ZFIN ID: ZDB-FIG-200426-16
Zhang et al., 2020 - Wnt-PLC-IP3-Connexin-Ca2+ axis maintains ependymal motile cilia in zebrafish spinal cord. Nature communications   11:1860 Full text @ Nat. Commun.
ADDITIONAL FIGURES
EXPRESSION / LABELING:
Genes:
Antibodies:
Fish:
Conditions:
Knockdown Reagents:
Anatomical Terms:
Stage Range: Prim-5 to Long-pec
PHENOTYPE:
Fish:
Conditions:
Knockdown Reagents:
Observed In:
Stage: Long-pec

Fig. 1 Wnt signaling is involved in the maintenance of ependymal motile cilia in zebrafish embryos.

a Transmission electron microscopy (TEM) of the spinal cords (SCs) of zebrafish embryos at 2 dpf. Arrowhead indicates a motile cilium with the 9 + 2 microtubule configuration, which is magnified to the right. Scale bar = 1 μm. b Immunofluorescence (IF) staining of an embryo at 1 dpf with anti-acetylated-α-tubulin antibody. Dorsal view anterior to the left. Arrowheads represent motile cilia. Scale bar = 20 μm. c, d IF staining of sonic-you (syut4) mutant embryos at 2 dpf with anti-acetylated-α-tubulin antibody. Dorsal view anterior to the left (c). d The cross-section image of the SC ventral to the bottom. Arrowheads represent motile cilia. Scale bars = 20 μm. e Embryos were treated with DAPT (50 μM) at 34–48 hpf and IF stained with anti-acetylated-α-tubulin antibody. Dorsal view anterior to the left. Arrowheads represent motile cilia. Scale bar = 20 μm. f Embryos were co-microinjected with wnt4b MO and wnt11 MO (wnt4b/11 MO) alone or along with wnt4b mRNA and wnt11 mRNA (wnt4b/11 mRNA), and IF stained at 2 dpf with anti-acetylated-α-tubulin antibody. Arrowheads represent motile cilia. Dorsal view anterior to the left. Scale bar = 20 μm. CO: Control. g Quantification of the number of cilia per frame in embryos in f. Data are presented as mean ± SD. **P < 0.01 and ****P < 0.0001 by one-way ANOVA with Tukey’s honest significant difference (HSD) post hoc test (control morphants: n = 12 embryos; wnt4b/11 double morphants: n = 9 embryos; wnt4b/11 double morphants + wnt4b/11 mRNA: n = 9 embryos; one frame per embryo). h A cross-section images of the SCs of control morphants and wnt4b/11 double morphants at 2 dpf probed with foxj1a riboprobes ventral to the bottom. Arrowheads represent ECs. Scale bar = 20 μm. CO: Control. i RNAs were extracted from each group (20 embryos in h) at 2 dpf and levels of foxj1a mRNAs were assessed by qPCR. Mean ± SD. ****P < 0.0001 by two-tailed unpaired Student’s t test from four biological replicates (three technical replicates each). j A cross-section image of the SC of a WT embryo at 2 dpf probed with fzd7b riboprobes ventral to the bottom. Arrowhead represents ECs. Scale bar = 15 μm. k Embryos were microinjected with control MO, fzd7b MO or fzd7b MO + fzd7b mRNA, and IF stained at 2 dpf with anti-acetylated-α-tubulin antibody. Arrowheads represent motile cilia. Dorsal view anterior to the left. Scale bar = 20 μm. CO: Control. l Quantification of the number of cilia per frame in embryos in k. Mean ± SD. ****P < 0.0001 by one-way ANOVA with Tukey’s HSD post hoc test (control morphants: n = 21 embryos; fzd7b morphants: n = 21 embryos; fzd7b morphants + fzd7b mRNA: n = 18 embryos; one frame per embryo). mTg(hsp70l:dkk1b-GFP) embryos at 24 hpf were subjected to heat shock at 39 °C for 1 h, and IF stained at 2 dpf with anti-acetylated-α-tubulin antibody. Arrowheads represent motile cilia. Dorsal view anterior to the left. Scale bar = 20 μm. n, o Embryos were treated with IWR-1 (10 μM) at 8–48 hpf and IF stained with anti-acetylated-α-tubulin antibody only (n) or double immunostained with anti-acetylated-α-tubulin antibody and anti-γ-tubulin antibody (o) at 2 dpf. Arrowheads represent motile cilia. Dorsal view anterior to the left. The boxed areas in o are magnified in the respective lower panels. Scale bar = 20 μm.

