Effects on Differentiation of Embryonic Ventral Midbrain Progenitors by Lmx1a, Msx1, Ngn2, and Pitx3

Maturation and neuronal differentiation of E14.5 rat midbrain progenitors after 8 d in vitro differentiation. A1–D2, In 4 d differentiated cultures, Lmx1a- and Msx1-transduced cells do not completely downregulate the expression of the immature neural marker SOX2 as opposed to Ngn2- and Pitx3-transduced cells. F–J, Transduced progenitors adopt a neuronal morphology and express MAP2 when overexpressing Lmx1a (F) and Ngn2 (H). Neuronal differentiation was not affected when cells were transduced with Msx1 (G) compared with control (J). Cells transduced with Pitx3 do not express MAP2 in combination with the transgene, even after 12 d differentiation (I). K, L, Bar diagrams representing the percentage of SOX2- and MAP2-positive cells of the number of transduced GFP-positive cells, after day 8 of in vitro differentiation. High magnification of the framed areas in A1, B1, C1, D1, and E1 are represented in A2, B2, C2, D2, and E2, respectively. Rectangular images on the bottom and right of A2, B2, C2, D2, and E2 represent a projection of 14 Z-stack images (total of 10–14 μm thick). The crosshairs in A2 were positioned to show a cell transduced (eGFP), positive for β-III-tubulin and negative for SOX2. Scale bars: A1, B1, C1, D1, E1, F–J, 50 μm; A2, B2, C2, D2, E2, 25 μm.

Glial phenotype of cells overexpressing Lmx1a, Msx1, Ngn2, and Pitx3. A–E, Ventral midbrain progenitors express the oligodendrocyte marker CNPase when transduced with Lmx1a (A), Msx1 (B), and control virus (E), but never when transduced with either Ngn2 (C) or Pitx3 (D). F–J, Ventral midbrain progenitors express the astrocyte marker GFAP when transduced with Lmx1a (F), Msx1 (G), and control virus (J), but not when transduced with either Ngn2 (H) or Pitx3 (I). K, L, Bar diagrams representing the percentage of CNPase- and GFAP-positive cells of the number of transduced GFP-positive cells, after day 8 in vitro differentiation. Scale bars, 50 μm.

Glial differentiation after dual transgene delivery. A1–B4, Ventral midbrain progenitors grew as neurospheres were double transduced using control retroviruses that drive the expression of DsRed2 or eGFP reporter genes. Using different MOI for each retrovirus (here 0.5 for DsRed control retrovirus and 1 for eGFP control retrovirus) allow us to confirm the specificity of the resulting fluorescence. C1–H2, Ventral midbrain progenitors coexpressing Lmx1a and Ngn2 (E1, E2) become MAP2-immunopositive neurons after 8 d differentiation. Low neuronal differentiation was achieved when overexpressing both Lmx1a and Msx1 (C1, C2) or Lmx1a and Pitx3 (G1, G2). Dual transgene delivery had not effect on promoting glial differentiation as shown after a combined immunocytochemistry using the same secondary antibody cyanine 5 to reveal the astrocyte and oligodendrocyte markers GFAP (G-) and CNPase (-C). Scale bars, 50 μm.

Ngn2 and Lmx1a but not Msx1 or Pitx3 induce neuronal postmitotic phenotype of ventral mesencephalic progenitors. A, Schematic representation of CldU pulse labeling assay. B1–F2, Cloro-deoxyuridine pulse labeling shows that most of Ngn2-transduced (D1, D2) and Lmx1a-transduced (B1, B2) cells have adopted a postmitotic fate before CldU exposure and express both the transgene (eGFP) and the neuronal marker β-III-tubulin. Important cell division was found in Pitx3-transduced cultures as shown by two dividing cells that had incorporated CldU (E2). Msx1-transduced (C1), Pitx3-transduced (E1), and control virus-transduced (F1) cells highly incorporated CldU. High magnification of the framed area in B1, C1, D1, E1, and F1 are represented in B2, C2, D2, E2, and F2, respectively. G1–K2, Immunocytochemistry for cleaved caspase3 (C-Casp3) shows the overexpression of the four genes and control mediated by the retrovirus is not toxic for the cells and does not induce them to undergo apoptosis. G3, G4, High magnification of the framed areas in G1 and G2, respectively. G1–K1, White arrowheads show cells immunopositive for cleaved caspase3. G3, G4, White arrowhead shows the phenotypic pyknotic nuclei of a cleaved caspase3-immunopositive cell undergoing apoptosis. L, Bar diagram representing the percentage of CldU-immunopositive cells of the number of transduced GFP-positive cells. M, Bar diagram representing the percentage of cleaved caspase3-immunopositive cells of the number of transduced GFP-positive cells after 4 d (black bars) and 8 d (gray bars) differentiation. Rectangular images on the bottom and right of B2–F2 represent a projection of 14, 14, 14, 20, and 14 Z-stack images, respectively (total of 10–14 μm thick). Scale bars: B1–F1, G1–K1, G2–K2, 50 μm; B2–D2, F2, 20 μm; E2, G3, G4, 10 μm.

