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First published online May 24, 2006
doi: 10.1242/10.1242/jcs.02955

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Differential actions of the proneural genes encoding Mash1 and neurogenins in Nurr1-induced dopamine neuron differentiation

Chang-Hwan Park^1,2,*, Jin Sun Kang^1,2,*, Jae-Sang Kim³, Seungsoo Chung⁴, Jin-Young Koh⁴, Eun-Hye Yoon^1,2, A. Young Jo^2,5, Mi-Yoon Chang^2,5, Hyun-Chul Koh^2,6, SeJin Hwang⁷, Haeyoung Suh-Kim⁸, Yong-Sung Lee^2,5, Kwang-Soo Kim⁹ and Sang-Hun Lee^2,5,

¹ Department of Microbiology, Hanyang University, Seoul 133-791, Korea
² Institute of Mental Health, Hanyang University, Seoul 133-791, Korea
³ Division of Molecular Life Sciences, Ewha Womans University, Seoul, 120-750, Korea
⁴ Department of Physiology, College of Medicine, Yonsei University, Seoul 120-752, Korea
⁵ Department of Biochemistry and Molecular Biology, Hanyang University, Seoul 133-791, Korea
⁶ Department of Pharmacology, Hanyang University, Seoul 133-791, Korea
⁷ Department of Anatomy and Cell Biology, College of Medicine, Hanyang University, Seoul 133-791, Korea
⁸ Department of Anatomy and Brain Disease Research Center, College of Medicine, Ajou University, Suwon 442-749, Korea
⁹ Molecular Neurobiology Laboratory; McLean Hospital/Harvard Medical School, Belmont, MA, 02478, USA

View larger version (118K): [in a new window]   Fig. 1. Expression of Mash1 and Ngn2 in the developing ventral midbrain. (A) Embryonic brains were cryosectioned along the red (B-D,G-H) and blue (E,F) lines, and the immunohistochemical images were taken from the areas in red (B-D,G-H) and blue (E,F) boxes with confocal microscopy. (B-D) Localization of Mash1 expression in AHD2-positive DA progenitor cells in the ventral midbrains at E11 (B,C) and E12 (D). The boxed area of B is magnified in C. Panel C shows micrographs of the z-stacked confocal picture along the y-axis (right) and the x-axis (upper). (E,F) Expression patterns of Mash1 and TH in the E13 ventral midbrain. Note that TH expression is confined to the mantle region in the ventral midbrain at E13, whereas Mash1 is expressed in the proliferating ventricular region. Panel F is a higher magnification image of the white boxed area in E. The expression pattern of Ngn2 in the embryonic ventral midbrain is shown in panels G-I. Ngn2 expression is not detected in the ventral midbrain at E12 (G). Inset, Ngn2 expression in the diencephalic area at E12 (positive control). Ngn2-expressing cells are detected in the ventral midbrain at E13 (H,I) and a subpopulation of AHD2-positive cells co-express Ngn2 (I). Bars, 10 µm.

View larger version (60K): [in a new window]   Fig. 2. Mash1-induced neuronal differentiation of Nurr1-TH cells. Mash1 or LacZ (control) was co-expressed with Nurr1 in the precursor cells isolated from rat embryonic cortices using a retroviral vector system. Differentiation of the precursors was induced and immunocytochemical, immunoblot and RT-PCR analyses were performed at day 3 (A-D,H) and at day 15 (E-G) of differentiation. (A,B) Representative images of TH-immunoreactive cells in the cultures transduced with Nurr1 and LacZ (A) and Nurr1 and Mash1 (B) at day 3 of in vitro differentiation. Insets, DAPI nuclear staining of the same field. (C) Morphometric analyses to compare the dendritic lengths of Nurr1-TH cells in the control and Mash1-transduced cultures. Box and bar in the graph C represent mean and s.e.m. of total dendritic lengths, respectively. (D) Western blot analyses combined with the findings in Fig. 5R,S demonstrate that Mash1 increased expression of generic neuronal marker TuJ1, without any effect on the Nurr1-induced TH expression. (E-G) Localization of the mature neuronal marker MAP2 in Nurr1-induced TH+ cells. Representative images of MAP2 and TH immunocytochemistry of control (E) and Mash1-transduced (F) cultures at in vitro differentiation day 15. Insets, DAPI nuclear staining of the same field. (G) Percentages of double-immunoreactive cells for TH and MAP2 (TH+/MAP2+) out of total TH+ cells. (H) Semi-quantitative RT-PCR analyses for the mRNAs specific to synaptic formation (synaptophysin and synapsin) and growth cone development (GAP43). *Significantly different from the controls with P<0.001. Bars, 10 µm.

