Astrocyte Primary Cilia are Dynamic during Development Open Access
Wei, Claire (Fall 2023)
Abstract
Astrocytes are the largest glial population in the brain, and their functions include regulating neuron homeostasis and synapses. Despite growing research about astrocytes over the past decade, astrocyte development remains a relatively unexplored field. Similar to neurons, astrocytes extend a primary cilium from the membrane of the cell soma. Primary cilia are singular projections critical to multiple developmental signaling pathways and are uniquely linked to the cell cycle. Abnormal cilia cause a class of disease, called ciliopathies, that are found to impact multiple cell types. However, little is known about primary cilia in astrocyte development. This project aims to characterize primary cilia and their relation to the cell cycle in the developing astrocyte lineage. To characterize astrocyte cilia, I used a genetic mouse model to mark cilia and determine the percentage of astrocytes with cilia. I used immunofluorescent staining to visualize the expression of specific ciliary proteins, ARL13B and AC3, in astrocyte primary cilia in vivo. I found changes in the percent of ciliated astrocytes as astrocytes progress through development. Furthermore, I noticed changes in ARL13B and AC3 expression within cilia over the same time period. Lastly, to explore the relationship between astrocyte primary cilia and the cell cycle, I used a genetic mouse model to label astrocyte cilia and stages of the cell cycle and live-imaged proliferating astrocytes in vitro. My results show the dynamic nature of primary cilia in developing astrocytes as cilia frequency and protein composition change over time, suggesting potential changes in primary cilia function over astrocyte development.
Table of Contents
1. Background………………………………………………………………………..1
1.1 Astrocytes……………………………..1
1.2 Primary cilia…………………….…….4
1.3 Astrocytes and primary cilia ……..…..6
1.4 Aim of study………………………….7
2. Methods…………………………………………………………………….…….7
2.1 Mouse lines……………………………..7
2.2 Tamoxifen injection…………………….8
2.3 Tissue harvesting …………..…………..8
2.4 Immunofluorescent staining…………....9
2.5 BaseScope/Immunofluorescence co-detection……………………….10
2.6 Imaging of IF stained slides…………….11
2.7 Quantification of IF images……….……11
2.8 Astrocyte primary cell culture…………..12
2.9 Live imaging of astrocyte cell culture…..12
3. Results…………………………………………………………..………………..13
3.1 Determining the frequency of astrocyte cilia over development…..13
3.2 Expression of ciliary proteins decreases as astrocytes mature……..16
3.3 Arl13b-Fucci system allows visualization of astrocyte primary cilia in the cell cycle………..18
4. Discussion………………………………………………………………….……..20
4.1 Percent of ciliated astrocytes remain the same over development…….20
4.2 Ciliary ARL13B and AC3 have greater function earlier in development…22
4.3 Arl13b-Fucci can be a useful system to live image primary cilia in proliferating astrocytes…….23
5. Conclusion………………………………………………………………….……..25
6. Appendix………………………………………………………………….……….26
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Astrocyte Primary Cilia are Dynamic during Development () | 2023-12-06 13:02:09 -0500 |
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