Uncovering the role of Importin9 in Hedgehog signaling - PROJECT SUMMARY The Hedgehog (Hh) pathway is a key signaling pathway for vertebrate development and adult tissue homeostasis. Precise control of Hh signaling is necessary as overactivation or suppression of the pathway causes severe developmental pathologies such as medulloblastoma and holoprosencephaly. Despite the need for targeted therapies, treatments to stabilize Hh signaling in disease states remain limited. Thus, uncovering novel regulatory mechanisms of Hh signaling will provide vital information for developing future therapeutic strategies. A key feature of vertebrate Hh signaling is its dependence on the primary cilium, a signaling organelle that serves as a hub for the trafficking and enrichment of Hh components. The ciliary enrichment of components such as SMO, the obligate Hh transducer, promotes activation of the pathway. SMO activation initiates a signaling cascade, resulting in the transport of GLI transcription factors from the cilium to the nucleus to facilitate the transcription of Hh targets. The field has dedicated decades to understanding how perturbations in the cilium impact Hh signaling. However, little is known about how the signal gets relayed from the cilium to the nucleus. My data identifies Importin9 (IPO9), a member of the karyopherin family, as a novel regulator of Hh signaling through an unknown mechanism. Karyopherins are known for their roles in nuclear trafficking in a RAN-dependent manner. However, RAN is also seen at the base of cilia, and karyopherin Importinβ2 is involved in ciliary trafficking, suggesting that other karyopherins may similarly function in ciliary trafficking. IPO9 binds to RAN and localizes to the base of cilia and nuclear periphery, suggesting it’s engaged in nuclear and/or ciliary trafficking. Additionally, IPO9 interacts and shares similar Hh signaling defects with ARL13B, a ciliary GTPase long studied by the Caspary lab. Like ARL13B, IPO9 regulates the Hh pathway downstream of activated SMO in vitro. In vivo, Ipo9 mouse mutants resemble ARL13B null (Arl13bhnn/hnn) embryos as they die embryonically and exhibit Hh-related phenotypes such as smaller embryo size, exencephaly, and craniofacial defects. These findings suggest that IPO9 and ARL13B function together to regulate Hh signaling. ARL13B regulates GLIs, which undergo several translocation and processing steps to become transcription repressors (GLIR) or activators (GLIA). ARL13B uncouples GLI regulation by specifically upregulating GLIA through an unknown mechanism. IPO9 loss does not impact GLIR production but still reduces the Hh response, suggesting that IPO9 may also regulate GLIA, similar to ARL13B. Thus, I hypothesize that during embryogenesis, IPO9 regulates vertebrate Hh signaling via GLIA, potentially as an effector of ARL13B. I will test this hypothesis by combining genetic, molecular, and biochemistry approaches. This work will uncover how IPO9 regulates Hh signaling by exploring IPO9’s relationship with ARL13B and GLIs. My work will identify new transport mechanisms between cellular organelles regulating the Hh pathway, offering potential strategies for targeting dysregulated Hh signaling in disease states.
