Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Fluids Barriers CNS
2020 Dec 11;171:72. doi: 10.1186/s12987-020-00234-z.
Show Gene links
Show Anatomy links
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
.
???displayArticle.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.
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) Lateralventricle: 3.5 µm/s, c2) III ventricle: 6.2 µm/s, c3) Midbrainventricle: 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: lateralventricle, III: 3rd ventricle, M: midbrainventricle, 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 lateralventricle 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: lateralventricle, III: 3rd ventricle, M: midbrainventricle, 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, midbrainventricle, anterior 4th and posterior 4th ventricle presented with before-after graphs. b Average compartmental cross-sectional area of the Lat-V, 3rd ventricle, midbrainventricle, 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: lateralventricle, III: 3rd ventricle, M: midbrainventricle, 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: lateralventricle, III: 3rd ventricle, M: midbrainventricle, 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
Abu-Daya,
Absence of heartbeat in the Xenopus tropicalis mutation muzak is caused by a nonsense mutation in cardiac myosin myh6.
2009, Pubmed,
Xenbase
Abu-Daya,
Absence of heartbeat in the Xenopus tropicalis mutation muzak is caused by a nonsense mutation in cardiac myosin myh6.
2009,
Pubmed
,
Xenbase
Adalis,
Cytotoxic effects of nickel on ciliated epithelium.
1978,
Pubmed
Alexandre,
The isthmic organizer links anteroposterior and dorsoventral patterning in the mid/hindbrain by generating roof plate structures.
2003,
Pubmed
Anderson,
A reinvestigation of dynein ATPase kinetics and the inhibitory action of vanadate.
1982,
Pubmed
Bachy,
The LIM-homeodomain gene family in the developing Xenopus brain: conservation and divergences with the mouse related to the evolution of the forebrain.
2001,
Pubmed
,
Xenbase
Banizs,
Dysfunctional cilia lead to altered ependyma and choroid plexus function, and result in the formation of hydrocephalus.
2005,
Pubmed
Boppart,
Optical coherence tomography imaging in developmental biology.
2000,
Pubmed
,
Xenbase
Chau,
Progressive Differentiation and Instructive Capacities of Amniotic Fluid and Cerebrospinal Fluid Proteomes following Neural Tube Closure.
2015,
Pubmed
Currie,
A behaviorally related developmental switch in nitrergic modulation of locomotor rhythmogenesis in larval Xenopus tadpoles.
2016,
Pubmed
,
Xenbase
Date,
Visualizing flow in an intact CSF network using optical coherence tomography: implications for human congenital hydrocephalus.
2019,
Pubmed
,
Xenbase
del Viso,
Generating diploid embryos from Xenopus tropicalis.
2012,
Pubmed
,
Xenbase
Dreha-Kulaczewski,
Respiration and the watershed of spinal CSF flow in humans.
2018,
Pubmed
Fame,
Directional cerebrospinal fluid movement between brain ventricles in larval zebrafish.
2016,
Pubmed
Fame,
Emergence and Developmental Roles of the Cerebrospinal Fluid System.
2020,
Pubmed
Faubel,
Cilia-based flow network in the brain ventricles.
2016,
Pubmed
Figdor,
Segmental organization of embryonic diencephalon.
1993,
Pubmed
Fujimoto,
Optical biopsy and imaging using optical coherence tomography.
1995,
Pubmed
Fujimoto,
Optical coherence tomography: an emerging technology for biomedical imaging and optical biopsy.
2000,
Pubmed
Furey,
De Novo Mutation in Genes Regulating Neural Stem Cell Fate in Human Congenital Hydrocephalus.
2018,
Pubmed
Gabridge,
Effects of heavy metals on structure, function, and metabolism of ciliated respiratory epithelium in vitro.
1982,
Pubmed
Gona,
Ultrastructural studies on the ventricular surface of the frog cerebellum.
