Dur AH et al. (2020), In Xenopus ependymal cilia drive embryonic CSF ...

XB-ART-57626

Fluids Barriers CNS 2020 Dec 11;171:72. doi: 10.1186/s12987-020-00234-z.

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

Dur AH , Tang T , Viviano S , Sekuri A , Willsey HR , Tagare HD , Kahle KT , Deniz E .

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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.

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Species referenced: Xenopus tropicalis
Genes referenced: cfap298 emx1 en2 lhx1

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	Fig. 1. Xenopus tropicalis ventricular development map by OCT imaging. Widefield and mid-sagittal plane in vivo OCT imaging of an individual embryo at various stages to track ventricular development. a Stage 19; the earliest stage at which ventricular space is visible. b Stage 26; rostral expansion of the ventricular space is shown. c Stage 32; caudal expansion and the earliest detectable intraventricular particle movement (Additional file 2: Movie S1). d Stage 39; continued caudal expansion and 4 distinct polarized flow fields are visible (Additional file 2: Movie S1). e Stage 46; further caudal expansion and 5 distinct polarized flow fields are visible (Additional file 2: Movie S1). f Stage 48; intraventricular particle density diminishes. g Stage 49; Anterior (red circle) and posterior (green circle) choroid plexus visible. h Rostrocaudal ventricular expansion progression. CSF: cerebrospinal fluid; CP: choroid plexus; OCT: optical coherence tomography, a: anterior; p: posterior; d: dorsal; v: ventral
	Fig. 2. Particle tracking with Gaussian process regression enables compartmental CSF flow speed measurements. a Mid-sagittal plane in vivo OCT imaging of a stage 46 tadpole outlining brain structures and ventricular spaces. b CSF polarity map based on temporally color-coded frames 1–1000 at the mid-sagittal plane delineates particle trajectories of 5 discrete flow fields (labeled 1–5). FF1: telencephalic, FF2: diencephalic, FF3: mesencephalic, FF4: anterior rhombencephalic, FF5: posterior rhombencephalic (red: clockwise, blue: counterclockwise). c Compartmentally-matched median CSF flow speed based on particle tracking using Gaussian process regression showing a caudo-rostral speed gradient. c1) Lateral ventricle: 3.5 µm/s, c2) III ventricle: 6.2 µm/s, c3) Midbrain ventricle: 6.6 µm/s, c4) Anterior IV ventricle: 11.8 µm/s, c5) Posterior IV ventricle: 33.2 µm/s. CSF: cerebrospinal fluid; a: anterior, p: posterior, d: dorsal, v: ventral, Lat-v: lateral ventricle, III: 3rd ventricle, M: midbrain ventricle, IV: 4th ventricle
	Fig. 3. Late pre-metamorphosis tadpole displays complex compartmental CSF circulation and bidirectional intercompartmental mixing. a Mid-sagittal plane in vivo OCT imaging of stage 30 tadpole before and after microbead injection. Temporal color-coded image shows suspended, static bead (Additional file 3: Movie S2). b Mid-sagittal plane in vivo OCT imaging of stage 49 tadpole before and after microbead injection. The temporal color-coded image shows multiplanar CSF circulation (Additional file 4: Movie S3). c Focused image shows the aqueduct between the lateral ventricle and 3rd ventricle. The trajectory of the CSF circulation is shown by the red and yellow arrows (Additional file 5: Movie S4). d Focused image shows the aqueducts between the 3rd, midbrain (MV) and 4th ventricles (IV). The trajectory of the CSF circulation is shown by the red and yellow arrows (Additional file 6: Movie S5). (E) Focused 4th ventricle OCT image shows the choroid plexus projections (white arrows). Post-NiCl intraventricular injection, OCT image allows visualization of the static ependymal cilia (white arrow—Additional file 6: Movie S6). Along the yellow line, the kymograph showing motile cilia’s beating along the choroid plexus surface, which stops post NiCl injection
