Supplemental Information. Ciliary Beating Compartmentalizes. Cerebrospinal Fluid Flow in the Brain. and Regulates Ventricular Development

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1 Current Biology, Volume Supplemental Information Ciliary Beating Compartmentalizes Cerebrospinal Fluid Flow in the Brain and Regulates Ventricular Development Emilie W. Olstad, Christa Ringers, Jan N. Hansen, Adinda Wens, Cecilia Brandt, Dagmar Wachten, Emre Yaksi, and Nathalie Jurih-Yaksi

2 Olstad et al, Supplemental Fig S1 Sagittal view hpf A cpiii * * o OT Horizontal view B Foxj1b:GFP gluttub Tg(foxj1b:gfp) gluttubdapi DAPI cpiii pg RV RV Tg(foxj1b:gfp) Foxj1b:GFP kda-ritc kda-ritc o C D Tg(foxj1a:gfp)nwTg gluttub DAPI hpf dpf ventral RV C D Tg(foxj1a:gfp)nwTg gluttub DAPI * o Tg(foxj1a:gfp)nwTg kda-ritc RV dpf D Tg(foxj1a:gfp)nwTg gluttub DAPI o RV Tg(foxj1a:gfp)nwTg kda-ritc R cp ventral o glutamylated tubulin D dorsal OT E merge T ventral o C cp dorsal pg * dorsal Tg(foxj1a:gfp)nwTg kda-ritc E ventral ventral cpiii * dorsal dorsal b-actin:arl1b-gfp DAPI # glutamylated tubulin b-actin:arl1b-gfp μm -1 μm OT - μm - μm - μm - μm Figure S1: Analysis of foxj1b:gfp and.kbfoxj1a:gfp transgenic lines. Related to Figure 1. (A-B) The Tg(foxj1b:gfp) transgenic line labels the cells located in the dorsal wall of the diencephalic ventricle (as indicated by a white arrowhead) before they become ciliated. Confocal microopy image of the sagittal midline of an anaesthetized larva injected with kda RITC-dextran (magenta) at hpf (A). The * indicate autofluoreence from the skin. GFP-positive cells are not yet ciliated at hpf, as shown by immunostaining with a glutamylated tubulin antibody (magenta) and confocal microopy (nuclei are stained with DAPI (blue); B). (C-D ) Tg(foxj1a:gfp)nwTg transgenic animal show a large portion of GFP-labeled ciliated cells in the dorsal wall of the

3 diencephalic ventricle (white arrowhead) at hpf, dpf and dpf and in the choroid plexus (cp) at dpf, in the same locations as in foxj1b:gfp transgenic line. The expression pattern is different from the foxj1a in situ hybridization, suggesting that the foxj1a. kb promoter is not sufficient to recapitulate the endogenous foxj1a expression pattern in the brain ventricles of the developing zebrafish larvae. (C-C ) Confocal microopy image of the sagittal midline of anaesthetized larvae injected with kda RITC-dextran (magenta) at hpf (C), dpf (C ) and dpf (C ). The * indicate autofluoreence from the skin. (D-D ) GFP-positive cells extend their cilia into the ventricular cavity at hpf (D), dpf (D ) and dpf (D ), as shown by immunostaining with a glutamylated tubulin antibody (magenta) and confocal microopy (nuclei are stained with DAPI (blue)). (E-E ) Immunostaining of a. dpf β-actin:arl1b-gfp larvae with a glutamylated tubulin antibody demonstrates that only a subset of cilia contain glutamylated tubulin. (E) Maximum Z-projection of a confocal stack showing glutamylated tubulin (magenta), transgenic β-actin:arl1b-gfp expression (green) and DAPI (blue) to indicate the nuclei. (E ) Montage of the inset indicated in (E). Glutamylated tubulin positive cilia are located in the dorsal and ventral walls along the midline of the diencephalic ventricle and in the dorsal roof of the telencephalic ventricle. Note that arl1b-gfp positive cilia are located throughout the brain tissue. Each image corresponds to an individual optical section from dorsal to ventral, with 1 µm between consecutive optical sections as indicated. Glutamylated tubulin also labels the neuropil as indicated by the #. Nuclei are labeled with DAPI (blue). Scale bars are µm. Anterior to the left. Posterior to the right. : telencephalic ventricle, : diencephalic ventricle, RV: rhombencephalic ventricle, : spinal cord, pg: pineal gland, o: subcommissural organ, cp: choroid plexus, OT: optic tectum, T: telencephalon, D: diencephalon, R:rhombencephalon.

