DEV024232 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 - Adobe PDF

• Supplemental Figure S1 -

  Fig. S1. Quantitative analysis of DA cells in control versus Dkk1-injected embryos. Recorded numbers of diencephalic DA cells in WT (white circles) and in Dkk1-injected (black diamonds) embryos at 48 hpf. Cell numbers are plotted separately for (A) group 2 (Gr. 2) and (B) group 3-6 (Gr. 3 to 6). Each data point represents a unilateral cell count (total number divided by two) in a single embryo, out of a total number (sample #) of 85 specimens. Note the invariant number of DA neurons in the WT population.

• Supplemental Figure S2 -

  Fig. S2. All TH-positive DA neurons express neurog1 in the diencephalon. (A-A′′′) Lateral view of neurog1::gfp transgenic reporter embryo (at 24 hpf), which was subjected to in situ hybridization with an antisense barhl2 probe followed by double immunofluorescence staining with antibodies against TH and GFP (A). High-magnification images of cells expressing neurog1, TH and barhl2 are shown in A′-A′′′, respectively. Arrows indicate neurog1⁺ TH⁺ double-positive cells. (B-B′′′) Dorsal view of double-transgenic neurog1::nRFP; dlx6a::GFP embryo (at 48 hpf), which was subjected to immunofluorescence staining with antibodies against TH (B). Arrowheads indicate neurog1⁺ TH⁺ double-positive neurons. Single-channel fluorescent images are presented in B′-B′′′. PT, posterior tuberculum; RFP, red fluorescent protein; Tel, telencephalon. Scale bars: 20 µm.

• Supplemental Figure S3 -

  Fig. S3. Positioning of diencephalic progenitor zones in the neural plate determined by the two-photon-based uncaging method. (A,A′) Embryos (anterior to the left) expressing GFP under the control of the neurog1 promoter (neurog1::gfp) were injected with caged dextran-fluorescein tracer dye at the 1-cell stage. At the 1- to 3-somite stage, the dye was uncaged at discrete domains (denoted I, II and III) of the diencephalic anlage. (A) Dorsal view; (A′) lateral view. (B,B′) Projected two-photon z-stack (anterior to the left) of transgenic neurog1::gfp embryo injected with caged fluorescein-dextran (B). The uncaging of the fluorescein tracer at a specific region of interest (dashed box) is visualized in B′. Dien, diencephalon; Tel, telencephalon; TG, trigeminal ganglion; VCC, ventrocaudal cluster.

• Supplemental Figure S4 -

  Fig. S4. The effect of cell division inhibitors on embryonic development. (A-D) Control embryos were incubated (A,C) in 4% DMSO or (B,D) in a solution containing the cell division inhibitors aphidicolin (APH, 1 µg/ml) and 5-hydroxyurea (5-HU, 50 mM) dissolved in 4% DMSO. Treatment was initiated at 6 hpf, and embryos were cultured overnight until 24 hpf. Lateral views of WT embryos immunostained for phospho-histone H3 (A,B) and of live embryos (C,D) demonstrating that embryogenesis proceeds relatively normally in the presence of cell division inhibitors. Scale bar: 50 µm.

• Supplemental Figure S5 -

  Fig. S5. Temporal inhibition of DA group 3-6 progenitor proliferation. Bar chart presenting average cell counts of Dat-expressing DA group 3-6 (Gr. 3-6) neurons in embryos injected with vehicle (WT) or dkk1 (+Dkk1). At the indicated time points, embryos were treated with either 4% DMSO or a cell cycle inhibitor cocktail containing 1 µg/ml aphidicolin (APH) and 50 mM 5-hydroxyurea (5-HU). DA neurons were scored at 48 hpf by whole-mount in situ hybridization with dat antisense probe. The number of embryos analyzed (n) in shown beneath.

• Supplemental Figure S6 -

  Fig S6. Wnt attenuation does not affect the barhl2 diencephalic expression domain. Lateral views (anterior to the left) of wild-type (A), lef1 MO-injected (B) or X8 deletion mutant (C) embryos. Embryos were fixed at 24 hpf and subjected to whole-mount in situ hybridization with antisense RNA probe directed against barhl2. All specimens were then subjected to immunostaining with an anti-tyrosine hydroxylase (TH) antibody to detect DA neurons. Scale bar: 50 µm.