Effect of neurog1hi1059/hi1059 and olig2m1317/m1317 mutants on dopaminergic development (A-F) th expression in (B,D,F) neurog1hi1059/hi1059 mutants compared to (A,C,E) wildtype siblings at (A,B) 30 hpf and (C–F) 72 hpf. (A,B) At 30 hpf in neurog1hi1059/hi1059 mutants no th+ cells are present (black arrowheads). (C–F) In the H/PT area, DAC2, DAC4 and DAC5 are missing (black arrowheads) and DAC6 is reduced (non-filled arrowhead). (G–L) th expression in (H,J,L) olig2m1317/m1317 mutants compared to (G,I,K) wildtype siblings at (G,H) 30 hpf and (I–L) 72 hpf. At 30 hpf in (H) olig2m1317/m1317 mutants, the number of th+ cells is reduced compared to (G) wildtype (non-filled arrowhead). At 72 hpf in (J,L) olig2m1317/m1317 mutants the numbers of th+ DAC2 (non-filled arrowheads) and DAC6 (black arrowheads) cells are reduced compared to (I,K) wildtype siblings. (M,N) Quantification of th+ cells in mutant neurog1 (M) and olig2 (N) embryos compared to wildtype siblings at 72 hpf. The number (n) of embryos analyzed for the cell counts are as indicated in the grey box; p-values were calculated using the Mann-Whitney test. p-values indicate significance if they are <0.05 (**, 0.001–0.01; ***, 0.0001–0.001; ****, <0.0001), if no asterisks are present, effects are not significant. For numbers see Supplemental Table 1. Abbreviations: numbers 1 through 6 for DAC1-6, dopaminergic cell cluster 1–6; H, hypothalamus; LC, locus coeruleus; MO, medulla oblongata; OB, olfactory bulb; PO, preoptic area; Pr, pretectum; PT, posterior tuberculum; SP, subpallium; TC, telencephalic cluster. (A-D, G-J) lateral views (enucleated embryos: C, D and I, J) and (E, F, K, L) dorsal views. All images show single optical planes. Anterior is to the left. Scale bars: 100 μm.

neurog1 and olig2 single and double mutant phenotypes and Notch inhibition reveal potential epistatic relationships. (A–H) th expression in (A,E) wildtype, (B,F) neurog1hi1059/hi1059 mutants, (C,G) olig2m1317/m1317 mutants and (D,H) neurog1hi1059/hi1059; olig2m1317/m1317 double mutants in (E–H) DAPT treated embryos compared to (A–D) control siblings at 72 hpf. The numbers 1-6 mark the corresponding (DAC1-DAC6) dopaminergic cell clusters (A). The number of th+ DAC5 and DAC6 cells is increased in WT embryos (E) after DAPT treatment, compared to (A) control embryos (filled black arrowheads). The number of th+ DAC6 cells in olig2m1317/m1317 mutant embryos (G) after DAPT treatment is increased compared to control olig2m1317/m1317 mutant embryos (C, open arrowheads). DAPT treatment does not alter the number of th+ cells in neurog1hi1059/hi1059 or double mutant embryos (B,F,D,H). (I) Quantification of th+ cells in single and double mutants compared to wildtype siblings at 72 hpf. The number of embryos analyzed for each genotype are indicated (n-numbers in grey box); p-values were calculated using the Mann-Whitney test. p-values indicate significance if they are <0.05 (*, 0.01–0.05; **, 0.001–0.01; ***, 0.0001–0.001; ****, <0.0001). For numbers see Supplemental Table 2. Abbreviations: numbers 1 through 6 for DAC1-6, dopaminergic cell cluster 1–6; LC, locus coeruleus; PO, preoptic area. All images show single optical planes of dorsal views. Anterior is to the left. Scale bars: 100 μm.

Heat-shock driven overexpression of neurog1 or olig2 induces supernumerary and ectopic DA neurons. th expression was analyzed following heat-shock in (B,D,F) Tg(hsp70:neurog1)ups1 compared to (A,C,E) wildtype siblings at (A,B) 30 hpf and (C–F) 72 hpf. (G–L) th expression in heat shocked (H,J,L) Tg(hsp70l:olig2)m1306 compared to (G,I,K) wildtype siblings at (G,H) 30 hpf and (I–L) 72 hpf. The numbers 1 through 6 mark the dopaminergic cell clusters DAC1-DAC6 (E). Overexpression of neurog1 induces supernumerary and ectopic th+ cells at 30 hpf and 72 hpf (B,D,F). Overexpression of olig2 results in supernumerary th+ cells at 30 hpf and 72 hpf, probably belonging to the DAC2 group (H,J,L). (M) Overview of effects of neurog1 and olig2 mutant and overexpression, as well as of Notch inhibition on the development of catecholaminergic neurons in the specific anatomical clusters. The specific experiments are shown in Fig. 1, Fig. 2 and this Figure. CC indicates that cells of the indicated CA groups were counted. EN indicates enhanced stain intensity or ectopic catecholaminergic neurons, which were not counted. Red colored boxes indicate significantly decreased numbers of CA neurons whereas green colored boxes indicate significantly increased cell numbers. Grey colored boxes indicate no change in cell numbers between the given genotypes and treatment conditions. For numbers see Supplemental Table 3. Abbreviations: numbers 1 through 6 for DAC1-6, dopaminergic cell cluster 1–6; H, hypothalamus; LC, locus coeruleus; PO, preoptic area; Pr, pretectum; PT, posterior tuberculum; TC, telencephalic cluster. (A-D, G-J) show lateral views (I,J show enucleated embryos) and (E,F,K,L) dorsal views. All images show single optical planes. Anterior is to the left. Scale bars: 100 μm.