1982,
Pubmed
Greitz,
Cerebrospinal fluid circulation and associated intracranial dynamics. A radiologic investigation using MR imaging and radionuclide cisternography.
1993,
Pubmed
Guirao,
Coupling between hydrodynamic forces and planar cell polarity orients mammalian motile cilia.
2010,
Pubmed
Gutiérrez-Chico,
Optical coherence tomography: from research to practice.
2012,
Pubmed
Hagenlocher,
Ciliogenesis and cerebrospinal fluid flow in the developing Xenopus brain are regulated by foxj1.
2013,
Pubmed
,
Xenbase
Hagiwara,
Cell biology of normal and abnormal ciliogenesis in the ciliated epithelium.
2004,
Pubmed
Hänzi,
Developmental changes in head movement kinematics during swimming in Xenopus laevis tadpoles.
2017,
Pubmed
,
Xenbase
Hemmati-Brivanlou,
Cephalic expression and molecular characterization of Xenopus En-2.
1991,
Pubmed
,
Xenbase
Hirota,
Planar polarity of multiciliated ependymal cells involves the anterior migration of basal bodies regulated by non-muscle myosin II.
2010,
Pubmed
Howden,
Three-dimensional cerebrospinal fluid flow within the human ventricular system.
2008,
Pubmed
Hukriede,
Conserved requirement of Lim1 function for cell movements during gastrulation.
2003,
Pubmed
,
Xenbase
Jaffe,
c21orf59/kurly Controls Both Cilia Motility and Polarization.
2016,
Pubmed
,
Xenbase
Kaltenbrun,
Xenopus: An emerging model for studying congenital heart disease.
2011,
Pubmed
,
Xenbase
Khokha,
Techniques and probes for the study of Xenopus tropicalis development.
2002,
Pubmed
,
Xenbase
Kurtcuoglu,
Mixing and modes of mass transfer in the third cerebral ventricle: a computational analysis.
2007,
Pubmed
Lee,
Riding the wave of ependymal cilia: genetic susceptibility to hydrocephalus in primary ciliary dyskinesia.
2013,
Pubmed
Lehtinen,
The cerebrospinal fluid provides a proliferative niche for neural progenitor cells.
2011,
Pubmed
Louvi,
Cilia in the CNS: the quiet organelle claims center stage.
2011,
Pubmed
Lupo,
Induction and patterning of the telencephalon in Xenopus laevis.
2002,
Pubmed
,
Xenbase
McMahon,
Mechanistic insights from the LHX1-driven molecular network in building the embryonic head.
2019,
Pubmed
,
Xenbase
Mestre,
Flow of cerebrospinal fluid is driven by arterial pulsations and is reduced in hypertension.
2018,
Pubmed
Milán,
Patterns of calretinin, calbindin, and tyrosine-hydroxylase expression are consistent with the prosomeric map of the frog diencephalon.
2000,
Pubmed
,
Xenbase
Mirzadeh,
Cilia organize ependymal planar polarity.
2010,
Pubmed
Mohun,
The morphology of heart development in Xenopus laevis.
2000,
Pubmed
,
Xenbase
Mukhopadhyay,
Effect of inhibition of axonemal dynein ATPases on the regulation of flagellar and ciliary waveforms in Leishmania parasites.
2018,
Pubmed
Nakamura,
Isthmus organizer for midbrain and hindbrain development.
2005,
Pubmed
Nelson,
The distribution, activity, and function of the cilia in the frog brain.
1974,
Pubmed
Ohata,
Planar Organization of Multiciliated Ependymal (E1) Cells in the Brain Ventricular Epithelium.
2016,
Pubmed
Olsen,
Effect of cadmium acetate, copper sulphate and nickel chloride on organ cultures of mouse trachea.
1979,
Pubmed
Olstad,
Ciliary Beating Compartmentalizes Cerebrospinal Fluid Flow in the Brain and Regulates Ventricular Development.
2019,
Pubmed
Puelles,
Expression patterns of homeobox and other putative regulatory genes in the embryonic mouse forebrain suggest a neuromeric organization.
