ZFIN ID: ZDB-FIG-200426-17
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:
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Conditions:
Knockdown Reagents:
Anatomical Terms:
Stage Range: Long-pec to Day 4
PHENOTYPE:
Fish:
Conditions:
Knockdown Reagents:
Observed In:
Stage Range: Long-pec to Day 4

Fig. 2 <italic>plcδ3a</italic> is a target gene of Wnt signaling and plays an important role in the maintenance of ependymal motile cilia.

a DMSO (vehicle control) or thapsigargin (4 nl at 5 μM) was microinjected into the hindbrain ventricles of zebrafish larvae at 4 dpf and the larvae were double immunostained with anti-acetylated α-tubulin antibody (red) and anti-γ-tubulin antibody (green) 4 h after injection. Arrowheads indicate motile cilia. Dorsal view anterior to the left. Scale bar = 20 μm. b Quantification of the number of cilia and basal bodies per frame in embryos in a. Mean ± SD. ****P < 0.0001 by two-tailed unpaired Student’s t test (n = 4 embryos per group; one frame per embryo). ns, not significant. c Embryos at 2 dpf were probed with plcβ4 or plcδ3a riboprobes and their SCs were cross-sectioned. Images are oriented ventral to the bottom. Arrowheads represent ECs. Scale bar = 20 μm. d DMSO (vehicle control) or U-73122 (4 nl at 10 μM) was microinjected into the hindbrain ventricles of zebrafish larvae at 4 dpf and the larvae were IF stained with anti-acetylated α-tubulin antibody 4 h after microinjection. Dorsal view anterior to the left. Scale bar = 20 μm. e Control morphants or wnt4b/11 double morphants were microinjected with plcβ4 or plcδ3a mRNA and immunostained with anti-acetylated α-tubulin antibody at 2 dpf. Arrowheads represent motile cilia. Dorsal view anterior to the left. Scale bar = 20 μm. f Quantification of the number of motile cilia per frame in embryos in e. Mean ± SD. **P < 0.01 and ****P < 0.0001 by one-way ANOVA with Tukey’s HSD post hoc test (control morphants: n = 12 embryos; wnt4b/11 double morphants: n = 9 embryos; control morphants + plcβ4 mRNA: n = 4 embryos; control morphants + plcδ3a mRNA: n = 3 embryos; wnt4b/11 double morphants + plcβ4 mRNA: n = 9 embryos; wnt4b/11 double morphants + plcδ3a mRNA: n = 5 embryos; one frame per embryo). ns: not significant. g Control morphants or wnt4b/11 double morphants at 2 dpf were probed with plcδ3a riboprobes and their SCs were cross-sectioned. Images are oriented ventral to the bottom. Arrowheads point to ECs. Scale bar = 20 μm. h RNAs were extracted from each group (20 embryos in g) at 2 dpf and levels of plcδ3a mRNA were assessed by qPCR. Mean ± SD. *P < 0.05 by two-tailed unpaired Student’s t test from four biological replicates (three technical replicates each). CO: Control. i Embryos at 45 hpf were treated with DMSO or BIO (5 μM) for 1–3 h and probed with plcδ3a riboprobes, and their SCs were sectioned. Images are oriented ventral to the bottom. hpt: hours post-treatment. Arrowhead indicates ECs. Scale bar = 20 μm. j Upon BIO treatment, RNAs were extracted from each group (20 embryos in (i)) and levels of plcδ3a mRNAs were assessed by qPCR. Mean ± SD. *P < 0.05 and ****P < 0.0001 by two-tailed unpaired Student’s t test from three biological replicates (eight technical replicates each). k Schematic of the plcδ3a promoter-firefly luciferase construct used in the luciferase reporter assay. Black rectangles represent three (1–3) Tcf binding elements (TBEs) and red rectangles TBEs with deletions. l HEK 293T cells were transfected with WT or mutant plcδ3a promoter-firefly luciferase constructs and Renilla luciferase plasmid with or without β-catenin plasmid, and processed for dual luciferase assay. Relative Light Units: firefly luciferase activity/Renilla luciferase activity. **P < 0.01 and ****P < 0.0001 by one-way ANOVA with Tukey’s HSD post hoc test (n = 3 culture replicates per group; each culture was assayed three times).

Gene Expression Details
Gene Antibody Fish Conditions Stage Anatomy Assay
plcb4 AB standard conditions Long-pec spinal cord ependymal cell ISH
plcd3a AB control Long-pec spinal cord ependymal cell ISH
AB standard conditions Long-pec spinal cord ependymal cell ISH
AB chemical treatment by environment: 6-bromoindirubin-3'-oxime Long-pec spinal cord ependymal cell ISH
AB + MO3-wnt4b + MO4-wnt11 standard conditions Long-pec spinal cord ependymal cell ISH
Long-pec whole organism RTPCR
Antibody Labeling Details
Antibody Assay Fish Conditions Stage Anatomy
Ab1-tuba IHC AB control Long-pec ependymal cell motile cilium
IHC Day 4 ependymal cell motile cilium
IHC AB chemical treatment by environment: thapsigargin Day 4 ependymal cell motile cilium
IHC AB chemical treatment by environment: U-73122 Day 4 ependymal cell motile cilium
IHC AB + MO3-wnt4b + MO4-wnt11 standard conditions Long-pec ependymal cell motile cilium
Ab1-tubg1 IHC AB control Day 4 ependymal cell ciliary basal body
IHC AB chemical treatment by environment: thapsigargin Day 4 ependymal cell ciliary basal body
Phenotype Details
Fish Conditions Stage Phenotype
AB chemical treatment by environment: 6-bromoindirubin-3'-oxime Long-pec spinal cord ependymal cell plcd3a expression increased amount, abnormal
AB chemical treatment by environment: thapsigargin Day 4 ependymal cell ciliary basal body organization normal occurrence, normal
Day 4 ependymal cell motile cilium assembly decreased occurrence, abnormal
Day 4 spinal cord has fewer parts of type ependymal cell motile cilium, abnormal
AB chemical treatment by environment: U-73122 Day 4 ependymal cell motile cilium assembly decreased occurrence, abnormal
Day 4 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 plcd3a expression decreased amount, abnormal
Long-pec spinal cord has fewer parts of type ependymal cell motile cilium, abnormal
Long-pec whole organism plcd3a expression decreased amount, 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.