Gene Expression Details
Gene Antibody Fish Conditions Stage Anatomy Assay
foxj1a AB control Long-pec spinal cord ependymal cell ISH
Long-pec whole organism RTPCR
AB + MO3-wnt4b + MO4-wnt11 standard conditions Long-pec spinal cord ependymal cell ISH
Long-pec whole organism RTPCR
fzd7b AB standard conditions Long-pec spinal cord ependymal cell ISH
Antibody Labeling Details
Antibody Assay Fish Conditions Stage Qualifier Anatomy
Ab1-tuba IHC AB control Long-pec ependymal cell motile cilium
IHC AB standard conditions Prim-5 ependymal cell motile cilium
IHC Prim-5 spinal cord ependymal cell
IHC AB chemical treatment by environment: DAPT Long-pec ependymal cell motile cilium
IHC Long-pec spinal cord ependymal cell
IHC AB chemical treatment by environment: IWR-1-endo Long-pec ependymal cell motile cilium
IHC AB + MO2-fzd7b standard conditions Long-pec ependymal cell motile cilium
IHC Long-pec spinal cord ependymal cell
IHC AB + MO3-wnt4b + MO4-wnt11 standard conditions Long-pec ependymal cell motile cilium
IHC Long-pec spinal cord ependymal cell
IHC shhaDf(Chr07)t4/t4 standard conditions Long-pec ependymal cell motile cilium
IHC Long-pec spinal cord ependymal cell
IHC w32Tg control Long-pec ependymal cell motile cilium
IHC Long-pec spinal cord ependymal cell
IHC w32Tg heat shock Long-pec Not Detected spinal cord ependymal cell
Ab1-tubg1 IHC AB control Long-pec ependymal cell ciliary basal body
IHC AB chemical treatment by environment: IWR-1-endo Long-pec ependymal cell ciliary basal body
Phenotype Details
Fish Conditions Stage Phenotype
AB chemical treatment by environment: DAPT Long-pec ependymal cell motile cilium assembly normal occurrence, normal
Long-pec spinal cord has normal numbers of parts of type ependymal cell motile cilium, normal
AB chemical treatment by environment: IWR-1-endo Long-pec ependymal cell ciliary basal body organization normal occurrence, normal
Long-pec ependymal cell motile cilium assembly decreased occurrence, abnormal
Long-pec spinal cord has fewer parts of type ependymal cell motile cilium, abnormal
AB + MO2-fzd7b standard conditions Long-pec ependymal cell motile cilium assembly decreased occurrence, abnormal
Long-pec spinal cord has fewer parts of type ependymal cell motile cilium, abnormal
AB + MO3-wnt4b + MO4-wnt11 standard conditions Long-pec ependymal cell motile cilium assembly decreased occurrence, abnormal
Long-pec spinal cord ependymal cell foxj1a expression increased amount, abnormal
Long-pec spinal cord has fewer parts of type ependymal cell motile cilium, abnormal
Long-pec whole organism foxj1a expression increased amount, abnormal
shhaDf(Chr07)t4/t4 standard conditions Long-pec ependymal cell motile cilium assembly normal occurrence, normal
Long-pec spinal cord has normal numbers of parts of type ependymal cell motile cilium, normal
w32Tg control Long-pec ependymal cell motile cilium assembly normal occurrence, normal
Long-pec spinal cord has normal numbers of parts of type ependymal cell motile cilium, normal
w32Tg heat shock Long-pec ependymal cell motile cilium assembly decreased occurrence, abnormal
Long-pec spinal cord ependymal cell ab1-tuba labeling absent, abnormal
Long-pec spinal cord has fewer parts of type ependymal cell motile cilium, abnormal
Acknowledgments:
ZFIN wishes to thank the journal Nature communications for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ Nat. Commun.