Neuronal VEGF-A and miR-9 Expression in Neural Stem Cells (NSCs) Are Conserved in the Human Brain

(A) Schematic representation of the method used to obtain primary human NSCs from fetal brain at 14 weeks of development. At 7 days in culture (A'), a majority of the TUJ1-expressing embryonic neurons express detectable levels of VEGF-A (98.5%, n = 201). NSCs (arrow) and neurons (arrowhead) express VEGF-A in primary human cell culture.

(B and C) Confocal sections of primary human cortical neurons (B) and iPSC-derived human cortical neurons (C) immunolabeled with VEGF-A. Young human cortical neurons show heterogeneous localization of VEGF-A (arrows show cytoplasmic and axonal localization; arrowhead indicates strong nuclear expression).

(D) Confocal section of double in situ/immunolabeling showing overlap in the expression of vegfaa and Tg(huc:egfp) post-mitotic neurons in the retina (arrowheads).

(E) iPSC-derived human cortical spheroid in culture. In situ hybridization showing MIR-9 expression in the ventricular-like zone of human cortical spheroid (E'). Confocal section of double in situ/immunolabeling showing co-localization of MIR-9 and SOX2 in iPSC-derived human NSCs (E'').

(F) Confocal section of double immunolabeling with glutamine synthetase (GS) and EGFP in Tg(hsa-MIR-9-2:egfp) retina at 72 hpf, showing miR-9 expression in retinal NSCs. Confocal section of double in situ/immunolabeling showing overlap in the expression of deltaA and EGFP in Tg(hsa-MIR-9-2:egfp) hindbrain at 72 hpf. Dorsal view of the brain with anterior up. Lateral view of the retina.

Scale bars: 25 μm in (B) and (C); 100 μm in (D), (E'), and (F); 4 mm in (E); and 10 μm in (A') and (E'').

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.

tlx and onecut Are Direct miR-9 Targets Regulating VEGF-A Transcriptional Expression In Vivo

(A) Alignments of vertebrate TLX and ONECUT2 orthologs show strong conservation in the 3′ UTR containing a putative miR-9 binding site.

(B) Schematic representation of the TP assay. In the presence of the TP, the miR-9 binding site is not accessible and the mRNA degradation is inhibited.

(C and D) Schematic representation of the zebrafish tlx (C) and ocl (D) mRNA indicating the sequence of the putative miR-9 binding site in the 3′ UTR (red) and the alignment with miR-9 or TP. tlx and ocl mRNA behave like direct miR-9 targets, showing an increase in the TP context.

(E) Schematic representation highlighting putative Tlx (NAGTCA/purple) (Qu et al., 2010; Yu et al., 2000) and Onecut (NN(A/G)TC(A/C)A(T/G)NN/orange) (Lannoy et al., 1998; Plaisance et al., 2006) binding sites in 1.5 kb of the vegfaa promoter.

(F) Whole-mount in situ hybridization against egfp driven by 1.5 kb of the vegfaa promoter. Embryos were injected with vegfaa:egfp only, hs:tlx, or hs:ocl and were heat-shocked at 7 hpf. Embryos display ectopic expression of the egfp mRNA 2 hr after treatment, showing that Tlx and Onecut have the ability to induce the transcription via 1.5 kb of the vegfaa promoter.

(G and H) Whole-mount in situ hybridization against vegfaa (G) and huc (H) in embryos at 9 hpf. Embryos were injected with hs:tlx or hs:ocl and heat-shocked at 7 hpf. Embryos display ectopic expression of vegfaa, but not huc, 2 hr after treatment, showing that Tlx and Onecut transcription factors have the ability to induce the transcription of the endogenous vegfaa gene independently of the neuronal fate.

(I) Mosaic expression of Tlx-p2A-EGFP or Ocl-p2A-EGFP by heat shock at 8.5 hpf induces endogenous vegfaa expression in as little as 30 min in a cell-autonomous manner, suggesting a direct regulation.