neurog1 and olig2 are coexpressed in defined domains during development. (A1-D3) Co-expression of neurog1 (magenta) and olig2 (green) at (A1-B3) 30 hpf and (C-D3) 48 hpf. (B1–B3) is a magnification of the corresponding highlighted area of a different embryo in (A3/AB), (D1-D3) of the corresponding highlighted area of a different embryo in (C3/CD). The white line in (A3) frames the outline of the imaged zebrafish larval head, neural tube and eyes; the line in (C3) frames the outline of the imaged zebrafish larval head, nose pits and eyes. At both stages some cells show coexpression of neurog1 and olig2 (B3,D3, asterisks). (AB, CD) Schemes show lateral head views of 30 and 48 hpf zebrafish larvae with schematic expression patterns of stained mRNAs and indicate approximate positions of planes and projections. Areas identified to express both genes are colored in orange (see also Supplemental Movies 1, 2). Abbreviations: H, hypothalamus; PT, posterior tuberculum. All images show dorsal views. (B1–B3,D1-D3) show single planes; (A1-A3,C1–C3) are 20–40 μm maximum intensity projections. Anterior is to the left. Scale bars: (A,C) 100 μm, (B,D) 10 μm.

Analysis of potential cross-regulation of neurog1 and olig2 expression. (A–D) olig2 expression in (B,D) neurog1hi1059/hi1059 mutants compared to (A,C) wildtype siblings at (A,B) 30 hpf and (C,D) 48 hpf. At 48 hpf, olig2 expression in the hindbrain is reduced in (D) neurog1hi1059/hi1059 mutants compared to (C) wildtype siblings (transparent arrowheads). (E–H) neurog1 expression in (F,H) olig2m1317/m1317 mutants compared to (E,G) wildtype siblings at (E,F) 30 hpf and (G,H) 48 hpf. At 30 and 48 hpf, neurog1 expression in the H/PT is reduced in (F,H) olig2m1317/m1317 mutants compared to (E,G) wildtype siblings (black arrowheads). (I–L) neurog1 expression upon heat-shock in (J,L) Tg(hsp70l:olig2)m1306 transgenic fish compared to (I,K) wildtype siblings. (K,L) are magnifications of (I,J) respectively. (J,L) Tg(hsp70l:olig2)m1306 transgenic fish show increased neurog1 expression in the H/PT when compared to (I,J) wildtype siblings (arrowheads). All olig2 overexpressing embryos of each genotype showed increased neurog1 expression in hypothalamus and posterior tuberculum. The phenotypes were observed in N/N (number of embryos with phenotype shown in figure panel/total number of embryos analyzed): (A) 8/8; (B) 6/6; (C) 10/10; (D) 7/7; (E) 7/7: (F) 7/7; (G) 10/10; (H) 8/8; (I,K) 8/8; (J,L) 8/8. Abbreviations: H, hypothalamus; HB, hindbrain; PT, posterior tuberculum; Tel, telencephalon. All images show lateral views (C,D and G,H show enucleated embryos) and single optical planes. Anterior is to the left. Scale bars: 100 μm.

olig2 expression is regulated by Shh signaling and acts upstream of her2. (A, B) her2 expression in (B) olig2m1317/m1317 mutants compared to (A) wildtype siblings at 48 hpf. The expression of her2 is increased in the hypothalamus/posterior tuberculum in olig2m1317/m1317 mutant embryos (black arrowheads). (C1-D3) her2 expression (green) relative to neurog1 expression (magenta) in (D1-D3) olig2m1317/m1317 mutants compared to (C1–C3) wildtype siblings at 48 hpf. her2 expression is increased whereas neurog1 expression is decreased in olig2m1317/m1317 mutant embryos (white arrowheads). (E–H) olig2 expression at 48 hpf in (F,H) embryos treated with Cyclopamine between 24 and 48 hpf, compared to (E,G) control embryos treated with ethanol. olig2 expression is reduced in the hypothalamus/posterior tuberculum and in the thalamus upon inhibition of Shh signaling by Cyclopamine. The phenotypes were observed in N/N (number of embryos with phenotype shown in figure panel/total number of embryos analyzed): (A) 4/4; (B) 7/7; (C1–C3) 4/4; (D1-D3) 4/4; (E,G) 15/15; (F,H) 15/15. Abbreviations: H, hypothalamus; HB, hindbrain; PT, posterior tuberculum; Tel, telencephalon. All images show lateral views, except E, F showing dorsal views; (A and B) show single optical planes; (C1-D3) are 340 μm maximum intensity projections. Anterior is to the left. Scale bars: 100 μm.

