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

Abstract

Motile cilia defects impair cerebrospinal fluid (CSF) flow and can cause brain and spine disorders. The development of ciliated cells, their impact on CSF flow, and their function in brain and axial morphogenesis are not fully understood. We have characterized motile ciliated cells within the zebrafish brain ventricles. We show that the ventricles undergo restructuring through development, involving a transition from mono- to multiciliated cells (MCCs) driven by gmnc. MCCs co-exist with monociliated cells and generate directional flow patterns. These ciliated cells have different developmental origins and are genetically heterogenous with respect to expression of the Foxj1 family of ciliary master regulators. Finally, we show that cilia loss from the tela choroida and choroid plexus or global perturbation of multiciliation does not affect overall brain or spine morphogenesis but results in enlarged ventricles. Our findings establish that motile ciliated cells are generated by complementary and sequential transcriptional programs to support ventricular development.

Keywords: brain; cerebrospinal fluid; choroid plexus; cilia; ependymal cell; foxj1; gmnc; multiciliated cells; scoliosis; zebrafish.

Graphical abstract

Figure 1

Multiciliation and ventricular/parenchymal expansion correlate during development (A1–A4) Brain ventricles expand during development as shown upon 3D reconstruction of brain ventricles injected with Rhodamine B isothiocyanate (RITC)-dextran. (A1) 4 dpf, n = 4; (A2) 14 dpf, length: 0.5 to 0.6 cm, n = 4; (A3) 28 to 32 dpf, length: 1–1.5 cm, n = 3; and (A4) 2 to 12 months, larger than 2 cm, n = 3. (B) At 4 dpf, single glutamylated tubulin-positive cilia are located on the forebrain choroid plexus (fChP), on the dorsal roof and ventral part of the tectal/diencephalic ventricle, and in the rhombencephalic ChP (rChP) (n = 5). Dashed lines label boundaries between confocal tiles. (C) At 14 dpf, cilia number increases along the dorsal telencephalon, rostral to the fChP, and in the rChP. Cells are monociliated throughout the brain (n = 3). (D1–D4) At 28 to 32 dpf, brushes of glutamylated tubulin-positive cilia appear on the dorsal telencephalon anterior to the ChP and in the ChP (n = 3; D1). (D2) The presence of monociliated (arrow) and MCCs (arrowhead) is shown upon co-staining with the membrane marker β-catenin. (D3) MCCs are located medially to monociliated cells in the tela choroida (TC; quantified in bottom panel; n = 9). (D4) The fChP comprises mono- and MCCs, arranged in an anterior-posterior manner (n = 3). (E1–E4) In the adult brain, MCCs are enriched in the medial part of the TC above the telencephalon (E1) and in the ChP (E4). (E2) Cilia do not cover the entire apical surface of MCCs as shown upon co-staining with β-catenin (n = 4). (E3) Adult fChP consists of multiple interconnected cavities, as shown upon 3D reconstruction of ventricles injected with RITC-dextran (n = 4), and contains mono- and MCCs (E4) (n = 3). (F1–F5) Mono- and MCCs are present on the adult telencephalic/diencephalic midline in the region surrounding the anterior commissure (Ca) highlighted in red. Scanning electron microscopy (n = 3) and immunostaining with glutamylated tubulin (n = 3) show the presence of MCCs (F2 and F4) and monociliated cells (F3 and F5). Location of (F2)–(F5) is indicated in (F1). A, anterior; P, posterior; D, dorsal; V, ventral; M, medial; L, lateral; Tel, telencephalon; TeO, optic tectum; Rhomb, rhombencephalon; CC, cerebellum; MCCs, multiciliated cells. Arrowheads show MCCs, and arrows show monociliated cells. See also Figure S1.

Figure 2

Cilia in the telencephalon, TC, and ChP are motile and generate CSF flow (A) Dorsal view of an adult brain explant. (B–D) Cilia in the forebrain ChP (B), TC (C), and telencephalic midline (D) are motile as shown by analysis of high-speed video microscopy using a pixel-based Fourier analysis. (B1–D1) Map of ciliary beating with ciliary beat frequency (CBF) color-coded in the ChP (B1), TC (C1), and hemisphere explant anterior and posterior to the Ca (D1). (B2–D2) Probability histogram showing the frequency of the pixel-based analysis and the average ± SEM for ChP (B2), TC (C2), and telencephalic hemisphere (D2). n = number of brain explants. (E) Dorsal view of an adult brain explant injected with 1 μm fluorescent beads. (F–H) Directional fluid flow in the ChP (F; n = 4), dorsal telencephalon (G; n = 5), and telencephalic midline (H; n = 5) is color-coded. Tel, telencephalon; A, anterior; P, posterior; D, dorsal; V, ventral; R, right; L, left. See also Videos S1, S2, and S3.

