Periodic Reporting for period 1 - CiliaryTip (The role of ciliary tip organization in hedgehog signaling)
Okres sprawozdawczy: 2020-07-01 do 2022-06-30
This project examined the role of ciliary microtubule organization in hedgehog signaling. Cilia are essential motile and sensory organelles, with a sophisticated microtubule-based core structure (called the axoneme). The axoneme is made of compound outer microtubules that transition from a triplet organization at the base (A-, B- and C-tubules), to doublet organization in the middle segment (A- and B-tubules) and singlet organization (A-tubules) near the distal end of the cilium. These structural changes define the cilium geometry and correlate with the compartmentalization of various processes. Three proteins are so far known to control the length of ciliary microtubules in the protist Tetrahymena (CEP104, ARMC9 and CHE-12). Defects in these proteins affect hedgehog signaling in cell culture and in humans they cause a subset of cases of Joubert Syndrome, a neurodevelopmental ciliopathy. This raises the possibility that the ciliary geometry is important for hedgehog signaling and embryonic development. To test this, we examined the ciliary microtubule organization in the absence and presence of hedgehog signaling and the role of CEP104 and ARMC9 in hedgehog signaling and Xenopus embryonic development. We found that B-tubules terminate closer to each other in the absence of hedgehog signaling confirming the presence of a link between the cilium geometry and signaling. We also found that the role of CEP104 and ARMC9 in controlling the length of ciliary microtubules is conserved in vertebrates. We uncovered non-ciliary roles for CEP104 and show that defects in CEP104 and ARMC9 lead to the development of hedgehog related phenotypes and neurodevelopmental abnormalities in Xenopus.
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.
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.
In almost all types of cilia the A-tubules are longer than the B-tubules creating a distinct distal segment (or ciliary tip). However, the functional significance of this microtubule organization remains elusive. We provide evidence supporting that this microtubule organization is important for cilia-based hedgehog signaling and embryonic development. Defects in CEP104 and ARMC9 underlie a subset of cases of Joubert Syndrome. Thus, our results contribute towards a better understanding of the disease etiology.
In addition to the research objectives, this project aimed at broadening the researcher’s expertise and skill set. The researcher was trained as a cellular biologist and through this project she gained substantial expertise in developmental biology and the use of the model organism Xenopus laevis. The covid19 pandemic limited the opportunities to communicate and disseminate this work. However, small progress was made through participation in the European Researcher’s Night event and the Cilia2022 conference.
In addition to the research objectives, this project aimed at broadening the researcher’s expertise and skill set. The researcher was trained as a cellular biologist and through this project she gained substantial expertise in developmental biology and the use of the model organism Xenopus laevis. The covid19 pandemic limited the opportunities to communicate and disseminate this work. However, small progress was made through participation in the European Researcher’s Night event and the Cilia2022 conference.