The primary cilium is a micron-scale protrusion from the cell surface that allows cells to receive and process a variety of signals from the external environment. The Hedgehog pathway is one of the best-characterized signaling cascades regulated by the primary cilium, with virtually all components of the pathway found to dynamically localize to cilia and to require cilia for their function. Consistent with the key role of Hh signaling in developmental patterning and tissue homeostasis, cilium dysfunction leads to birth defects and to a group of pediatric disorders known collectively as ciliopathies. Critically, while primary cilia are now recognized to have an essential role in Hh signaling, the molecular mechanisms by which Hh pathway components traffic to and from cilia and are regulated by cilia remain unknown. Gli family proteins are the ultimate effectors of the Hh pathway, undergoing cilium-dependent post- translational processing into transcriptional activator and repressor forms. Here I will investigate the role of cilia in the Hh pathway by dissecting the ciliary trafficking and processing of Gli proteins through a combination of biochemical, live imaging and functional genomics approaches. Leveraging new tools developed in my post- doctoral studies, I will: 1) use microscopy assays to monitor the ciliary entry and exit of Gli proteins and determine how these events are regulated by Hh signaling; 2) dissect the functional role of the ciliary trafficking machinery in Gli protein trafficking and regulation; and3) apply functional genomics tools to identify factors that link the activity of upstream Hh pathway proteins to dynamic changes in Gli protein transport and processing. The mentored phase of this award will be carried out at Stanford University under the mentorship of Dr. Maxence Nachury, an expert in ciliary trafficking, and Dr. Matthew Scott, a leader in the Hh field. During this time, I will use imaging assays to dissect Gli protein trafficking and develop biochemical tools to assess the contribution of ciliary trafficking components to Gli3 regulation. Additionally I will work with Dr. Michael Bassik, an expert in functional genomics, to initiate an RNAi-based screen aimed at identifying new regulators of ciliary Hh signaling. After moving to an independent position, I will further investigate the mechanisms of Gli protein trafficking and post translational regulation. I will also continue my functional genomics studies, using powerful genetic interaction analysis tools to validate and characterize hit genes isolated from my primary screen. In summary, this project will improve our understanding of Hh signaling in primary cilia, give new insights into cilium-based developmental disorders, and provide the foundation for a successful independent research program. This award will allow me to gain needed training in functional analysis of the Hh pathway and in RNAi-based screening technologies, thereby ensuring the success of the proposed research and accelerating my transition to independence.
The primary cilium is an antenna-like cellular protrusion that allows cells to receive and respond to a variety of external signals that instruct cell growth, differentiation, and proliferation. One of the signals processed by the primary cilium, known as Hedgehog, has critical roles in embryonic development and cancer, with disruptions to cilia or Hedgehog signaling causing birth defects and childhood cancers. This proposal will determine how Hedgehog signals are processed in primary cilia, with the goal of improving our understanding of ciliary diseases and identifying new avenues for therapeutic intervention.
|Breslow, David K; Hoogendoorn, Sascha; Kopp, Adam R et al. (2018) A CRISPR-based screen for Hedgehog signaling provides insights into ciliary function and ciliopathies. Nat Genet 50:460-471|