Neural progenitors isolated from E14.5 rat midbrain have a limited capacity to become dopaminergic neurons in response to Lmx1a, Msx1, Ngn2, or Pitx3 overexpression. A1, A2, Cells immunopositive for TH (red) are the result of spontaneous differentiation of nontransduced eGFP-negative progenitors. A2 represents high magnification of the framed area in F1, showing a TH-immunopositive cell that does not express eGFP. A1 is a nonrepresentative picture showing three TH-immunopositive neurons (white arrowheads) among hundreds of Lmx1a transduced eGFP-positive cells. B1, B2, En1 is not induced by Lmx1a overexpression (<1% of the transduced cells express En1). High magnification of the framed area in B1 is represented in B2. C, RT-PCR shows the ventral midbrain progenitors have the capability to differentiate into dopaminergic neurons regardless of Lmx1a, Msx1, Ngn2, or Pitx3 transgene overexpression. D–H, Msx1-transduced cells express the midbrain dopaminergic subtype marker calbindin at higher frequency 8 d after in vitro differentiation than cultures transduced with either Lmx1a, Ngn2, Pitx3, or control retroviruses (although <1% of the total number of transduced cells). White arrowhead in D–H point out single calbindin-immunopositive cells, either transduced or not. I–M, VMAT2 expression increases in cultures transduced with Ngn2 (K), whereas neither Msx1 (J) nor Pitx3 (L) have such an effect, when compared with control cultures transduced with the empty vector (M). A trend of increase VMAT2 expression was observed in Lmx1a-transduced cultures (I, N). Bar diagrams represent the percentage of VMAT2 (N)-positive cells of the number of GFP-positive cells, after 8 d (black bars) and 12 d (gray bars) of in vitro differentiation. Rectangular images on the bottom and right of A2 represent a projection of 14 stack images on z-axis (total of 14 μm thick). Scale bars: A1, 200 μm; A2, 25 μm; B1, D–M, 50 μm; B2, 10 μm.

Dual transgene delivery does not promote TH expression. A1–D3, Ventral midbrain progenitors infected with Lmx1a virus labeled with the DsRed2 reporter from an internal ribosome entry site (Lmx1a:DsRed) and Msx1 labeled with eGFP reporter (Msx1:eGFP) do not become TH immunopositive. E1–H3, Ventral midbrain progenitors transduced with Lmx1a:DsRed2 and Ngn2:eGFP do not become TH immunopositive. I1–L3, Ventral midbrain progenitors transduced with Lmx1a:DsRed2 and Pitx3:eGFP do not become TH immunopositive. Addition of 1.7 nm SHH (data not shown) or no growth factor did not induce TH expression after 8 d (A1, A2, E1, E2, I1, I2) and 12 d (C1, C2, G1, G2, K1, K2) in vitro differentiation. Addition of both 1.7 nm SHH and 100 ng/ml FGF8 induced TH expression (<1% of the cells), regardless of which transgenes were overexpressed (B1–B3, D1–D3, F1–F3, H1–H3, J1–J3, L1–L3). Right column represents a high-magnification picture of the white frames areas. White arrowheads point at the TH-immunopositive neurons. Scale bar: A1–L2, 100 μm; B3–L3, 50 μm.