ZFIN ID: ZDB-FIG-200426-18
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:
Knockdown Reagent:
Anatomical Terms:
Stage: Long-pec
PHENOTYPE:
Fish:
Knockdown Reagent:
Observed In:
Stage Range: Long-pec to Day 4

Fig. 3 Cx43 is implicated in the maintenance of ependymal motile cilia.

a WISH of a WT embryo at 2 dpf with cx43 riboprobes (left). Lateral view anterior to the left. Right panel shows the cross-section image of the SC ventral to the bottom. Arrowhead indicates ECs. Scale bars = left: 650 μm; right: 20 μm. b Embryos at one-cell stage were microinjected with cx43 MO and imaged at 2 dpf. Lateral view anterior to the left. Scale bar = 650 μm. c Embryos at one-cell stage were microinjected with either control MO or cx43 MO or cx43 MO + mouse Cx43 mRNA, and IF stained with anti-acetylated α-tubulin antibody at 2 dpf. Arrowheads represent spinal motile cilia. Dorsal view anterior to the left. Scale bar = 20 μm. dTg(bactin2:Arl13b-GFP) embryos expressing GFP in motile cilia were microinjected at one-cell stage with either control (CO) MO or cx43 MO and subjected to time-lapse imaging for 2 min (6.7 frames s−1) with an intravital multiphoton microscope. Dorsal view anterior to the left. Scale bar = 7.5 μm. e Embryos at one-cell stage were microinjected with control MO or cx43 MO and Qdots were microinjected into the hindbrain ventricles of larvae at 4 dpf, which were then imaged at 10 min and 60 min after the microinjection. Dashed lines indicate migration of Qdots. Arrowheads mark the caudal end of Qdot flow. Lateral view anterior to the left. Scale bar = 650 μm. Insets represent magnifications of the dotted areas. CO: Control. f Quantification of Qdot migration distance in the larvae shown in d. Mean ± SD. ***P < 0.001 by two-tailed unpaired Student’s t test (n = 3 larvae per group). g Embryos at one-cell stage were microinjected with either control MO or cx43 MO and immunostained with anti-γ-tubulin antibody at 2 dpf. Arrowheads indicate motile cilia. Dorsal view anterior to the left. Scale bar = 20 μm. hTg(foxj1a:eGFP) embryos expressing GFP in ECs were microinjected with either control MO or cx43 MO and imaged at 2 dpf. Arrowheads represent spinal ECs. Dorsal view anterior to the left. Scale bar = 650 μm.

Gene Expression Details
Gene Antibody Fish Conditions Stage Anatomy Assay
EGFP hsc16Tg control Long-pec spinal cord ependymal cell IFL
hsc16Tg + MO6-gja1b standard conditions Long-pec spinal cord ependymal cell IFL
GFP hsc5Tg control Long-pec ependymal cell motile cilium IFL
hsc5Tg + MO6-gja1b standard conditions Long-pec ependymal cell motile cilium IFL
gja1b AB standard conditions Long-pec spinal cord ependymal cell ISH
Antibody Labeling Details
Antibody Assay Fish Conditions Stage Anatomy
Ab1-tuba IHC AB control Long-pec ependymal cell motile cilium
IHC AB + MO6-gja1b standard conditions Long-pec ependymal cell motile cilium
Ab1-tubg1 IHC AB control Long-pec ependymal cell ciliary basal body
IHC AB + MO6-gja1b standard conditions Long-pec ependymal cell ciliary basal body
Phenotype Details
Fish Conditions Stage Phenotype
AB + MO6-gja1b standard conditions 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
Day 4 central canal cerebrospinal fluid circulation decreased process quality, abnormal
hsc16Tg control Long-pec spinal cord has normal numbers of parts of type ependymal cell, normal
hsc16Tg + MO6-gja1b standard conditions Long-pec spinal cord has normal numbers of parts of type ependymal cell, normal
hsc5Tg + MO6-gja1b standard conditions Long-pec ependymal cell cilium movement decreased occurrence, 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.