ZFIN ID: ZDB-FIG-130801-32
Wang et al., 2013 - miR-34b regulates multiciliogenesis during organ formation in zebrafish. Development (Cambridge, England)   140(13):2755-2764 Full text @ Development
ADDITIONAL FIGURES
EXPRESSION / LABELING:
Genes:
Antibody:
Fish:
Condition:
Knockdown Reagents:
Anatomical Terms:
Stage Range: Prim-25 to Protruding-mouth
PHENOTYPE:
Fish:
Knockdown Reagents:
Observed In:
Stage Range: Prim-25 to Protruding-mouth

Fig. 5 miR-34b regulates multiciliogenesis and kidney morphogenesis through cmyb. (A) WISH analysis of cmyb expression at 36 hpf. The arrow and dashed line show the region of MCCs. (B) QRT-PCR analysis of cmyb expression in MCCs that were isolated from 3-dpf Tg(cmyb:EGFP) embryos by flow cytometry. The expression level was normalized to that of β-actin 1. (C) A 2-dpf Tg(cmyb:EGFP) embryo shows enhanced fluorescence in MCCs (arrow) after miR-34b knockdown. (D) Statistical analysis of A and C showing normal versus high expression of cmyb. (E) The percentage of cmyb:EGFP+ cells with cilia bundles was calculated for each embryo and analyzed statistically at 36 hpf. (F,G) A magnified lateral view of 3-dpf embryos after in situ hybridization with rfx2 or pax2 probe shows the distribution of MCCs and the state of proximal tubule convolution. Arrows (F) indicate rfx2+ MCCs. The DT angle was measured. (H) The percentage of cmyb:EGFP+ cells with cilia bundles was calculated for each Tg(hsp70:cmyb) embryo and analyzed statistically at 36 hpf (I) A magnified lateral view of 2-dpf Tg(hsp70:cmyb) embryos after in situ hybridization with rfx2 probe shows the distribution of MCCs. Arrows indicate rfx2+ MCCs. The percentage of embryos with accumulated rfx2+ MCCs (Accumulated) in F and I was calculated. (J) WISH analysis of cmyb expression at 2 dpf in the olfactory placode (arrows). A representative image of a 3-dpf Tg(cmyb:EGFP) embryo shows enhanced fluorescence of the olfactory placode (arrows) after miR-34b knockdown. The number of embryos with normal (top pictures) or high expression level (bottom pictures) of cmyb among all analyzed embryos is indicated (bottom right). (K) The ciliary (anti-acetylated-tubulin staining, red) morphology of the olfactory placode at 3 dpf in normal embryos, miR-34b morphants and embryos injected with both miR-34b MO and cmyb MO. Cilia length was analyzed at three positions (arrows) for each embryo, and the average cilia length is listed. Each circle/triangle/cross represents an embryo. Mean ± s.d. ***P<0.0001 (unpaired t-test). Scale bar: 10 μm.

Gene Expression Details
Gene Antibody Fish Conditions Stage Anatomy Assay
EGFP zf169Tg standard conditions Long-pec pronephros multi-ciliated epithelial cell IFL
Protruding-mouth peripheral olfactory organ IFL
zf169Tg + MO1-mir34b standard conditions Long-pec pronephros multi-ciliated epithelial cell IFL
Protruding-mouth peripheral olfactory organ IFL
myb WT standard conditions Prim-25 pronephros multi-ciliated epithelial cell ISH
Long-pec peripheral olfactory organ ISH
WT + MO1-mir34b standard conditions Prim-25 pronephros multi-ciliated epithelial cell ISH
Long-pec peripheral olfactory organ ISH
pax2a WT standard conditions Protruding-mouth pronephros epithelium ISH
WT + MO1-mir34b standard conditions Protruding-mouth pronephros epithelium ISH
WT + MO1-mir34b + MO2-myb standard conditions Protruding-mouth pronephros epithelium ISH
rfx2 WT standard conditions Protruding-mouth pronephros multi-ciliated epithelial cell ISH
WT + MO1-mir34b standard conditions Protruding-mouth pronephros multi-ciliated epithelial cell ISH
WT + MO1-mir34b + MO2-myb standard conditions Protruding-mouth pronephros multi-ciliated epithelial cell ISH
hkz03tTg standard conditions Long-pec pronephros multi-ciliated epithelial cell ISH
hkz03tTg heat shock Long-pec pronephros multi-ciliated epithelial cell ISH
Antibody Labeling Details
Antibody Assay Fish Conditions Stage Anatomy
Ab1-tuba IHC WT standard conditions Protruding-mouth peripheral olfactory organ cilium
IHC WT + MO1-mir34b standard conditions Protruding-mouth peripheral olfactory organ cilium
IHC WT + MO1-mir34b + MO2-myb standard conditions Protruding-mouth peripheral olfactory organ cilium
Phenotype Details
Fish Conditions Stage Phenotype
WT + MO1-mir34b standard conditions Prim-25 multi-ciliated epithelial cell cilium physical object quality, abnormal
Protruding-mouth multi-ciliated epithelial cell accumulation pronephric duct, abnormal
Protruding-mouth olfactory epithelium cilium shortened, abnormal
Protruding-mouth pronephric proximal convoluted tubule closed, abnormal
WT + MO1-mir34b + MO2-myb standard conditions Prim-25 multi-ciliated epithelial cell cilium normal, normal
Protruding-mouth multi-ciliated epithelial cell accumulation pronephric duct, normal
Protruding-mouth olfactory epithelium cilium shortened, normal
Protruding-mouth pronephric proximal convoluted tubule closed, normal
hkz03tTg standard conditions Prim-25 multi-ciliated epithelial cell cilium physical object quality, abnormal
Long-pec multi-ciliated epithelial cell accumulation pronephric duct, abnormal
Acknowledgments:
ZFIN wishes to thank the journal Development (Cambridge, England) for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ Development