4 Figure S: Ciliary beating parameters change from to dpf. Related to Figure. (A) Motile cilia were identified in the dorsal telencephalic ventricle (; green arrow), dorsal diencephalic ventricle (; blue arrow) and ventral (yellow arrow) at and dpf upon light-sheet microopy of β-actin:arl1b-gfp larvae. Pie charts indicate the numbers of analyzed cilia in the different ventricular locations (color-coded). (B) Quantification of beating frequencies (CBF) from individual cilia revealed a significant difference between and dpf when comparing the mean of individual larvae. (C) The ciliary beating frequency decreases significantly in all ventricular regions from to dpf when comparing the mean of individual larvae. (D) There is no significant difference in the beating frequency for cilia located in proximity to the ducts as compared to the ones located in the middle of the dorsal wall of the. The dorsal wall of the was segmented into four bins, as indicated in the upper panel, and the CBFs were plotted according to their location. (E) The width but not the angle of ciliary beating (as represented in FigD) is slightly different when comparing data obtained from horizontal (top) and sagittal (side) recordings at dpf. (F) The width, angle and beating frequency significantly decrease from to dpf when the ventricular space becomes tighter. Mean ± s.d of all data is indicated. The mean for each larva is indicated by a red cross. p-value by the Wilcoxon rank-sum test. The data obtained at dpf are the same as those presented in Fig.

5 A -hpf:/ without flow A -hpf: / with flow A -hpf Pulsatile flow 1 Flow fields μm/s.hz Pulsatile flow 1 Flow fields μm/s frequency (Hz). 1. B Pulsatile flow Flow fields C Flow velocity dorsal D Frequency pulsations dpf min BDM before BDM.Hz.Hz μm/s μm/s velocity (μm/s) Figure S: The directional CSF flow and the pulsatile movement are established by - hpf, and the pulsatile movement is contributed by the heartbeat. Related to Figure. CSF flow patterns and pulsations along the sagittal midline of the diencephalic ventricle () are obtained by confocal microopy upon ventricular injection of fluoreent beads. The pulsatile CSF movement is visualized by displaying the relative power (%) at the most abundant periodic frequency identified by Fast Fourier Transform and indicated for each representative example. The flow fields are analyzed by particle image velocimetry (PIV). (A-A ) Fourier transform and PIV analysis revealed a pulsatile movement (left) and a directional flow along the dorsal (right) in / embryos at hpf (A, example without flow) and / embryos at hpf (A, example with flow). The pulsatile CSF movement, when detected, has a frequency of.1 ±.11 Hz at hpf (A ). (B) Upon ablation of the heartbeat by treatment with BDM, the directional, near-wall flow persists, while the pulsatile CSF movement is absent. Representative example of a dpf larvae before (top) and after (bottom) min of mm BDM treatment. (C) Quantification of the PIV velocities along the dorsal wall of the before and after heart microdissection, revealed that the heartbeat does not contribute to a net flow at dpf (left). Note that upon min treatment with BDM the velocities are significantly reduced (right), suggesting that long incubation time or off-target effects of BDM may affect the flow velocities. P-values according to the Wilcoxon signed-rank test. (D) The frequencies of the pulsatile movement are not affected by the absence or immotility of cilia. No significant difference detected with the Kruskal-Wallis test. All individual values are plotted in a atter plot together with the mean and standard deviation. Anterior to the right, posterior to the left. Scale bars are µm. heartbeat p=. heartstop p=.1 before BDM min BDM frequency (Hz) Control elipsa oval smh foxj1a nw

6 Figure S: D analysis of CSF flow patterns and pulsations in the telencephalic and diencephalic ventricles at dpf. Related to Figure. CSF flow patterns and pulsations in the telencephalic () and diencephalic ventricles () were obtained by confocal microopy upon ventricular injection of fluoreent beads. Sequential recordings were obtained every µm from a sagittal viewing angle. The pulsatile CSF movement is visualized by displaying the relative power (in %) at the most abundant periodic frequency identified by Fast Fourier Transform (indicated in Hz). The flow fields were analyzed by particle image velocimetry (PIV). (A) Z-projection of an anatomical stack of the ventricles from a horizontal viewing angle. Recordings were obtained every µm from the midline to the extremities of the ventricles as indicated by the red lines. (B) Pulsatile and PIV analyses of the CSF flow patterns revealed that in the, the pulsations are restricted to the - duct and that the directional flow is strongest along the dorsal wall of the across most of its width. (C) In the, the pulsations are observed in the -RV duct and in the middle of the ventricle. The directional flow is only observed at the midline along the dorsal and ventral wall of the. Scale bars are µm. A: anterior, P: posterior, L: left, R: right, D: dorsal, V: ventral, : telencephalic ventricle, : diencephalic ventricle, OT: optic tectum.

