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ZFIN ID: ZDB-FIG-170921-15
Madelaine et al., 2017 - MicroRNA-9 Couples Brain Neurogenesis and Angiogenesis. Cell Reports   20:1533-1542 Full text @ Cell Rep.
ADDITIONAL FIGURES
EXPRESSION / LABELING:
Genes:
Fish:
Condition:
Knockdown Reagent:
Anatomical Terms:
Stage Range: Long-pec to Protruding-mouth
PHENOTYPE:
Fish:
Condition:
Knockdown Reagent:
Observed In:
Stage Range: Long-pec to Protruding-mouth

Fig. 2

miR-9 Controls Brain Vasculature Development by Limiting VEGF-A Expression

(A) Schematic representation of the miR-9 MO and miR-9 RNA mimic effects. In the presence of the miR-9 MO, miR-9 is paired with the MO, inhibiting the mRNA degradation. The miR-9 RNA mimic is ubiquitously expressed to allow the degradation of the miR-9 targets.

(B) Whole-mount in situ hybridization against vegfaa in embryos at 48 hpf. vegfaa mRNA behaves like a miR-9 target, showing an increase in the miR-9 knockdown and a decrease in the miR-9 gain of function.

(C and D) Whole-mount in situ hybridization against miR-9 in control (C) and miR-9 morphant (D) shows that miR-9 knockdown does not affect brain and eye morphogenesis at 72 hpf. In miR-9 morphant Tg(kdrl:mCherry) brain at 72 hpf, the mesencephalic central artery (MMCtA), hindbrain and retinal blood vessels are affected (asterisks in D). In the hindbrain, primordial hindbrain channels (PHBCs) are thicker, and central arteries (CtAs) are disorganized and thinner and show aberrant multidirectional sprouting.

(E) Confocal projections of EGFP labeling in Tg(kdrl:egfp) at 72 hpf showing ECs (E'; EGFP+/DAPI+) in posterior PHBCs in control or miR-9 morphant larvae.

(F) Quantification of the size, in microns, of PHBCs (the region of interest [ROI] indicated in E) (control, n = 33; or miR-9 MO, n = 28) and the total number of ECs recruited to form the posterior PHBCs (control, n = 12; or miR-9 MO, n = 10) at 72 hpf.

(G) Quantification of the number of CtAs found on each hemi-hindbrain in the control (n = 20) or the miR-9 MO dilution series (ns = 34, 28, and 56 for croissant dilutions) at 72 hpf.

(H and I) Tg(kdrl:mCherry) brain at 72 hpf showing blood vessel formation in the hindbrain (H), and quantification of the number of CtAs found on each hemi-hindbrain (I), of control larvae (n = 40), SU5416-treated larvae (0.06 μM; n = 48), miR-9-morphant larvae (n = 48), and miR-9-MO larvae treated with 0.06 μM SU5416 (n = 30). Dorsal view of the brain with anterior up. Lateral view of the retina.

Scale bars: 100 μm for in situ hybridization (ISH) in (B)–(D) and 10 μm for immunolabeling in (C)–(E') and (H). Error bars represent SD. ∗∗∗p < 0.0005, determined by t test, two-tailed; n.s, not significant.

Gene Expression Details
Gene Antibody Fish Conditions Stage Anatomy Assay
GFP la116Tg control Protruding-mouth basilar artery IHC
Protruding-mouth central artery IHC
Protruding-mouth primordial hindbrain channel IHC
la116Tg + MO1-mir9 standard conditions Protruding-mouth basilar artery IHC
Protruding-mouth central artery IHC
Protruding-mouth primordial hindbrain channel IHC
mCherry s896Tg control Protruding-mouth basilar artery IHC
Protruding-mouth central artery IHC
Protruding-mouth middle mesencephalic central artery IHC
Protruding-mouth posterior communicating artery IHC
Protruding-mouth primordial hindbrain channel IHC
Protruding-mouth retina blood vessel IHC
s896Tg chemical treatment by environment: semaxanib Protruding-mouth central artery IHC
Protruding-mouth primordial hindbrain channel IHC
s896Tg + MO1-mir9 control Protruding-mouth basilar artery IHC
Protruding-mouth central artery IHC
Protruding-mouth middle mesencephalic central artery IHC
Protruding-mouth posterior communicating artery IHC
Protruding-mouth primordial hindbrain channel IHC
Protruding-mouth retina blood vessel IHC
s896Tg + MO1-mir9 chemical treatment by environment: semaxanib Protruding-mouth central artery IHC
Protruding-mouth primordial hindbrain channel IHC
vegfaa WT control Long-pec brain ISH
Long-pec retina ISH
WT + MO1-mir9 standard conditions Long-pec brain ISH
Long-pec retina ISH
Antibody Labeling Details No data available
Phenotype Details
Fish Conditions Stage Phenotype
WT + MO1-mir9 standard conditions Long-pec brain vegfaa expression increased amount, abnormal
Long-pec retina vegfaa expression increased amount, abnormal
la116Tg + MO1-mir9 standard conditions Protruding-mouth central artery vascular sprouts increased amount, abnormal
Protruding-mouth primordial hindbrain channel increased thickness, abnormal
Protruding-mouth primordial hindbrain channel endothelial cell increased amount, abnormal
s896Tg chemical treatment by environment: semaxanib Protruding-mouth central artery vascular sprouts decreased amount, abnormal
s896Tg + MO1-mir9 control Protruding-mouth brain blood vessel malformed, abnormal
Protruding-mouth central artery decreased thickness, abnormal
Protruding-mouth central artery disorganized, abnormal
Protruding-mouth central artery vascular sprouts decreased amount, abnormal
Protruding-mouth central artery vascular sprouts disorganized, abnormal
Protruding-mouth hindbrain blood vessel malformed, abnormal
Protruding-mouth middle mesencephalic central artery malformed, abnormal
Protruding-mouth primordial hindbrain channel increased thickness, abnormal
Protruding-mouth retina blood vessel malformed, abnormal
s896Tg + MO1-mir9 chemical treatment by environment: semaxanib Protruding-mouth central artery vascular sprouts amount, ameliorated
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.