	Fig. 4. In Xenopus tropicalis polarized embryonic CSF circulation forms independent of cardiac forces. a, g Widefield image of the heart vicinity and OCT image of the dorsal cardinal vein of a stage 46 tadpole. White circle outlines the cardiac sac. Black dotted line marks the heart and the outflow tract of the tadpole pre-cardiac ablation. The absence of the heart is shown post-cardiac ablation. White arrows point to the outer vein walls and a temporally color-coded image indicates the presence or absence of blood flow. b, h Mid-sagittal plane in vivo OCT image of the ventricular space. c, i CSF polarity flow map. d, j Post-Gaussian processing CSF flow map. e, k Median compartmental median CSF flow speed. f, l Average compartmental area. (Additional file 10: Movie S8). CSF: cerebrospinal fluid; OCT: optical coherence tomography, Lat-v: lateral ventricle, III: 3rd ventricle, M: midbrain ventricle, IV: 4th ventricle
	Fig. 5. Pre/post cardiac ablation CSF flow speed and ventricular area measurements (Stage 46). a Median compartmental CSF flow speed of the Lat-V, 3rd ventricle, midbrain ventricle, anterior 4th and posterior 4th ventricle presented with before-after graphs. b Average compartmental cross-sectional area of the Lat-V, 3rd ventricle, midbrain ventricle, anterior 4th and posterior 4th ventricle presented with before-after graphs. c Median compartmental flow speed along the aqueducts between the 3rd and midbrain ventricles, and the 4th and midbrain ventricles shown with before-after graphs. Red area outlines the cross section of where the particle velocimetry is applied, yellow-dotted arrow indicates the direction of flow. Lat-v: lateral ventricle, III: 3rd ventricle, M: midbrain ventricle, IV: 4th ventricle
	Fig. 6. Xenopus tropicalis ventricular cilia distribution in brain explants (Stage 46). a Mid-sagittal OCT image showing flow fields 4 and 5. a1 Brain stained with an anti-GT335 antibody (red) which labels cilia, and with phalloidin (green) which labels actin to mark cell borders. The dorsal fourth ventricle roof is populated with MCCs that display translational polarity. a2 The lateral walls of the 4th ventricle displays monociliated cells. a3 The ventral surface of the 4th ventricle populated with monociliated cells. b The lateral, third, and midbrain ventricles show a dense population of monociliated cells. Lat-v: lateral ventricle, III: 3rd ventricle, M: midbrain ventricle, IV: 4th ventricle, CA: cerebral aqueduct, FF: flow field
	Fig. 7. Ependymal cilia driven flow is most impactful on rostral development. Mid-sagittal plane in vivo OCT imaging of stage 46 control and c21orf59 morphant tadpoles. a, f Midsagittal ventricular space, yellow arrow points the aqueductal stenosis. b, g CSF polarity flow map. c, h Post-Gaussian processing flow map. d, i Median compartmental flow speed. e, j Average compartmental area. p < 0.01, p < 0.001, p < 0.001, **p < 0.0001
	Fig. 8. Ependymal cilia driven flow is most impactful on rostral development and cardiac forces have no effect. a Normal expression of emx1 mRNA in a stage 46 wild-type tadpole. Expression is confined to the dorsal telencephalic area. White arrows in the magnified region mark the dorsal telencephalic region where emx1 is strongly expressed. b c21orf59 knockdown results in expanded emx1 expression caudally to the diencephalon and mesencephalon regions indicated by red arrows. c Normal expression of lhx1 mRNA in a stage 46 wild-type tadpole. Expression is strong around the thalamic region as indicated by a white arrow and extends to the diencephalon. d c21orf59 knockdown results in a loss of the lhx1 expression in the telencephalon, indicated by a red arrow. e Normal expression of en2 mRNA in a stage 46 tadpole. Expression localizes to the midbrain-hindbrain boundary outlined with dotted white line. f c21orf59 knockdown results in no observed change in the en2 expression pattern. g, i, k Normal expression of emx1, lhx1, and en2 mRNA when compared with h, j, l heartless tadpoles shows no observed changes in expression patterns
	Fig. 9. Development of Xenopus embryonic CSF circulation

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Abu-Daya, Absence of heartbeat in the Xenopus tropicalis mutation muzak is caused by a nonsense mutation in cardiac myosin myh6. 2009, Pubmed, Xenbase