Final Report Summary - CILIARYDISEASE (Deciphering mechanisms of ciliary disease)
Cilia are microtubule-based hair-like structures protruding from the surface of most mammalian cells.
They serve numerous physiological functions such as fluid transport, sensory perception, or signal transduction. Ciliary dysfunction underlies multiple human congenital syndromes, causing blindness, chronic respiratory disorders, kidney and heart disease, obesity, and diabetes. It is assumed that 1 in 1000 people is affected. Prerequisite for the development of novel therapeutic approaches, is a better understanding of ciliary structure, assembly, disassembly, and function. In our studies, we identified two novel ciliary genes: Pitchfork (Pifo) and Flattop (Fltp).
In functional analyses in knock-out (KO) mouse embryos and cell culture, we found that Pifo is required for several aspects of cilia disassembly. As cilia retraction is imperative for proper cell division, insufficient Pifo function led to severe mitotic defects. Because cilia motility at the embryonic node is required for the correct establishment of left-right-asymmetry, ciliary defects by Pifo mutation led to left-right-patterning defects and consequently fatal heart failure. In patients diagnosed with ciliopathies and left-right-asymmetry defects, we found a specific Pifo mutation and demonstrated that this was indeed unable to trigger cilia disassembly (Kinzel et al., 2010).
We established the Pifo protein interactome and showed that Pifo with Gprasp2 and Smoothened forms a heterotrimeric complex. This hints to the existence of a novel mechanism of Hedgehog signal transduction and links these newly discovered components to clinically observed phenotypes obesity and diabetes. We anticipate that the newly identified complex will be a novel important drug target (Jung, Padula, et al., under revision; Jung, Messias, in prep.).
For Fltp, our genetic lineage tracing and KO analyses showed that the protein is involved in transportation, docking, or positioning of the basal body, the nucleation site for cilia growth. However, we found that Fltp deletion does not only result in severely impaired ciliogenesis, but also in misplacement of an interacting protein (Dlg3, discs large) which is involved in apical-basal and planar cell polarity (PCP) establishment. Our data suggest that Fltp plays a role at the interface between ciliogenesis and PCP by translating positional information to precise basal body localization and docking, which is essential for proper ciliary and cellular function in the inner ear. Our findings might have broader implications for other cell types, ciliary disease and asymmetric cell division (van Campenhout et al., 2011, Dev. Cell; Gegg et al., 2014, eLife).
We also investigated the impact of the acquisition of tissue polarity on beta-cell development and determination of functional heterogeneity in the islet niche of the pancreas. Our studies not only reveal 3D architecture and PCP as underlying principal of functional beta-cell heterogeneity, but also puts forward Fltp as a biomarker to study islet heterogeneity and plasticity for regenerative therapy (Bader et al., in prep.; patent application filed).
Further to the findings related to the pancreas we identified Fltp-high cells as Wnt/PCP activated intestinal stem cells (ISC) which retain self-renewal potential and constitute a subpopulation of ISCs that give rise to the secretory lineage (Böttcher et al., in prep.).
In summary, our studies provide novel insights into the complex processes of primary cilia assembly, disassembly, and signaling, as well as into interrelations between planar cell polarity and ciliogenesis. The identification of Fltp as a biomarker to explore beta-cell regeneration could allow for a transformative breakthrough in diabetes research. Using this marker will clarify the existence of proposed multipotent progenitors, provide new biomarkers and molecular targets for regenerative therapy and unravel pathomechanisms of diabetes. Exploring proliferative and mature beta-cells opens complete new avenues for regenerative therapy.
They serve numerous physiological functions such as fluid transport, sensory perception, or signal transduction. Ciliary dysfunction underlies multiple human congenital syndromes, causing blindness, chronic respiratory disorders, kidney and heart disease, obesity, and diabetes. It is assumed that 1 in 1000 people is affected. Prerequisite for the development of novel therapeutic approaches, is a better understanding of ciliary structure, assembly, disassembly, and function. In our studies, we identified two novel ciliary genes: Pitchfork (Pifo) and Flattop (Fltp).
In functional analyses in knock-out (KO) mouse embryos and cell culture, we found that Pifo is required for several aspects of cilia disassembly. As cilia retraction is imperative for proper cell division, insufficient Pifo function led to severe mitotic defects. Because cilia motility at the embryonic node is required for the correct establishment of left-right-asymmetry, ciliary defects by Pifo mutation led to left-right-patterning defects and consequently fatal heart failure. In patients diagnosed with ciliopathies and left-right-asymmetry defects, we found a specific Pifo mutation and demonstrated that this was indeed unable to trigger cilia disassembly (Kinzel et al., 2010).
We established the Pifo protein interactome and showed that Pifo with Gprasp2 and Smoothened forms a heterotrimeric complex. This hints to the existence of a novel mechanism of Hedgehog signal transduction and links these newly discovered components to clinically observed phenotypes obesity and diabetes. We anticipate that the newly identified complex will be a novel important drug target (Jung, Padula, et al., under revision; Jung, Messias, in prep.).
For Fltp, our genetic lineage tracing and KO analyses showed that the protein is involved in transportation, docking, or positioning of the basal body, the nucleation site for cilia growth. However, we found that Fltp deletion does not only result in severely impaired ciliogenesis, but also in misplacement of an interacting protein (Dlg3, discs large) which is involved in apical-basal and planar cell polarity (PCP) establishment. Our data suggest that Fltp plays a role at the interface between ciliogenesis and PCP by translating positional information to precise basal body localization and docking, which is essential for proper ciliary and cellular function in the inner ear. Our findings might have broader implications for other cell types, ciliary disease and asymmetric cell division (van Campenhout et al., 2011, Dev. Cell; Gegg et al., 2014, eLife).
We also investigated the impact of the acquisition of tissue polarity on beta-cell development and determination of functional heterogeneity in the islet niche of the pancreas. Our studies not only reveal 3D architecture and PCP as underlying principal of functional beta-cell heterogeneity, but also puts forward Fltp as a biomarker to study islet heterogeneity and plasticity for regenerative therapy (Bader et al., in prep.; patent application filed).
Further to the findings related to the pancreas we identified Fltp-high cells as Wnt/PCP activated intestinal stem cells (ISC) which retain self-renewal potential and constitute a subpopulation of ISCs that give rise to the secretory lineage (Böttcher et al., in prep.).
In summary, our studies provide novel insights into the complex processes of primary cilia assembly, disassembly, and signaling, as well as into interrelations between planar cell polarity and ciliogenesis. The identification of Fltp as a biomarker to explore beta-cell regeneration could allow for a transformative breakthrough in diabetes research. Using this marker will clarify the existence of proposed multipotent progenitors, provide new biomarkers and molecular targets for regenerative therapy and unravel pathomechanisms of diabetes. Exploring proliferative and mature beta-cells opens complete new avenues for regenerative therapy.