1993,
Pubmed
Raimondi,
A unifying theory for the definition and classification of hydrocephalus.
1994,
Pubmed
Rekate,
The definition and classification of hydrocephalus: a personal recommendation to stimulate debate.
2008,
Pubmed
Sawamoto,
New neurons follow the flow of cerebrospinal fluid in the adult brain.
2006,
Pubmed
Schindelin,
The ImageJ ecosystem: An open platform for biomedical image analysis.
2015,
Pubmed
Schindelin,
Fiji: an open-source platform for biological-image analysis.
2012,
Pubmed
Scholpp,
Hedgehog signalling from the zona limitans intrathalamica orchestrates patterning of the zebrafish diencephalon.
2006,
Pubmed
Schwarz,
Human Cerebrospinal fluid promotes long-term neuronal viability and network function in human neocortical organotypic brain slice cultures.
2017,
Pubmed
Seeley,
The perennial organelle: assembly and disassembly of the primary cilium.
2010,
Pubmed
Sena,
An Evolutionarily Conserved Network Mediates Development of the zona limitans intrathalamica, a Sonic Hedgehog-Secreting Caudal Forebrain Signaling Center.
2016,
Pubmed
,
Xenbase
Shawlot,
Lim1 is required in both primitive streak-derived tissues and visceral endoderm for head formation in the mouse.
1999,
Pubmed
Simeone,
Two vertebrate homeobox genes related to the Drosophila empty spiracles gene are expressed in the embryonic cerebral cortex.
1992,
Pubmed
Siyahhan,
Flow induced by ependymal cilia dominates near-wall cerebrospinal fluid dynamics in the lateral ventricles.
2014,
Pubmed
Stadlbauer,
Insight into the patterns of cerebrospinal fluid flow in the human ventricular system using MR velocity mapping.
2010,
Pubmed
Sweetman,
Three-dimensional computational prediction of cerebrospinal fluid flow in the human brain.
2011,
Pubmed
Sweetman,
Cerebrospinal fluid flow dynamics in the central nervous system.
2011,
Pubmed
Tang,
Gaussian process post-processing for particle tracking velocimetry.
2019,
Pubmed
,
Xenbase
Thouvenin,
Origin and role of the cerebrospinal fluid bidirectional flow in the central canal.
2020,
Pubmed
Tinevez,
TrackMate: An open and extensible platform for single-particle tracking.
2017,
Pubmed
Ting,
A robust ex vivo experimental platform for molecular-genetic dissection of adult human neocortical cell types and circuits.
2018,
Pubmed
Tsang,
Lim1 activity is required for intermediate mesoderm differentiation in the mouse embryo.
2000,
Pubmed
Wagshul,
The pulsating brain: A review of experimental and clinical studies of intracranial pulsatility.
2011,
Pubmed
Wallmeier,
De Novo Mutations in FOXJ1 Result in a Motile Ciliopathy with Hydrocephalus and Randomization of Left/Right Body Asymmetry.
2019,
Pubmed
Willsey,
Katanin-like protein Katnal2 is required for ciliogenesis and brain development in Xenopus embryos.
2018,
Pubmed
,
Xenbase
Willsey,
The neurodevelopmental disorder risk gene DYRK1A is required for ciliogenesis and control of brain size in Xenopus embryos.
2020,
Pubmed
,
Xenbase
Wullimann,
Postembryonic neural proliferation in the zebrafish forebrain and its relationship to prosomeric domains.
1999,
Pubmed
,
Xenbase
Yasuoka,
Evolutionary origins of blastoporal expression and organizer activity of the vertebrate gastrula organizer gene lhx1 and its ancient metazoan paralog lhx3.
2009,
Pubmed
,
Xenbase
Yoshida,
Emx1 and Emx2 functions in development of dorsal telencephalon.
1997,
Pubmed
Youn,
Primary Cilia in Brain Development and Diseases.
2018,
Pubmed