(J) Whole-mount in situ hybridization against vegfaa in embryos at 48 hpf injected with the control MO, miR-9 MO, or miR-9 MO with tlx and ocl MOs. Dorsal view with anterior up. Lateral view of the retina.

Scale bars: 100 μm in (C), (D), (F)–(H), and (J) or 10 μm in (I).

Tlx- and Oc-Dependent Neuronal Expression of VEGF-A Affect Brain Vasculature Development

(A) Whole-mount in situ hybridization against vegfaa at 48 hpf in controls or embryos expressing uas:tlx, uas:ocl, or uas:tlx and uas:ocl in a pan-neuronal manner in Tg(alpha-tubulin:gal4).

(B) Quantification of the number of CtAs found on each hemi-hindbrain in controls (n = 34) or larvae expressing uas:tlx (n = 46), uas:ocl (n = 46) or uas:tlx and uas:ocl (n = 46) in a pan-neuronal manner in Tg(alpha-tubulin:gal4) at 72 hpf.

(C) Whole-mount in situ hybridization against vegfaa in embryos at 48 hpf injected with the control MO, tlx TP, ocl TP, or tlx and ocl TP.

(D) Quantification of the number of CtAs found on each hemi-hindbrain in the control MO (n = 32), tlx TP (n = 34), ocl TP (n = 34), or tlx and ocl TP (n = 34) larvae at 72 hpf.

(E) Neuronal expression of VEGF-A in the Tg(alpha-tubulin:gal4) line leads to a global increase in ECs and/or blood vessel formation in the brain at 72 hpf. While most of the vessels appear to be thicker, the mesencephalic central artery (MMCtA) and retinal blood vessels are reduced or missing (arrow and asterisk in E and E'). In the hindbrain, PHBCs are thicker, while CtA sprouting is reduced, suggesting that their development is affected following neuronal VEGF-A expression (E”).

(F) Quantification of the number of CtAs found on each hemi-hindbrain in controls (n = 30) and larvae expressing vegfaa (n = 20) in neurons at 72 hpf.

(G) During the normal development of the neurovascular system, miR-9 represses the expression of Tlx and Oc to limit the level of neuronally derived VEGF-A. A reduction of miR-9 expression and/or an overexpression of Tlx/Oc increases the level of neuronal VEGF-A and affects the development of the brain vasculature. Dorsal view of the brain with anterior up.

Scale bars: 100 μm in (A), (C), (E), and (E') or 10 μm in (F) and (E”). Error bars represent SD. ^∗p < 0.05; ^∗∗∗p < 0.0005, determined by t test, two-tailed.

Neurons expressing VEGF-A are closely associated with blood vessels (related to Figure 1)

(A) Confocal section of double in situ/immunolabelling with vegfaa and EGFP in the hindbrain of Tg(huc:egfp) line at 48 hpf shows neuronal expression of vegfa in the zebrafish brain. Close up of vegfaa expression in the hindbrain (A'). (B, C) Confocal section of double immunolabelling showing the physical interaction/close proximity between axons (Acetyl Tubulin, green) and blood vessels (red) in Tg(kdrl:mCherry) hindbrain at 36 and 48 hpf. (D-H) Confocal section of double immunolabelling showing the physical interaction/close proximity between axons (Acetyl Tubulin, green) and blood vessels (red) in Tg(kdrl:mCherry) retina at 36 hpf, 48 hpf or in the adult retina. Close up on a blood vessel in the adult retina (H'). Arrowheads show co-localization. Dorsal view of the brain with anterior up. Lateral view of the retina. Scale bars: 100 μm (A and D-H) or 10 μm (A', B, C and H').

miR-9 expression in NSCs in the zebrafish brain (related to Figure 1)

(A, B) Confocal section of double in situ/immunolabelling showing extensive overlap in the expression of endogenous miR-9 and EGFP protein in hindbrain at 72 hpf or in the ventricular zone of the adult zebrafish telencephalon in the Tg(gfap:gal4), Tg(uas:egfp) line, revealing miR-9 expression in embryonic and adult NSCs. (C) Whole-mount in situ hybridization against miR-9 showing the time course of miR-9 expression in the developing embryo at 24, 36 and 48 hpf. Dorsal view of the brain with anterior up. Lateral view of the retina. Scale bars: 10 μm (A, B) or 100 μm (C).

