Fig 5. Forced expression of ND1 in at embryonic stage induces complete ectopic differentiation of OB neurons in the SVZ.

A- Cartoon illustrating the approach used for in utero delivery of ND1, Ngn2 and GFP. B- Absence of cells transduced with ND1 retrovirus in the RMS 3 weeks post-injection revealed by immunohistochemistry for GFP. Right panels represent higher magnification images of areas framed on left panels. Insets show DAPI staining (in blue). C- Rare GFP⁺ cells are observed in the OB of animals injected with ND1 retrovirus compare to animals injected with Ngn2 (107 ± 3.1 cells) and control retrovirus (97 ± 6.4 cells). D- Bilateral protrusions in the SVZ revealed by DAPI staining on coronal sections of a 2-week old animal injected with ND1 retrovirus. Black arrowheads pinpoint at the bulges. E- Total number of cells in the bulge of animals injected with ND1 (106453 ± 11715 cells) and animals injected with Ngn2 (43621 ± 4566 cells) retroviruses. F- Absence of ND1 transduced cells outside the bulge revealed by immunohistochemistry for GFP. G- Anatomical structure of the bulge revealed by immunohistochemistry for GFP and NeuN on sagittal and coronal sections; DAPI staining (in blue) on serial coronal sections shows the bulge specifically locates in the SVZ region. H- Transduced cells located in the bulge express ND1 and GFP. I- Absence of nestin⁺/GFP⁺ cells in the bulge of ND1 transduced animals indicates transduced SVZ progenitors have matured. J- Nearly all SVZ neural progenitors tranduced with ND1 virus (99.1% ± 0.3%) become NeuN⁺ neurons. SVZ neural progenitors tranduced with Ngn2 virus become NeuN⁺ neurons (78.6% ± 2.3%) and GFAP⁺ astrocytes (22.4% ± 2.5%). K- None of the SVZ neural progenitors transduced with ND1 (and Ngn2; data not shown) become DARPP32⁺ striatal medium spiny neurons. L- Neurites of ND1-transduced neurons (bright GFP⁺ cells) stain immune-positive for VGluT2. M- SVZ neural progenitors transduced with ND1 differentiate into NPY⁺ (7.4% ± 1.2%), ParV⁺ (3.4% ± 0.3%), CalR⁺ (30.9% ± 2%) and CalB⁺ (13.9% ± 2.4%) neurons. Forced expression of ND1 in SVZ progenitors accelerate neuronal differentiation without modifying neuronal identity (here shown for CalR neurons). Like ND1, Ngn2 did not modify neuronal identity. A to M- Hip = hippocampus, VM = ventral mesencephalon, MGE = medial ganglionic eminence, Cc = corpus callosum, Str = striatum, Sp = septum, B = bulge, SVZ = subventricular zone, MCL = mitral cell layer, RMS = rostral migratory stream, GL = glomeruli, EPL = external plexiform layer, V = ventricle, OB = olfactory bulb. Values shown as mean ± s.e.m., n = 3 animals. Scale bars: 25 μm (H, J, K and M), 50 μm (K, B and C) and 100 μm (G).