In Xenopus ependymal cilia drive embryonic CSF circulation and brain development independently of cardiac pulsatile forces

Abstract

Background Hydrocephalus, the pathological expansion of the cerebrospinal fluid (CSF)-filled cerebral ventricles, is a common, deadly disease. In the adult, cardiac and respiratory forces are the main drivers of CSF flow within the brain ventricular system to remove waste and deliver nutrients. In contrast, the mechanics and functions of CSF circulation in the embryonic brain are poorly understood. This is primarily due to the lack of model systems and imaging technology to study these early time points. Here, we studied embryos of the vertebrate Xenopus with optical coherence tomography (OCT) imaging to investigate in vivo ventricular and neural development during the onset of CSF circulation. Methods Optical coherence tomography (OCT), a cross-sectional imaging modality, was used to study developing Xenopus tadpole brains and to dynamically detect in vivo ventricular morphology and CSF circulation in real-time, at micrometer resolution. The effects of immobilizing cilia and cardiac ablation were investigated. Results In Xenopus, using OCT imaging, we demonstrated that ventriculogenesis can be tracked throughout development until the beginning of metamorphosis. We found that during Xenopus embryogenesis, initially, CSF fills the primitive ventricular space and remains static, followed by the initiation of the cilia driven CSF circulation where ependymal cilia create a polarized CSF flow. No pulsatile flow was detected throughout these tailbud and early tadpole stages. As development progressed, despite the emergence of the choroid plexus in Xenopus, cardiac forces did not contribute to the CSF circulation, and ciliary flow remained the driver of the intercompartmental bidirectional flow as well as the near-wall flow. We finally showed that cilia driven flow is crucial for proper rostral development and regulated the spatial neural cell organization. Conclusions Our data support a paradigm in which Xenopus embryonic ventriculogenesis and rostral brain development are critically dependent on ependymal cilia-driven CSF flow currents that are generated independently of cardiac pulsatile forces. Our work suggests that the Xenopus ventricular system forms a complex cilia-driven CSF flow network which regulates neural cell organization. This work will redirect efforts to understand the molecular regulators of embryonic CSF flow by focusing attention on motile cilia rather than other forces relevant only to the adult.

Diversity and function of motile ciliated cell types within ependymal lineages of the zebrafish brain

“…Impaired motile cilia are also associated with hydrocephalus both in mice and human ciliopathy patients ( Brody et al., 2000 ; Ringers et al., 2020 ; Lee, 2013 ; Jiménez et al., 2014 ; Wallmeier et al., 2019 ; Ibañez-Tallon et al., 2004 ; Karimy et al., 2020 ; Boon et al., 2014 ; Wallmeier et al., 2014 ). To test for such a hydrocephalus phenotype, we used optical coherent tomography (OCT) ( Date et al., 2019 ; Dur et al., 2020 ) and visualized the ventricular size in anesthetized adult zebrafish in vivo , since dissected brain explants often caused the ventricular system to collapse ( Figure S6 A). In line with data from mammalian studies, we observed that foxj1b and gmnc mutants have significantly enlarged ventricles.…”

Sustained glymphatic transport and impaired drainage to the nasal cavity observed in multiciliated cell ciliopathies with hydrocephalus

“…The mechanism underlying development of communicating hydrocephalus in ciliopathy is still poorly understood. However, an increasing number of reports in mice, Xenopus and zebrafish have shown that the MCC (ependymal cells) lining the cerebral ventricles are important for: (i) CSF 'near wall' circulation [19,20,22], (ii) transport of nutrients and (iii) secretion of neuropeptides important for directional neural stem cell (NSC) migration (reviewed in Spassky and Meunier, 2017 [69]). Furthermore, genetic studies in humans with hydrocephalus and ciliopathy mouse models have uncovered defective neural stem cell proliferation including impaired cortical neurogenesis [60][61][62]70].…”

“…Another contributor to CSF movement within the CNS involves the motile multi-ciliated cells (MCCs) lining the cerebral ventricles [ 18 , 19 ]. Ex situ studies using organotypic cerebral ventricle explant cultures from rodents and pigs [ 19 ], as well as in vivo experiments in Xenopus [ 20 , 21 ] and zebrafish [ 22 ], have revealed intricate dynamic patterns of CSF flow through the ventricles orchestrated by the MCCs. However, the role of MCCs in CSF transport and CNS fluid regulation is incompletely understood [ 23 ].…”

The regulatory roles of motile cilia in CSF circulation and hydrocephalus

“…These models offer many advantages over other animal models, such as easy handling and maintenance, a short developmental period, and easy genetic manipulation, making them ideal for elucidating the developmental mechanisms underlying human congenital diseases [ 88 – 90 ]. A recent visualization study of CSF circulation within the ventricular system of Xenopus [ 92 , 93 ] and zebrafish larva [ 94 , 95 ] indicated that these lower vertebrates are promising experimental models for further investigation of the early roles of CSF in body axis formation and brain development and several brain diseases associated with CSF circulation [ 92 , 95 , 96 ]. Xenopus foxj1 was previously shown to play a significant role in MCC differentiation as well as ciliogenesis.…”