CFAP45, a heterotaxy and congenital heart disease gene, affects cilia stability.

Congenital heart disease (CHD) is the most common and lethal birth defect, affecting 1.3 million individuals worldwide. During early embryogenesis, errors in Left-Right (LR) patterning called Heterotaxy (Htx) can lead to severe CHD. Many of the genetic underpinnings of Htx/CHD remain unknown. In analyzing a family with Htx/CHD using whole-exome sequencing, we identified a homozygous recessive missense mutation in CFAP45 in two affected siblings. CFAP45 belongs to the coiled-coil domain-containing protein family, and its role in development is emerging. When we depleted Cfap45 in frog embryos, we detected abnormalities in cardiac looping and global markers of LR patterning, recapitulating the patient's heterotaxy phenotype. In vertebrates, laterality is broken at the Left-Right Organizer (LRO) by motile monocilia that generate leftward fluid flow. When we analyzed the LRO in embryos depleted of Cfap45, we discovered "bulges" within the cilia of these monociliated cells. In addition, epidermal multiciliated cells lost cilia with Cfap45 depletion. Via live confocal imaging, we found that Cfap45 localizes in a punctate but static position within the ciliary axoneme, and depletion leads to loss of cilia stability and eventual detachment from the cell's apical surface. This work demonstrates that in Xenopus, Cfap45 is required to sustain cilia stability in multiciliated and monociliated cells, providing a plausible mechanism for its role in heterotaxy and congenital heart disease.

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

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.