Adusumilli et al., 2020 - miR-7 Controls the Dopaminergic/Oligodendroglial Fate through Wnt/β-catenin Signaling Regulation. Cells   9(3) Full text @ Cells

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Figure 1

miR-7 negatively regulates TCF7L2, encoding the Wnt/β-catenin transducer, and the basic helix–loop–helix (bHLH) genes TCF4 and TCF12. (A) Various seed match regions (7_m8, 6mer*, or 8mer types) for miR-7 are detectable in the human (h), mouse (m), and zebrafish (z) 3′UTRs of Tcf4 and Tcf12 transcripts. A seed match region for miR-7 is detectable in the 5′UTR of Tcf7l2 transcripts in the three species; an additional 7_A1 seed region is detectable in the 3′UTR of the zebrafish tcf7l2 transcript. (B) TCF4 and TCF12 are down regulated by miR-7, following miR-7 over-expression (miR-7 duplex (7DP)). PAX6 is used as a positive control being a known target of miR-7. Normalization was done to GAPDH and presented as fold change ± SEM (n ≥ 3) relative to the expression of control duplex NCDP; (*) p < 0.05 and (**) p < 0.01. (C) Luciferase reporter assay: the 3′UTR region of TCF12 and the 3′UTR of b-fragment of TCF4 were cloned downstream of the Renilla luciferase gene in the psiCheck2 vector. Repression of the luciferase activity was observed for miR-7 miRNA response element (MRE) (75%), TCF4 3′UTR (40%), and TCF12 3′UTR (~40%). Every Renilla luciferase reading was normalized to that of the control firefly luciferase. The luciferase activities of miR-7 transfected cells were presented as percentage relative to the level of luciferase in NCDP transfected cells (this control luciferase level is considered as 100%). The values represent average ± SEM (n ≥ 8). Two-tail t-test results are indicated by (**) p < 0.01 and (*) p < 0.05, relative to NCDP. (D) miR-7 represses the endogenous TCF7L2 protein levels in neural progenitor cells 48 h after transfection with 7DP at two tested doses: 50 nM and 80 nM (control duplex: NCDP50). Antisense down-regulation of miR-7 (7AS-100) leads to increased levels of TCF7L2 protein, compared with control antisense (NCAS-100) and untransfected controls (analysis at 96 h after transfection). (E) The TCF7L2 protein levels were quantified from the Western blot bands, normalized to the β-actin (ACTB) levels and presented as fold change ± SEM (n ≥ 3). Two tailed t-tests are indicated by (*) p < 0.05 and (**) p < 0.01.

Figure 2

miR-7 negatively regulates Wnt signaling in zebrafish embryos and HEK293T cells. (A,B) miR-7 knockdown in zebrafish (mir7a MO) resulted in an increase of Wnt reporter expression in the ventral brain (B, green signals indicated by the white arrowhead) when compared with embryos injected with the control mismatch morpholino (mismMO) (A). Both panels display lateral views of 42 hpf Wnt:GFP transgenic embryos, anterior to the left; di: diencephalon. (C) HEK293T cells were co-transfected with TOPflash vector, NCDP, or 7DP, as well as Renilla luciferase control vector (pRL-TK). Similarly, the FOPflash vector (with mutated Tcf binding sites) was co-transfected with NCDP and 7DP, as well as pRL-TK. Luciferase activity was measured at 48 h post-transfection. About a 60% reduction in luciferase activity was observed in the TOPflash vector, whereas not much significant change was observed in the FOPflash vector. Luciferase readings were normalized to the co-transfected control Renilla luciferase vector pRL-TK plasmid. The error bars represent luciferase activity average ± SEM, where the experiment was done four times with two biological replicates each time (n = 6). Two-tailed t-tests are described as (**) where p < 0.01, and (*) where p < 0.05.