7 A -μm -μm -1μm μm Telencephalic-to-diencephalic duct Heartbeat pulsatile analysis particle tracking A -μm -μm -1μm μm Telencephalic-to-diencephalic duct pulsatile analysis No heartbeat particle tracking Hz 1. Hz 1 B -μm -μm -1μm μm Diencephalic-to-rhombencephalic duct Heartbeat pulsatile analysis particle tracking RV RV. Hz B -μm -μm -1μm μm Diencephalic-to-rhombencephalic duct No heartbeat pulsatile analysis particle tracking. Hz 1 Figure S: D analysis of the CSF flow patterns and pulsations in the ventricular ducts at dpf. Related to Figure. CSF flow patterns and pulsations in the ducts were obtained by confocal microopy upon ventricular injection of fluoreent beads. Sequential recordings were obtained every 1 µm from a sagittal viewing angle. The pulsatile CSF movement is visualized by displaying the relative power (in %) at the most abundant periodic frequency identified by Fast Fourier Transform (indicated in Hz). Particle tracking was done manually. The arrows represent the direction from the first point to the last point of tracking and the number indicate the tracking time in seconds. (A-B) The Fourier Transform-based analysis and particle tracking revealed pulsations in the - and -RV ducts, as shown for a representative example (n = ) with heartbeat. In the - duct, there is no

8 net movement of particles across the duct, instead the particles coming from the are pushed caudally back into the (A). In the -RV duct, some of the particles coming from the remain in the and are pushed dorsally (B). (A -B ) In absence the of heartbeat after heart microdissection, the pulsations are gone. As in the larvae with heartbeat, there was no directional flow across the - (A ) and -RV (B ) ducts observed in the larvae without heartbeat at any investigated depths. Scale bars are µm. Anterior to the left, posterior to the right.

9 Figure S: The directional CSF flow in the diencephalic ventricle is abolished in oval mutants. The brain ventricular system is not significantly altered upon loss of primary and/or motile cilia at dpf. Related to Figure and. (A-F) Immunostaining for cilia with a glutamylated tubulin antibody (magenta) show that oval mutants retain about % of cilia at dpf (A-C) and % at dpf (D-F) in the dorsal wall of the diencephalic ventricle (). Nuclei are stained with DAPI (blue). (A -F ) The directional CSF flow is abolished in many oval mutant larvae as compared to their control siblings when analyzed by PIV at dpf (A -C ) and dpf (D -F ). Notably, a few larvae retained some unidirectional flow at dpf (novalflow = ). (G) Oval mutants have a higher probability than control

10 larvae of developing enlarged ventricles at dpf, indicated by significantly increased ventricle heights and numbers of outliers (encircled in red). (H-K) The brain ventricular system is not significantly altered upon loss of primary and/or motile cilia at dpf. Various ventricular hallmarks were measured on confocal z- stacks of larvae injected with kda RITC-dextran at dpf. These included the height of the telencephalic (; H) and (I) and the width of the - duct (J) and the -RV duct (K). The controls of all mutants are pooled into one group and compared to mutants lacking all cilia, elipsa and oval (green), or mutants with immotile cilia only, smh and foxj1a nw (blue). Outliers (encircled in red) were defined as measurements outside the 1. interquartile ranges of the pooled control group (indicated by a light blue box). No significant differences or outliers were detected across the different mutants compared to controls. Plots shown in panels (C), (C ), (F), (F ), and (G) contain all controls including the ones presented in Fig and Fig. Horizontal lines indicate the mean of the respective sample groups, vertical lines represent the mean ± s.d. All p-values were calculated with the Kruskal-Wallis test followed by a pairwise comparison on the whole dataset (that included mutants shown in Fig and Fig). Scale bars are µm (flow fields) or µm (stainings). White areas in the flow field representations were not included in PIV analysis, due to the presence of brain tissue and absence of particles.

11 Figure S: Upon bodily movement at dpf, the stringent compartmentalization of the larval ventricular system is temporarily eliminated. Related to Figure. (A-A ) Prior to bodily movement (t = -. s), the directional CSF flow along the ventricular walls is evident, referred to as the baseline flow. (A ) After the bodily movement (t =.. s), a strong surge of CSF backwards from the telencephalic ventricle () to the rhombencephalic ventricle (RV) is prominent. (A ) About. s (t =.. s) later, the baseline flow as seen in (A) is re-established. Representative example of n = 1. (B-F) In order to quantify the impact of bodily movement on the CSF flow directionality, we measured the flow direction in all consecutive PIV analyzed frames in the (B-B ), ventral diencephalic ventricle (; C-C ), dorsal (D-D ) and RV (E-E ). Bodily movements affect the directionality of CSF flow across the ventricles as indicated by polar histograms of multiple frames preceding movement (-. s; B -E ), shortly after movement (.. s, B -E ) and following movement (. s, B -E ). In the, the polar histogram shows little flow directionality before movement (B ), a strong directionality of - o pointing caudally toward the shortly after movement (B ), and re-establishment of baseline flow directionality. s after the bodily movement (B ). In the ventral and RV, the CSF flow is highly pulsatile before and after bodily movement as indicated by the axial distribution of flow direction pointing towards 1 o and o (C, C, E, E ). Just after the movement, the flow is primarily oriented caudally towards o (C, E ). In the dorsal, the flow direction is oriented rostrally with an angle of 1 o (D, D ), but upon bodily movements, the flow is directed caudally (D ). Scale bar is µm. Each individual movement is represented in different color (1 bodily movement for larvae). Polar histograms represent the probability distribution (ale bar is 1 %). The mean is indicated in grey.