ZFIN ID: ZDB-FIG-200426-19
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:
Gene:
Antibody:
Fish:
Conditions:
Knockdown Reagent:
Anatomical Terms:
Stage: Long-pec
PHENOTYPE:
Fish:
Conditions:
Knockdown Reagent:
Observed In:
Stage: Long-pec

Fig. 4 The electrochemical coupling function of Cx43 plays an important role in the maintenance of ependymal motile cilia.

a Domain structure of Cx43. TM, transmembrane. b, d Embryos at one-cell stage were microinjected with control MO, cx43 MO or cx43 MO + mRNA encoding mouse Cx43 lacking the C-terminal domain (b) or cx43 MO + mRNA that encodes mouse mutant Cx43 lacking the channel function (d), and IF stained with anti-acetylated α-tubulin antibody at 2 dpf. Arrowheads represent motile cilia. Dorsal view anterior to the left. Scale bar = 20 μm. c, e Quantification of the number of ependymal motile cilia in b, d, respectively. Mean ± SD. **P < 0.01 and ****P < 0.0001 by one-way ANOVA with Tukey’s HSD post hoc test: cn = 20 embryos per each group; en = 10 embryos per each group except cx43 MO + cx43 T154A mRNA (n = 7 embryos). ns, not significant. f Embryos were treated with carbenoxolone (1 μM) at 18–48 hpf and immunostained with anti-acetylated α-tubulin antibody at 2 dpf. Arrowheads point to motile cilia. Dorsal view anterior to the left. Scale bar = 20 μm. g Embryos at one-cell stage were microinjected with cx43 MO, treated with either DMSO (vehicle control) or BIO (5 μM) at 12–48 hpf, and IF stained with anti-acetylated α-tubulin antibody at 2 dpf. Arrowheads represent motile cilia. Dorsal view anterior to the left. Scale bar = 20 μm. h Quantification of the number of ependymal motile cilia per frame in embryos in g. Mean ± SD. ****P < 0.0001 by two-tailed unpaired Student’s t test (cx43 MO + DMSO: n = 18 embryos; cx43 MO + BIO: n = 20 embryos; one frame per embryo). iTg(foxj1a:GCaMP6s) embryos expressing a calcium indicator (GCaMP6s) in ECs were microinjected at one-cell stage with control MO or cx43 MO, and cx43 morphants were treated with BIO (5 μM) at 12–48 hpf. Subsequently, they were subjected to time-lapse imaging for 3 min (20 frames/min) with a confocal microscope. Dorsal view anterior to the left. Scale bar = 20 μm. j The GFP fluorescence intensity in ten cells (1–10) in each embryo in i was individually assessed at 0 (F0), 60 (F60), 120 (F120) and 180 s (F180), and presented as F60/F0, F120/F0 and F180/F0.

Gene Expression Details
Gene Antibody Fish Conditions Stage Anatomy Assay
GCaMP cms5Tg standard conditions Long-pec spinal cord ependymal cell IFL
cms5Tg + MO6-gja1b standard conditions Long-pec spinal cord ependymal cell IFL
Antibody Labeling Details
Antibody Assay Fish Conditions Stage Anatomy
Ab1-tuba IHC AB control Long-pec ependymal cell motile cilium
IHC AB standard conditions Long-pec ependymal cell motile cilium
IHC AB chemical treatment by environment: Carbenoxolone sodium Long-pec ependymal cell motile cilium
IHC AB + MO6-gja1b standard conditions Long-pec ependymal cell motile cilium
IHC AB + MO6-gja1b chemical treatment by environment: 6-bromoindirubin-3'-oxime Long-pec ependymal cell motile cilium
Phenotype Details
Fish Conditions Stage Phenotype
AB chemical treatment by environment: Carbenoxolone sodium 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 + MO6-gja1b 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 + MO6-gja1b chemical treatment by environment: 6-bromoindirubin-3'-oxime Long-pec ependymal cell motile cilium assembly occurrence, ameliorated
Long-pec spinal cord has number of ependymal cell motile cilium, ameliorated
cms5Tg + MO6-gja1b standard conditions Long-pec ependymal cell calcium-mediated signaling decreased occurrence, abnormal
Long-pec spinal cord ependymal cell GCaMP expression decreased amount, abnormal
cms5Tg + MO6-gja1b chemical treatment by environment: 6-bromoindirubin-3'-oxime Long-pec ependymal cell calcium-mediated signaling occurrence, ameliorated
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.