The Wnt antagonist Dkk1 causes an increase in DA cell number. (A,B) Dorsal views of zebrafish embryos injected with (A) vehicle solution or (B) an in vitro synthesized capped mRNA encoding Dkk1. Embryos were fixed at 72 hours post-fertilization (hpf) and subjected to immunostaining with an anti-tyrosine hydroxylase (TH) antibody to detect dopaminergic (DA) neurons. Diencephalic DA groups [as annotated by Rink and Wullimann (Rink and Wullimann, 2002)] are marked. (C-F) Projected confocal z-stack images of DA neurons (red) in transgenic embryos expressing GFP under the control of the dlx6a promoter (dlx6a::gfp; lateral view). Embryos were injected with either a vehicle solution (C,E) or dkk1 mRNA (D,F), and were fixed at either 24 (C,D) or 48 (E,F) hpf. The insets in E and F show bubble-plot graphic representations of the x, y, z cell position of all dlx6a+ (green) and TH+ (red) cells in the ventral diencephalon. Bubble size was derived from the position of the cells along the z-axis to give the impression of depth. The positions of DA group 2 (Gr. 2) and groups 3-6 (Gr. 3-6) are indicated. (G) Bar chart showing average counts of dopamine transporter (Dat)-expressing DA group 2 (Gr. 2) cells in WT (control) and dkk1-injected embryos. Dat+ neurons were detected by whole-mount in situ hybridization with dat probe at 22, 28 and 48 hours post-injection (n=25). (H) Bar chart showing average counts of dlx6a+ cells in the basal diencephalon of WT and dkk1-injected dlx6a::gfp transgenic embryos (n=5). (I) Schematic depicting the major diencephalic DA cell groups that appear in the developing embryo at 48 hpf, relative to the dlx6a::gfp expression domain. Dien, diencephalon; HB, hindbrain; MB, midbrain; PT, posterior tuberculum; Tel, telencephalon; TDB, telencephalic-diencephalic boundary. Scale bars: 25 μm.
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