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ZFIN ID: ZDB-FIG-161122-25
Katz et al., 2016 - A Nuclear Role for miR-9 and Argonaute Proteins in Balancing Quiescent and Activated Neural Stem Cell States. Cell Reports   17:1383-1398 Full text @ Cell Rep.
ADDITIONAL FIGURES
EXPRESSION / LABELING:
Genes:
Antibodies:
Fish:
Knockdown Reagent:
Anatomical Terms:
Stage Range: Long-pec to Adult
PHENOTYPE:
Fish:
Knockdown Reagent:
Observed In:
Stage: Adult

Fig. 4

miR-9 Is Nuclear in Mature qNSCs and Is Significantly Associated with the Nuclear Localization of the RISC Effector Ago

(A) Representative image of ISH for miR-9 (magenta) and double immunostaining for PCNA (light blue) and GS (green) with nuclear DAPI counterstaining (dark blue) highlighting the presence of miR-9 in the nucleus of qNSCs (yellow arrow) and not in the nucleus of aNSCs (red arrow). Scale bar, 10 μm.

(B) Sections through the medial region of the dorsal telencephalic ventricle of a 48 hpf embryo, a 1 mpf juvenile, and a 3 mpf adult zebrafish illustrating an ISH of miR-9 (magenta) alone (top) and together with DAPI counterstaining in gray (bottom). Scale bar, 5 μm.

(C) Proportion of cells showing strong nuclear localization of Ago (blue) or mostly cytoplasmic localization of Ago (light gray) in miR-9+ versus miR-9 NSCs populations, illustrating the association between nuclear Ago localization and miR-9 expression.

(D) ISH of miR-9 (magenta) and double fluorescent immunostaining for gfap:GFP (green) and Ago (ab57113, light blue) with nuclear DAPI counterstaining (dark blue). Strong nuclear Ago localization is observed in miR-9+ NSCs (yellow arrows) in contrast to cytoplasmic Ago localization in miR-9 NSCs (white asterisk and gray arrow). Scale bar, 20 μm.

(E) Double fluorescent immunostaining for gfap:GFP (green) and Ago (light blue) with nuclear DAPI counterstaining (dark blue) in conditions of miR-9 kd (bottom) when compared to controls (top). NSCs with strong nuclear Ago staining (yellow arrowheads) are lost in miR-9 MO-treated brains, while the nuclear signal for Ago in neuronal cells is not affected by miR-9 kd (white asterisks). Scale bar, 10 μm.

(F) Quantification of the percentage of NSCs displaying a strong nuclear Ago signal 2 dpi of the control or miR-9 vivoMO. ∗∗p < 0.01; one-way ANOVA with Bonferroni post hoc correction. Data are represented as mean ± 95% CI; n = 3 brains per condition.

Gene Expression Details
Gene Antibody Fish Conditions Stage Qualifier Anatomy Assay
GFP mi2001Tg control Adult Not Detected neuronal stem cell nucleus IHC
Adult radial glial cell nucleus IHC
Adult Not Detected telencephalon neuronal stem cell IHC
Adult telencephalon radial glial cell IHC
mi2001Tg standard conditions Adult Not Detected neuronal stem cell nucleus IHC
Adult radial glial cell nucleus IHC
Adult Not Detected telencephalon neuronal stem cell IHC
Adult telencephalon radial glial cell IHC
mi2001Tg + MO1-mir9 standard conditions Adult radial glial cell nucleus IHC
Adult telencephalon radial glial cell IHC
mir9-2 AB standard conditions Long-pec telencephalon ventricular zone ISH
Long-pec ventricular zone cytoplasm ISH
Long-pec Not Detected ventricular zone nucleus ISH
mir9-6 AB standard conditions Days 30-44 telencephalon ventricular zone ISH
Days 30-44 ventricular zone cytoplasm ISH
Days 30-44 Not Detected ventricular zone nucleus ISH
Adult Not Detected neuronal stem cell nucleus ISH
Adult radial glial cell nucleus ISH
Adult Not Detected telencephalon neuronal stem cell ISH
Adult telencephalon radial glial cell ISH
Adult telencephalon ventricular zone ISH
mi2001Tg standard conditions Adult Not Detected neuronal stem cell nucleus ISH
Adult radial glial cell nucleus ISH
Adult Not Detected telencephalon neuronal stem cell ISH
Adult telencephalon radial glial cell ISH
Adult telencephalon ventricular zone ISH
Antibody Labeling Details
Antibody Assay Fish Conditions Stage Qualifier Anatomy
Ab1-ago IHC mi2001Tg control Adult Not Detected neuronal stem cell nucleus
IHC Adult radial glial cell nucleus
IHC Adult Not Detected telencephalon neuronal stem cell
IHC Adult telencephalon radial glial cell
IHC mi2001Tg standard conditions Adult Not Detected neuronal stem cell nucleus
IHC Adult radial glial cell nucleus
IHC Adult Not Detected telencephalon neuronal stem cell
IHC Adult telencephalon radial glial cell
IHC mi2001Tg + MO1-mir9 control Adult radial glial cell nucleus
Ab1-glul IHC AB standard conditions Adult telencephalon neuronal stem cell
IHC Adult telencephalon radial glial cell
IHC Adult telencephalon ventricular zone
Ab2-pcna IHC AB standard conditions Adult neuronal stem cell nucleus
IHC Adult Not Detected radial glial cell nucleus
IHC Adult telencephalon neuronal stem cell
IHC Adult Not Detected telencephalon radial glial cell
Phenotype Details
Fish Conditions Stage Phenotype
mi2001Tg + MO1-mir9 control Adult radial glial cell nucleus Ab1-ago labeling decreased amount, abnormal
Acknowledgments:
ZFIN wishes to thank the journal Cell Reports for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ Cell Rep.