Exploring ciliary function and cerebrospinal fluid (CSF) flow in the functioning of the brain. Lessons learned from Zebrafish

Abstract

Cilia are slender hair-like structures extending from cell surfaces which play essential roles in various physiological processes. Within the nervous system, primary cilia contribute to signaling and sensory perception, while motile cilia facilitate cerebrospinal fluid (CSF) flow. Although cilia are abundant in various organs, including the brain, the development of ciliated cells and their impact on CSF flow, as well as their function in brain and axial morphogenesis, remain incompletely understood. Additionally, the precise role of cilia-mediated flow remains poorly defined. In my doctoral thesis, I aimed to investigate how ciliary beating and fluid flow contribute to the overall development of the brain. To investigate this, my PhD thesis comprises of 4 papers, 1 protocol paper and 3 research papers. In paper 1, I enlisted a methodology to monitor and study ciliated cells in the zebrafish brain. In paper 2, I investigated the development of multiciliated cells (MCCs) over time and their significance in brain and axial morphogenesis using zebrafish as a model which provides easy access to study ventricular development and fluid flow. In paper 2, we found that the zebrafish ventricles undergo restructuring during development with transition from mono to multiciliated cells driven by the transcription factors gmnc. All together in paper 2 we showed that ventricular development is supported by complimentary and sequential transcriptional programs including gmnc and the two zebrafish paralogues foxj1a and foxj1b. Furthermore, I investigated different zebrafish cilia models to study the function of cilia and cerebrospinal fluid dynamics and its contribution to brain development. In paper 3 together with my colleagues, we explored the impact of ciliary defects including defective primary and motile cilia (elipsa/traf3ip1/ift54 mutation) on the development of neural circuits. We found that cilia defects had significant effects on neurogenesis and brain morphology, particularly in the cerebellum. We performed whole-brain calcium imaging and observed reduced light-evoked and spontaneous neuronal activity across all brain regions, suggesting that cilia are involved in neural circuit function. In paper 4 we specifically studied the function of motile cilia mediated flow in a zebrafish mutant with paralyzed motile cilia (ccdc103/smh mutation). Our results showed that loss of motile cilia mediated flow had no obvious impact on brain morphology or cell proliferation unlike the effects seen in the elipsa mutant. Whole-brain calcium imaging did show reduced light evoked neuronal responses and loss of asymmetry in the brain. Additionally, we observed reduced spontaneous and sensory-driven astroglial calcium signals. All together in paper 4 we found that loss of motile cilia-generated flow affects brain physiology in larval zebrafish, most likely through altered astroglial function. In summary, my PhD thesis encompasses fundamental research that explores the diversity of ciliated cells and its relationship with ventricular development within the developing zebrafish brain. Moreover, it explores cilia function through the characterization of different cilia models. The work presented in this PhD thesis opens new opportunities for investigating the molecular mechanisms that unravel how distinct ciliated lineages control specific aspects of brain development. These mechanisms are particularly relevant to understanding cilia-related brain dysfunction in ciliopathy and identify potential therapeutic targets.