Figure 3

Ciliated cells in the TC, telencephalon, and ChP express foxj1a, foxj1b, and gmnc to different extents (A–D) Expression of foxj1a, foxj1b, and gmnc in the brain using multiplex HCR (A1–D1) and two transgenic lines, Tg(foxj1a:gfp)BAC^vcc41 (A2–D2) and Gt(foxj1b:GFP)^tsu10Gt (A3–D3). (A1–A3) At 4 dpf, foxj1b is expressed in the dorsal telencephalon, nose, neuromasts, and midbrain; foxj1a is expressed in the nose, neuromasts, and midbrain; and no gmnc HCR signal is detected in the brain. Cells with a solitary glutamylated tubulin-labeled cilium (magenta in A2 and A3 insets) on the dorsal telencephalon express primarily foxj1b. (B1–B3) At 14 dpf, there is an expansion of foxj1b-expressing cells anterior to the ChP (B1 and B3). foxj1a expression remains low in the dorsal telencephalon and ChP (B1 and B2). Few gmnc puncta are present in the anterior part of the ChP (arrowhead in B1). (C1–C3) At 1 month, gmnc and foxj1a are highly expressed in the anterior ChP and dorso-medial TC (C1 and C2). (C1 and C3) foxj1b is expressed in both the anterior and posterior ChP. (C2 and C3) foxj1a:GFP and foxj1b:GFP cells bear glutamylated tubulin-positive cilia. (D1–D3) On the midline of the adult telencephalon/diencephalon, foxj1a and foxj1b are mainly expressed in the ventral part of the brain, surrounding the Ca. (D1) foxj1a- and foxj1b-expressing domains are not fully overlapping. The dotted line in inset indicates a sharp boundary for foxj1a expression but not for foxj1b. There is very low gmnc expression. (D2 and D3) Immunostaining with glutamylated tubulin shows that foxj1a:GFP- and foxj1b:GFP- expressing cells harbor multiple cilia (arrowhead) or a solitary cilium (arrow). (D3) Ciliated cells with multiple cilia (white arrowhead) or a single cilium (white arrow) are foxj1b:GFP-negative. Number of datasets: A1 = 4, B1 = 4, C1 = 4, D1 = 3, A2 = 4, B2 = 3, C3 = 4, D3 = 4, A3 = 4, B3 = 3, C3 = 4, and D3 = 3. A, anterior; P, posterior; D, dorsal; V, ventral; M, medial; L, lateral; Tel, telencephalon; TeO, optic tectum; OB, olfactory bulb; nm, neuromast. Note that there is unspecific HCR signal associated with blood cells and vasculature (indicated by the hashtag symbol). See also Figure S2.

Figure 4

gmnc is required for multiciliation in ependymal cells (A and B) Absence of MCCs in 1-month-old (A1 and A2; n = 3 ctrl; 3 mutants) and adult brains of gmnc mutants (B1–B3; n = 3 ctrl; 3 mutants). Arrowheads show MCC in controls. All ciliated cells in gmnc mutant are monociliated (arrow). (A1) 1-month dorsal telencephalon and (A2) ChP. (B1) Adult dorsal telencephalon. (B2) Anterior portion of the adult ChP. (B3) Adult midline above the Ca. (C1–C3) gmnc enhances the expression of foxj1a and foxj1b in the TC and ChP. (C1) HCR revealed a gmnc-dependent foxj1a expression, particularly in the anterior ChP (arrow in insets). n = 4 controls; 6 mutants. The hashtag symbol shows nonspecific signal in blood vessels. (C2) foxj1b:GFP remained expressed in gmnc mutant with reduced and more homogeneous levels (n = 4 controls; 7 mutants). β-catenin and glutamylated tubulin staining was used for quantification of GFP levels in C3. (C3) Monociliated cells express less foxj1b:GFP than MCCs and monociliated cells in gmnc mutant express similar foxj1b:GFP levels as control monociliated cells. All data points are plotted; mean and SD of n fish; ^∗p < 0.05 calculated on the average fluorescence per fish using rank sum test. (D) HCR shows that foxj1a and foxj1b remained expressed in the telencephalic midline of gmnc mutant (n = 3). Asterisk indicates axons. A, anterior; P, posterior; R, right; L, left; Tel, telencephalon. See also Figure S3.