View larger version (31K): [in a new window]   Fig. 3. Acquisition of presynaptic neuronal function by Nurr1-induced DA cells with Mash1. Precursor cells from E14 cortices were transduced with Nurr1 and Mash1 (N+M) or Nurr1 and LacZ (N, control), and the functional analyses were performed after 10 days of in vitro differentiation. (A) HPLC quantification of DA release. The graph shows DA levels in the medium conditioned for 24 hours (24 hr medium) and released by KCl-evoked depolarization in N2 with 56 mM KCl for 15 minutes (KCl 15min), n=3. (B) DA uptake. The graph shows the specific DA uptake of the cells transduced with Nurr1 and Mash1 or Nurr1 and LacZ (n=5 for each value). Specific DA uptake was calculated by subtracting non-specific uptake (with nomifensine) from value without nomifensine. *Significantly different from the controls at P<0.001. (C-E) Electrophysiological properties of cells differentiated from the precursor cells transduced with Nurr1 and Mash1. (C) Typical recordings of electrophysiological properties of a differentiated neuron. Left panel, Current-clamp recordings during prolonged depolarizing current injections. Top traces represent current injections, whereas bottom traces indicate voltage recordings. Depolarizing current injections elicited fast action potentials. Right panel, voltage-dependent membrane currents. Depolarizing voltage steps (top traces) elicited outward K+ currents and fast inward Na+ currents (bottom traces). (D) Effect of tetrodotoxin (TTX) on action potential and voltage-dependent Na+ currents. TTX completely inhibited the action potential evoked by depolarizing current injections. Inset, Na+ current by depolarizing voltage steps. (E) Current-clamp recordings during prolonged hyperpolarizing current injections. The cells display the time-dependent anomalous rectification that is a characteristic of midbrain DA neurons after a hyperpolarizing pulse.

View larger version (108K): [in a new window]   Fig. 4. Inhibitory activity of the atonal-related bHLH proteins on Nurr1-induced expression of DA neuronal phenotype. The cortical precursor cells, at day 3 of in vitro expansion, were transduced with Nurr1 plus Ngn1, Ngn2, NeuroD or LacZ (control) using retroviral infection. After 3 days of differentiation, the cultures were subjected to immunocytochemical (A-E), immunoblot analyses (F) for TH and RT-PCR analyses (G) for TH, AADC (DA synthesis), VMAT2 (DA storage) and DAT (DA uptake). (A-D) Representative images of TH/TuJ1 immunocytochemistry on the cells transduced with LacZ (A), Ngn1 (B), Ngn2 (C), and NeuroD (D) along with Nurr1. Note that TH+ cell numbers were reduced by the bHLH co-expression, with increases of TuJ1+ neurons. Insets, DAPI nuclear staining of the same fields. The graph in E represents percentage of TH+ cells out of total (DAPI+) cells. *Significantly different from the control at P<0.001. Bars, 10 µm.

View larger version (98K): [in a new window]   Fig. 5. Essential structural components of Ngn1 and NeuroD required for the inhibitory actions on Nurr1-induced TH expression. (A-H) Effect of the abolishment of DNA-binding capacity of Ngn1 and NeuroD. Each of the wild types and the DNA-binding mutants of Ngn1 and NeuroD was introduced into Nurr1-transduced precursors by co-infection as described in the Materials and Methods, and TH+ cell numbers and TH protein levels were determined at day 3 of differentiation using immunocytochemical (A-G) and western blot analyses (H), respectively. Note that the abolishment of the DNA-binding capacity in Ngn1 and NeuroD abrogates not only their generic neurogenic activities (TuJ1 protein level in H), but also the inhibitory activity on Nurr1 and TH expression. (I) Schematic diagrams of the chimeric proteins of NeuroD (D) and Mash1 (M) or NeuroD and MyoD (Y) used to identify the domains for the differential roles of Mash1 (or MyoD) and NeuroD. Co-transduction of each of the chimeric proteins with Nurr1 was carried out as described above, and the effects of the chimeric bHLHs on Nurr1-TH expression were determined at day 3 of differentiation by immunochemical (J-R) and immunoblot (S) analyses. (J-S) Analyses of the chimeric proteins reveal that the HLH domains are responsible for the differentiation actions of Mash1 and NeuroD. Note that the effects of the chimera YD and MD, which hold NeuroD HLH domain, on Nurr1-induced TH+ cell numbers and TH protein levels are not distinguishable from those of wild-type NeuroD, whereas the inhibitory activity of NeuroD is abolished by substituting the HLH domain with that of Mash1 or MyoD (in chimera DM or DY). Graphs G and R represent the mean percentage (± s.e.m.) of TH+ cells out of total DAPI+ cells. *Significantly different from the controls at P<0.001. Bars, 10 µm.

View larger version (26K): [in a new window]   Fig. 6. Atonal-related bHLHs repress Nurr1-induced transactivation of TH promoter activity. Promoter assays using the reporter vectors driven by TH6.0 (A) and NL3 (B) were performed as described in Materials and Methods. The data represent mean percentages (± s.e.m.) of luciferase activities relative to that of the control (sample transfected with pEF1-Nurr1 plus pEF1-EGFP). Titles of the treatments including the bHLH mutants and chimeras were abbreviated as shown in Fig. 5. Luciferase activity was determined at day 2 of post-transfection and normalized to the activity of the ß-galactosidase. *Significantly different from the control at P<0.001 in the duplicates of three independent experiments.