Fig. 4

Fig. 4 Nr4a2 is essential during the development of DA neurons in the PT area. (A) Western blot analysis of 48 hpf embryo lysates with Nr4a2 monoclonal antibody (upper panel), as well as β-actin monoclonal antibody as a loading control (lower panel). Protein lysates from 10 embryos are loaded in each lane. (B) Western blot analysis of Nr4a2 and TH level of the 5-mm injected, Nr4a2-deficient and rescued embryo lysates at 48 hpf; β-actin monoclonal antibody is used as a loading control. (C) In the Nr4a2a-, Nr4a2b- and Nr4a2-deficient embryos, TH staining is significantly decreased not only in Pr, PO and OB (black arrows), but also in the PT area (white arrows). Anterior is to the left. (D) Percentage of Nr4a2a-, Nr4a2b- and Nr4a2-deficient embryos that have the significantly decreased TH expression in Pr, PO, OB and the PT area. (E) Exogenous mouse Nr4a2 transcripts can partially rescue the loss-of-function of endogenous Nr4a2. Fewer TH⁺ and DAT⁺ neurons in the PT area are shown in nr4a2^mo-injected embryos at 48 hpf and the TH⁺ and DAT⁺ neurons in the PT area can be partially rescued after injection with mouse Nr4a2 mRNA (white arrows), with the TH⁺ neurons in the LC area as control (black arrowheads). (F) Bar scale of the HPLC analysis for DA content. The level of DA is decreased by 79.5% (p = 0.0027) in the embryo after nr4a2^mo treatment and it is recovered by 55.5% (p = 0.0029) after mouse Nr4a2 mRNA injection. DAT, dopamine transporter; LC, locus coeruleus; OB, olfactory bulb; PO, preoptic area; Pr, pretectum area; TH, tyrosine hydroxylase.