Characterization of ion channels in primary cilia: deciphering the fundamental principles controlling left-right axis formation

• Applicant: Dr. Nadine Mundt
• Subject Area: Anatomy and Physiology ; Cell Biology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 520195806

Primary cilia are solitary, non-motile tubular structures that protrude from the surface of all mammalian cells and function as cellular signaling hubs. Although expressed ubiquitously, primary cilia are particularly important during early embryogenesis. Our basic body plan relies on asymmetrically positioned organs along the central left-right axis. The key organizing structure in left-right asymmetry formation during embryogenesis is the embryonic node, a transiently expressed, oval-shaped depression. The embryonic node is decorated with motile cilia in the pit of the node, surrounded by 20-30 primary cilia located along the rim. It remains a fundamental unsolved question in physiology how the initial symmetry of the node becomes asymmetric with respect to gene expression, which consequently determines the organism's left-right axis. Unexpectedly, polycystin ion channels (PC1-L1/PC2) residing on primary cilia appear to be the most upstream signaling complex that controls asymmetric gene expression. The 'two-cilia model' postulates that motile cilia in the node generate a leftward-directed fluid flow that is "sensed" by immotile primary cilia in the periphery of the node by an unknown mechanism involving polycystin channels. How can a cation influx into the primary cilium control gene expression, and how are the polycystin channels regulated? In other words, what "asymmetry signals" do polycystin channels sense within the node? These fundamental questions are currently unknown and will be addressed in this proposal. We aim to solve this question by 1) the development of a functional cell assay that targets the polycystin ion channel complex to the plasma membrane of mammalian cells. This approach will allow high-throughput characterization of the biophysical properties and activation mechanism(s) of PC1-L1/PC2. 2) We successfully established in vivo ciliary electrophysiology in primary cilia of the embryonic node. Here, I propose to look carefully for in vivo PC1-L1/PC2 currents of the embryonic node and compare channel activity on either side of the node before and after flow onset. Putative ligands, identified in subproject 1, will eventually be tested on endogenous polycystin ion channels. 3) We seek to determine the causality between ciliary Ca2+ and asymmetric gene expression across the embryonic node. Flow-induced and PC2-dependent cytoplasmic and ciliary Ca2+ signaling in the embryonic node has been reported previously. However, a causative dependency of asymmetric gene expression and correct left-right patterning on ciliary Ca2+ signals has not been shown. Therefore, we will use a newly developed chemogenetic approach to study the effects of selectively upregulated ciliary Ca2+ in the embryonic node during embryogenesis.

• DFG Programme: WBP Fellowship
• International Connection: USA
• Host: Professor Dr. Markus Delling