miR-9 controls the neurovasculature formation in a dose dependent manner (related to Figure 2)

(A) Schematic representation of the miR-9 MO binding to microRNA-9. With the control MO, miR-9 is available, allowing the degradation of the mRNA target and revealing the miR-9 expression pattern with the specific miR-9 LNA probe. In presence of the miR-9 MO, miR-9 is bound by the MO, inhibiting the mRNA degradation and the binding of the LNA probe. (B) Whole-mount in situ hybridization against miR-9 at 72 hpf shows that miR-9 knockdown does not affect trunk morphogenesis.mCherry immunolabelling in Tg(kdrl:mCherry) at 72 hpf showing blood vessels formation in the trunk of control or miR-9 morphant larvae. In the trunk, miR-9 knockdown does not affect blood vessels development. (C) Confocal projections of mCherry immunolabelling in Tg(kdrl:mCherry) at 72 hpf showing blood vessels formation in control or miR-9 morphant larvae in the brain. The affected neurovasculature in the midbrain, hindbrain and retina is highlighted by an asterisk. (D, E) Whole-mount in situ hybridization against miR-9 at 72 hpf in larvae injected with the control MO or the miR-9 MO in the retina (D) and brain (E). Confocal projections of mCherry immunolabelling in Tg(kdrl:mCherry) at 72 hpf showing blood vessels formation in control MO and miR-9 MO injected larvae. Higher is the quantity of miR-9 MO injected in the egg, stronger is inhibition of miR-9 expression. The severity of the neurovasculature defects is correlated to the level of miR-9 inhibition. (F) The formation of the hyaloid vasculature in the retina of control MO (n=25) or miR-9 MO dilution series (n=24, 30 and 28 for croissant dilutions respectively) at 72 hpf was classified in four groups and quantified. Group 1: The blood vessels form a complex vascular network. Group 2: The hyaloid vasculature shows a reduced branching complexity. Group 3: In addition to the reduction of the branching, the caliber is affected and some vessels appear to be thicker. Group 4: The hyaloid vasculature is absent. Dorsal view of the brain with anterior up. Lateral view of the retina. Box1 is a close up of the retina. Box2 is a close up of the hindbrain. Box3 is a close up of the hyaloid vasculature in the retina. Scale bars: 10 μm for immunolabelling and 100 μm for whole mount ISH.

Validation of miR-9 targets conserved across vertebrates (related to Figure 3)

(A, B) Whole-mount in situ hybridization against tlx (A) or ocl (B) in larvae at 72 hpf injected with the control MO, the miR-9 MO or the miR-9 RNA mimic. tlx and ocl behave like a miR-9 target showing an increase in the miR-9 knockdown and a decrease in the miR-9 gain of function. (C, D) Schematic representation of the zebrafish oc1 (C) and oc2 (D) mRNA indicating the sequence of the putative miR-9 binding sites in the 3'UTR (red). Whole-mount in situ hybridization against oc1 (C) and oc2 (D) in larvae at 72 hpf injected with the control MO, the miR-9 MO or the miR-9 RNA mimic. onecut family mRNAs behave like miR-9 targets showing an increase in the miR-9 knockdown and a decrease in the miR-9 gain of function. (E, F) Fluorescent sensor assay to test the functionality of miR-9 binding sites in the 3'UTR of oc1 (E) and ocl (F) showing that miR-9 mediates oc1 and ocl inhibition via the 3'UTR. TagRFP and EGFP protein expression in embryos at 24 hpf: embryos were co-injected with the egfp mRNA containing the 3'UTR of oc1 (n=7) or ocl (n=3) fused to the SV40pA and the internal control, tagrfp mRNA (n=8 and 3 respectively) in the presence or absence of miR-9 mimic. The ratio between the level of EGFP and TagRFP proteins fluorescence shows a decrease in EGFP expression, but not TagRFP, in the presence of the miR-9 RNA mimic. Dorsal view of the brain with anterior up. Lateral view of the retina. Scale bars: 100 μm. Error bars represent s.d. *P<0.05, **P<0.001, ***P<0.0005, determined by t-test, two-tailed.