EXPRESSION / LABELING:
Gene:
Fish:
Knockdown Reagent:
Anatomical Term:
Stage: High-pec
PHENOTYPE:
Fish:
Knockdown Reagent:
Observed In:
Stage: High-pec

Figure 3

miR-7 knockdown increases Wnt signaling in the zebrafish diencephalon. (A,B,C) Morpholino-mediated knock-down of miR-7 (mature form) increases Wnt-reporter GFP (Green Fluorescent Protein) in the diencephalon (arrowheads) of miR-7 morphants (B) compared with not injected (A) and mismatched MO-injected (C) controls. Increased Wnt-reporter GFP is confirmed in loop7a1/2/3 (immature miR-7) morphants (D). (E,F) Two-color in situ hybridization for id3 (landmark) and Wnt:gfp mRNAs shows that most of the diencephalic Wnt-responsive cells (arrowheads) are located in the ventral posterior tuberculum (PTv) and hypothalamic (H) regions of 44 hpf embryos. Loop7a1/2/3 morphants display increased id3 and gfp expression, compared with controls. All panels show 44 hpf embryonic heads in lateral view, from anterior to the left. Tel: telencephalon; T: tectum; v: vessels. (G,H,I) Quantitative analysis of Wnt reporter in the zebrafish ventral diencephalon. Chart G refers to A–D experimental series; charts H and I refer to E–F experiments. Error bars represent ± SEM. The experiments were repeated thrice. Sample size n = 5 for quantification using Volocity software. Unpaired t-test results are indicated by (**) p < 0.01 and (***) p < 0.001. All panels zoom on the dorsal diencephalic (dd) region of 2 dpf embryos in lateral view, from anterior to the left. Displayed images represent the average phenotype from batches of n = 20 embryos per condition; the experiment was performed in duplicate.

Figure 4

miR-7 gene editing decreases miR-7 expression and increases Wnt signaling in the zebrafish diencephalon. (A) Position of the three Crispr/Cas9 guides used to perform gene editing in the zebrafish miR-7a locus. (B) Agarose gel electrophoretic analysis of the Crispr/Cas9-targeted region in the miR-7a gene, PCR-amplified from genomic DNA of control (ctrl) or guide-injected (g) embryos. Smears indicate Crispr/Cas9-induced gene modification. M: size marker; nc: PCR negative control. (CE) Decreased miR-7 expression is observed in crispants injected with the gU + gI1 Crispr/Cas9 guides, compared with the control (ctrl). Signal quantification (R.I.: relative intensity) is shown in E; (***), p < 0.001, n = 6. (FH) Analysis of diencephalic Wnt signaling (green cells within the white dashed ellipse) in the control (ctrl, F) and guide-injected (gU + gI1, G) Wnt:GFP transgenic embryos (G). Signal quantification is shown in (H). The highest Wnt signal increase (80% more than the control level) is reached with the gU + gI1 guide combination; (*) p < 0.05 and (***) p < 0001; (n.s.), not significant; n = 6. All embryonic heads are at 2 dpf and in dorsal (C,D) or lateral (F,G) view, with anterior to the left. te: telencephalon; di: diencephalon.

Figure 5

A subset of diencephalic Wnt-responsive cells express the dopaminergic (DA) marker tyrosine hydroxylase (TH). (A,B) Virtual anatomical reconstruction of a 72 hpf zebrafish brain, generated by ViBE-Z software, merging Wnt:GFP (green) and tyrosine hydroxylase (th, magenta) expression domains, compared with reference anatomical areas (blue lines). Virtual co-localizations (yellow arrowheads) are detected at the boundary between the ventral posterior tuberculum (PTv) and hypothalamus (H). (A,B) are dorsal and lateral views, respectively, with the anterior to the left, across the PTv/H boundary (section planes shown by white lines in the insets). (C,D) Co-localization of th-positive and Wnt-responsive areas (yellow arrowheads) is confirmed by dual color in situ hybridization in 24 and 48 hpf embryos. (C,D) are dorsal and lateral head views, respectively, with the anterior to the left.