Figure 5

foxj1a and foxj1b play diverse roles in the formation of motile ciliated cells (A) Lack of cilia in the dorsal telencephalon in foxj1b mutant (middle; n = 9) and throughout the larval ventricular system in double foxj1a/b mutant (n = 6) at 4 dpf. Maximum projection at two different depths is shown. (B1 and B2) MCCs (arrowhead) are not affected in the foxj1b mutant on the 1-month dorsal telencephalon (B1; n = 6) and ChP (B2; n = 6). Cilia remain absent in the monociliated cells (arrow) located laterally to the MCCs as shown in the magnified inset. (C1–C3) MCCs are not affected in the adult medial TC (C1; n = 3), ChP (C2; n = 4), and midline of the telencephalon/diencephalon (C3; n = 3). (C2) Monociliated cells are particularly affected in the ChP. (D1–D3) Loss of cilia in the medial TC (D1) and ChP (D2) of adult gmnc;foxj1b double mutants (n = 4). (D3) Single cilia remained in the telencephalic midline of double mutants (arrow) (n = 4). (E) Diverse genetic programs instruct the formation of ciliated cells in the TC/ChP and in the telencephalon midline. TeO, optic tectum; sco, sub-commissural organ. Arrows indicate monociliated cells, arrowheads indicate MCCs, and asterisks indicate axon tracts. Cilia loss is indicated by hashtag symbols. See also Figures S4 and S5.

Figure 6

Diversity of motile ciliated cells in the forebrain (A and B) Single-cell RNA sequencing analysis of all cells (A1) or all progenitor-like cells (PCs; B1) revealed the presence of two potential ependymal clusters, dkk (A2) and PC14 (B2), based on the expression of foxj1a/b (foxj1a-only cells in cyan, foxj1b-only cells in magenta, and foxj1a/b-co-expressing cells in black) gmnc, enkur, and mia. (C) Scatterplot representing the expression levels of foxj1a and foxj1b in all cells. (D) Selection of differentially regulated genes between the dkk and PC14 ependymal clusters. (E) The dkk cluster markers dkk3b (n = 5), clu (n = 13), rbp4 (n = 4), and igfbp2a (n = 8) are expressed in the 1-month-old TC and ChP, similar to foxj1a and enkur, as shown by HCR. (F) The PC14 markers gfap (n = 4) and her4 (n = 3) are excluded from the ChP at 1 month. (Top) HCR for gfap shows expression in radial glia and absence of signal in ChP. (Bottom) Immunostaining of Tg(her4:gfp) with membrane marker β-catenin and cilia marker glutamylated tubulin revealed the absence of her4:gfp signal in ciliated cells of the ChP. (G) Molecular cartography on an adult telencephalic cryosection (DAPI; left) showed that foxj1a and foxj1b colocalized with gfap in the midline (inset 1) and not in the ChP (inset 2) and that igfbp2a and dkk3b are enriched in the ChP (inset 2) as compared to the midline (inset 1). n = 7 sections from one brain. (H1–H4) Pseudotime analysis of the PCs plotted using Monocle algorithm showed a progression from quiescent (s100b; H1) to a proliferative stage (pcna; H2). (H3) foxj1b is expressed more in quiescent progenitors, while foxj1a is expressed more in proliferative progenitors. (H4) PC14 ependymal cells (indicated in blue) branch out from quiescent progenitors. See also Figure S6 and Table S1.

Figure 7

gmnc−/−, foxj1b−/−, foxj1a+/−;foxj1b−/−, and gmnc−/−;foxj1b−/− mutants do not display major body and brain malformations but have enlarged telencephalic ventricles (A) Pictures of adult zebrafish showing absence of scoliosis. foxj1a^nw3 allele was analyzed. (B) Occurrence of scoliosis in some of the progeny of triple foxj1a; foxj1b; gmnc heterozygous incross. Note that the phenotype is not fully penetrant. The numbers indicate the ratio of animals showing the phenotype among all genotyped individuals. foxj1a^sq5717 allele was analyzed. (C) OCT images of anaesthetized adult zebrafish revealed enlarged telencephalic ventricles (highlighted with dashed red line) in animals with cilia defects. Transverse section taken as indicated by red dashed line in (D). (D) Quantification of ventricular size in all animals as a function of body length. (E) SD projection of OCT images of adult brain explants showing absence of overt brain malformations in cilia mutants. (F and G) Quantification of the telencephalic width (F) and length (G) as a function of body length. (D, F, and G) R-squared indicate the linear relationship between body length and the measurements as shown in red. Left: all controls pooled. Others: all controls in gray, sibling controls in black, and mutants in blue. p value based on rank sum between sibling controls indicated in black and blue. Examples shown are highlighted in the scatterplot with a hashtag symbol. D, dorsal; V, ventral; A, anterior; P, posterior; Tel, telencephalon; TeO, optic tectum. See also Figure S7 and Table S2.