neurog1 and olig2 are coexpressed with otpa. (A1-A3) otpa mRNA expression relative to Th protein expression. (B1–C4) co-expression of otpa (magenta) and neurog1 (green). (D1-E4) co-expression of otpa (magenta) and olig2 (green). (C1–C4) is a magnification of the corresponding highlighted area of a different embryo in (B3/BC), (E1-E4) of the corresponding highlighted area of a different embryo in (D3/DE). The white line in (A3) frames the outline of the imaged zebrafish larval head and eyes. (C3,4) and (E3,4) show TOTO3 nuclear stain. (A, BC, DE) Schemes show lateral head views of 30 hpf zebrafish larvae with schematic expression patterns of stained mRNAs and proteins and indicate approximate positions of planes and projections. Areas with coexpression are colored in orange. Abbreviations: PT, posterior tuberculum. All images show dorsal views. (C1–C4, E1-E4) show single confocal planes; (A1-B3, D1-D3) are 30–40 μm maximum intensity projections. Anterior is to the left. Scale bars: (A) 100 μm, (B,D) 50 μm, (C,E) 10 μm.

otpa and sim1a expression is reduced in the H/PT region upon loss of either Neurog1 or Olig2. (A–D) otpa expression in (B,D) neurog1hi1059/hi1059 mutants compared to (A,C) wildtype siblings at (A,B) 30 hpf and (C,D) 48 hpf. In neurog1hi1059/hi1059 mutants otpa expression in the H/PT is reduced if compared to wildtype siblings (black arrowheads). (E–H) otpa expression in (F,H) olig2m1317/m1317 mutants compared to (E,G) wildtype siblings at (E,F) 30 hpf and (G,H) 48 hpf. At 30 hpf in (F) olig2m1317/m1317 mutants, the otpa expression in the H/PT is reduced compared to (E) wildtype (black arrowhead). At 48 hpf in (H) olig2m1317/m1317 mutants otpa expression in the caudal H/PT is reduced (black arrowheads) and unchanged in the rostral H/PT (non-filled arrowheads). (I–L) sim1a expression in (J,L) neurog1hi1059/hi1059 mutants compared to (I,K) wildtype siblings at (I,J) 30 hpf and (K,L) 48 hpf. In neurog1hi1059/hi1059 mutants sim1a expression in the H/PT is reduced if compared to wildtype siblings at 30 hpf but not at 48 hpf (black arrowheads). (M–P) sim1a expression in (N,P) olig2m1317/m1317 mutants compared to (M,O) wildtype siblings at (M,N) 30 hpf and (O,P) 48 hpf. In olig2m1317/m1317 mutants, the sim1a expression in the H/PT is reduced compared to wildtype at 30 hpf as well as 48 hpf (black arrowheads). The phenotypes were observed in N/N (number of embryos with phenotype shown in figure panel/total number of embryos analyzed): (A) 10/10; (B) 15/15; (C) 9/9; (D) 12/12; (E) 18/18: (F) 12/12; (G) 17/17; (H) 6/6; (I) 6/6; (J) 7/7; (K) 5/5; (L) 10/10; (M) 4/4; (N) 11/11; (O) 3/3; (P) 6/6. Abbreviations: H, hypothalamus; PO, preoptic area; PT, posterior tuberculum. All images show lateral views (48 hpf: enucleated embryos) and are single planes. Anterior is to the left. Scale bar: 100 μm.

Transcriptional network integrating patterning and neurogenesis signals to control posterior tubercular/hypothalamic A11-type DA neuron development in zebrafish The network model shows olig2 and her2 as components that selectively contribute to DAC6 cluster development, while all other components control DAC2, 4, 5 and 6 cluster development, which are the only glutamatergic DA clusters in zebrafish embryos. Published evidence (numbers in brackets refer to references listed below) reveals Nodal, Wnt and Shh patterning signals to control early nkx2.1/4, fezf2 and otpa expression in progenitors. In DAC6, Olig2 is regulating the expression of neurog1 through inhibition of her2, which is a Notch-dependent Hes/Her gene. Olig2 and Neurog1 regulate the expression of otpa, which is crucial for the formation of the A11-type DA neurons, whereas sim1a is mainly regulated by Olig2 alone. Shh signaling contributes to the control of olig2 expression. Black arrows indicate positive genetic interactions. Red lines indicate inhibitory genetic interactions. Blue arrows indicate signaling pathways contributing to control of gene expression. Dashed lines indicate partial or unclear genetic/signaling interactions. References: (1) Takke et al. (1999); (2) Hashimoto et al. (2000); (3) Rohr et al. (2001); (4) Tyurina et al. (2005); (5) Ryu et al. (2007); (6) Jeong et al. (2006); (7) Blechman et al. (2007); (8) Russek-Blum et al. (2008); (9) Borodovsky et al. (2009); (10) Löhr et al., 2009; (11) Chapouton et al. (2011); (12) Manoli and Driever (2014); (13) Cheng et al. (2015).