Figure 6

miR-7a negatively regulates Wnt signaling and diencephalic th-positive cells in the zebrafish brain (AG’’): The knock-down of miR-7a activity by two independent morpholinos, targeting the immature and mature forms (loop7a1/2/3 MO, shown in E,E’,E’’, and mir7a MO, shown in F,F’,F’’) increases the amount of th-positive brain cells (in green) and the activity of a Wnt reporter (in red), compared with controls not injected (A,A’,A’’), co-injected with non-functional morpholino (mismMO) + non-functional miR-7a (NCDP) (B,B’,B’’), or co-injected (rescued) either with loop7a1/2/3 MO + 7DP (C,C’,C’’) or with miR-7a MO + 7DP (D,D’,D’’). On the contrary, over-expression of 7DP reduces the amount of th-positive brain cells and the activity of the Wnt reporter (G,G’,G’’). All pictures show the head region of 40 hpf embryos in lateral view, with the anterior to the left. te: telencephalon; di: diencephalon; hi: hindbrain. (H,I): Charts showing the th-positive cell counting (H), and the measure of Wnt reporter activity (I), expressed as fluorescence relative intensity (R.I.), in the seven conditions. Sample size n = 5 measures/condition; (*) p < 0.05; (**) p < 0.01; (***) p < 0.001.

Figure 7

miR-7 negatively regulates the number of TH+ DA neurons derived from H9NPC cells. (A,B,C) Control H9NPC cells (A) showing TH staining at day 10 of DA neurogenesis (schematized in the top of the figure). BDNF: Brain Derived Neurotrophic Factor; GDNF: Glial cell Derived Neurotrophic Factor; cAMP: cyclic Adenosine MonoPhosphate; TGFβ3: Transforming Growth Factor beta 3. Decrease of TH+ cells (red) was observed when cells were transfected with miR-7 duplex (C), compared with the control duplex (B) and untransfected cells (A). (D,E) Increase of TH+ cells was observed upon knockdown of miR-7 (E), when compared with the control antisense. (F) Ratio of TH+/TUJ1 cells was quantified by manual counting of the neurons from images captured from 10 different areas on a random basis. Error bars represent average% ± SEM; the experiment was repeated thrice with n = 3 different biological replicates; (**) p < 0.01.

Figure 8

miR-7 positively regulates Shh signaling responsiveness and olig2+ cells in the zebrafish diencephalon. (AD) Shh:GFP reporter embryos, injected with NCDP and mismatch morpholino, were used as controls (A). Injection of miR-7 7DP (B) or miR-7a morpholino alone (D) elicits opposite effects on Shh-responsive cells located in the diencephalic region, between the telencephalic-diencephalic boundary (TDB), the diencephalic-mesencephalic boundary (DMB), and the hypothalamus (H), compared with injected controls (A). Co-injection of 7DP and miR-7a MO rescues both conditions (C). All panels show the head region of 60 hpf embryos in lateral view, with the anterior to the left. (E) Chart refers to A–D experimental series. Error bars represent SEM; the experiments were repeated thrice. Sample size n = 5 measures/condition for quantification using Volocity software; (***) p < 0.001. (FI) olig2:EGFP embryos, injected with NCDP and mismatch morpholino, were used as controls (F). Telencephalic (te), diencephalic (di) and hindbrain (hi) expression of olig2-dependent EGFP appears increased in embryos overexpressing miR-7 7DP (G), and reduced in miR-7a morphants (I). Co-injection of miR-7a MO and 7DP rescues the phenotype (H). All panels are lateral views of the head region at 40 hpf, with the anterior to the left. (J) Chart refers to F–I experimental series. Error bars represent SEM; the experiments were repeated thrice. Sample size n = 5 measures/condition for quantification using Volocity software; (*) p < 0.05; (**) p < 0.01.

Figure 9

miR-7 regulates Wnt/Shh responsiveness and the balance between DA neurons and glia. The model summarizes the in vivo (zebrafish) and in vitro (human cells) findings from this work on miR-7 function, showing the direct regulation of Wnt signaling (by the Wnt-transducer TCF7L2) and possibly indirect regulation of Shh responsiveness, with consequences on the relative amount of DA neurons and glial cells generated during neural progenitor differentiation.

Acknowledgments:
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