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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.

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