Neural expression of tlx and ocl (related to Figure 3)

(A, B) Whole-mount in situ hybridization showing the time course of tlx (A) and ocl (B) expression in the developing embryo at 24, 48 and 72 hpf. During development, when miR-9 expression becomes broader and stronger after 24 hpf, we observe a correlative decrease in tlx and ocl expression. (C-H) Confocal section of double in situ/immunolabelling with tlx or ocl mRNAs and EGFP in the Tg(gfap:gal4); Tg(uas:egfp) line labelling NSCs in the hindbrain. When miR-9 is not expressed broadly throughout the brain at 24 hpf, we observed an overlap in the expression of tlx and ocl with the NSCs marker GFAP (C, D). At 48 hpf, when miR-9 is strongly expressed in the brain ocl is excluded from the NSCs domain (E, F). At 72 hpf, both tlx and ocl are not detected in NSCs (G, H). Arrowheads show co-localization with EGFP. Dorsal view of the brain with anterior up. Scale bars: 100 μm.

TLX and OC activity control neural stem cells fate (related to Figure 4)

(A) In vitro effect of transfecting primary human embryonic neural stem cells with a control EGFP plasmid, TLXp2A- EGFP or OC2-p2A-EGFP. Overexpression of TLX (n=646) and OC2 (n=528) significantly depletes the existing neural stem cell population in the dish compared to the EGFP control (n=408). Furthermore, in comparison with EGFP transfection (n=344), expression of TLX (n=99) and OC2 (n=69) slightly increases the percentage of primary human NSCs that differentiate into cortical neurons. (B) Triple immunolabelling against TUJ1, EGFP and VEGF-A after transfection of primary human embryonic NSCs with the TLX-p2A-EGFP plasmid (nuclear marker DAPI is in blue). Embryonic cortical neuron express high detectable level of nuclear (arrowhead) and cytoplasmic (arrow) VEGF-A after TLX expression (EGFP). (C) Confocal projection of immunolabelling with endogenous GS protein in the brain of control MO or miR-9 MO injected larvae and larvae expressing uas:tlx or uas:ocl in NSCs using Tg(gfap:gal4) line at 72 hpf. Embryonic NSCs are reduced in the miR-9 depleted and tlx or ocl expressing brain. (D) Whole-mount in situ hybridization against deltaA, ascl1a, ascl1b, neurog1 or huc in embryos at 48 hpf injected with the control MO, miR-9 MO, tlx TP or ocl TP. miR-9 inhibition increases neural progenitor cells (deltaA and ascl1a) and promote a neuronal fate (huc). tlx or ocl mRNA protection also leads to an increase in deltaA+ neural progenitors in the zebrafish brain. Of note, miR-9 morphant shows an increase in deltaA+ and ascl1a+ NPCs but not ascl1b+ or neurog1+ NPCs. Dorsal view, Anterior up. Scale bars: 10 μm (B) or 100 μm (C, D). Error bars represent s.d. *P<0.05, **P<0.001, ***P<0.0005, determined by t-test, two-tailed.

Tlx and Ocl neuronal expression affect vasculature development (related to Figure 4)

(A) Confocal projections of mCherry immunolabelling in Tg(kdrl:mCherry) at 72 hpf showing blood vessels formation in the hindbrain of controls or larvae expressing uas:tlx, uas:ocl or uas:tlx and uas:ocl in a pan-neuronal manner in Tg(alpha-tubulin:gal4) line. (B) Confocal projections of mCherry immunolabelling in Tg(kdrl:mCherry) at 72 hpf showing blood vessels formation in the hindbrain of control MO, tlx TP, ocl TP or tlx and ocl TP larvae. Dorsal view of the brain with anterior up. Scale bars: 10 μm.