilia coordinate Hh signaling in stem cell differentiation and cardiogenesis. In support of this, preliminary chimera mouse studies showed that primary cilia are important for heart development. This was judged by the distribution of enzymatically tagged wt and Ift88-/- ES cells in the developing heart at E8.5, where only the wt cells are localized to the heart chambers. This signifies that primary cilia are needed for the formation of the heart chambers.

The secondary thesis objective was to investigate the role of progesterone signaling in the female reproduction organs in addition to the role of primary cilia in human pancreatic development and cancer. The findings of the progesterone receptor in the lower half of the motile cilia in the oviduct, suggest a sensory role of motile cilia in progesterone signaling where they might coordinate post ovulatory events. In tissue sections of the developing human pancreas we found up to 20μm long primary cilia projecting into the duct lumen of the exocrine duct, which have increased ciliary localization of Gli2 and Smo after initiation of fetal development, i.e., at weeks 14 and 18. In contrast, ciliary localization of these Hh components was absent at the embryonic stage of development, i.e., at week 7.5. This suggests a role of primary cilia in coordinating Hh signaling in human pancreatic development and postnatal tissue homeostasis. In cultures of human pancreatic duct adenocarcinoma cell lines PANC-1 and CFPAC-1, Ptc in addition to Gli2 and Smo localize to primary cilia. These findings are consistent with the idea that the primary cilium continues to coordinate Hh signaling in cells derived from the mature pancreas. The fact that the Hh signaling pathway is active in the CFPAC-1 and PANC-1 cell lines without Hh stimulation suggests that ciliary Hh signaling plays a potential role in tumorigenesis.

In conclusion, this thesis supports the idea that both motile and primary cilia are critical organelles in the coordination of developmental processes and tissue function.