Our first aim was to explore a potential role of the ciliary microtubule organization in Hedgehog signaling. To do this we generated evidence for the specificity of ARMC9 to localize to the ends of B-tubules in Xenopus. We examined the termination points of B-tubules, in the primary cilia of the neural tube in Xenopus embryos, in the absence and presence of a hedgehog signaling chemical inhibitor. When Hedgehog signaling was blocked the spread of ARMC9 signal at the ciliary tip was decreased and the overall protein levels were increased. The change in ARMC9 localization suggests that B-tubules terminate closer to each other in the absence of hedgehog signaling confirming that there is a link between the ciliary tip organization and hedgehog signaling.
Our second aim was to determine the role of CEP104 and ARMC9 in hedgehog signaling and embryonic development. We first confirmed that the localization and function of CEP104 and ARMC9 are conserved in the cilia of Xenopus. In the primary cilia of the neural tube both proteins localize to the tip of the cilium suggesting the presence of a very short ciliary tip that cannot be resolved using fluorescence microscopy. Downregulation of CEP104 and ARMC9 led to the development of embryos with microphthalmia, fewer melanocytes and neuronal abnormalities suggesting defects in hedgehog signaling. In addition, embryos showed defects in the pronephros that suggested defects in cilia generated fluid flow. Thus, we examined the effect of CEP104 and ARMC9 on cilia generated fluid flow using the embryonic epidermis as a model. Downregulation of CEP104 caused a delay in neural tube closure which is likely caused by defects in cytoplasmic microtubules.
Overall, we show that 1) there is a link between the ciliary tip organization in neural tube cilia and hedgehog signaling, 2) we show that CEP104 and ARMC9 localization and function are conserved in vertebrates, 3) CEP104 and ARMC9 play a role in Hedgehog signaling and neurodevelopment in vivo and 4) uncovered novel roles of CEP104 on cytoplasmic microtubules.
This work will be presented in the Cilia2022 conference (October 2022, Germany). In terms of public engagement, I presented a poster introducing the public to our general laboratory work along with some results of this project in the European Researcher’s Night in 2021 (online event) where I received the “favorite poster” award.
