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First published online February 20, 2009
doi: 10.1242/10.1242/dev.033878

Development 136, 1007-1017 (2009)
Published by The Company of Biologists 2009
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Zebrafish diencephalic A11-related dopaminergic neurons share a conserved transcriptional network with neuroendocrine cell lineages

Heiko Löhr1, Soojin Ryu1,* and Wolfgang Driever1,2,{dagger}

1 Developmental Biology, Institute Biology I, Faculty of Biology, University of Freiburg, Hauptstrasse 1, D-79104 Freiburg, Germany.
2 Freiburg Institute for Advanced Studies, University of Freiburg, Albertstrasse 19, D-79104 Freiburg, Germany.


Figure 1
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  		Fig. 1. A mutation in arnt2 affects DA neuron groups in the ventral diencephalon of zebrafish. (A-F) Reduction of th-expressing DA neuron groups in the ventral diencephalon of m1055 mutants (B,D,F) compared with wild-type siblings (A,C,E) at 24 hpf (A,B) and 72 hpf (C-F). (G) Genetic mapping of the m1055 allele. SSLP markers (MGH panel black and T51 panel red) and genetic distances are shown. (H) Sequencing of arnt2 in m1055 mutants reveals a T to A exchange at position 1869. (I) Protein structure of the PAS family member arnt2. The premature stop codon in the m1055 allele disrupts the essential transactivation domain (TAD). (K,L) arnt2 is broadly expressed in wild type (K) at 24 hpf, but almost absent in m1055 mutants (L). (M,N) Fluorescent whole-mount in situ hybridization for arnt2 (green) combined with immunohistochemistry for TH (red) at 36 hpf. Arrowheads indicate co-expression. (A-D,K-L) Lateral views, (E,F,M,N) dorsal views, anterior towards the left. Scale bars: in F, 100 µm for E,F; in L, 100 µm for A-D,K,L; in N, 25 µm for M,N. AAC, arch associated cluster; d, diencephalon; DC, diencephalic cluster; H, hypothalamus; Hm, medial hypothalamus; LC, locus coeruleus; MO, medulla oblongata; PO, preoptic region; Pr, pretectum; PT, posterior tuberculum; sym, sympathetic CA neurons; t, telencephalon; TC, telencephalic cluster; VT, ventral thalamic cluster. Numbers in E,F indicate DA clusters in VT (1) and PT/H (2-6) according to Rink and Wullimann (Rink and Wullimann, 2002Go).

 

Figure 2
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  		Fig. 2. sim1b is expressed in a subset of DA neurons in the ventral diencephalon. (A-G") Expression of sim1b (A,C) and co-expression of sim1b (fluorescent whole-mount in situ hybridization, green) and TH (B,D-G"; immunohistochemistry red). (A,B) 1 dpf. (C) sim1b expression at 3 dpf. (D-D") Higher magnification of area framed in B. All TH-positive cells in this region co-express sim1b (arrows), whereas some sim1b-expressing cells are negative for TH (arrowheads). (E-E") Higher magnification of area framed in C. DA neuron groups 2, 4-6 colocalize with sim1b; DA neuron groups 1 and 3 do not co-express sim1b (arrowheads). (F-G") Dorsal views at 3 dpf in more dorsal (F-F") and more ventral (G-G") coronal optical sections. (A,B,F-G") Dorsal views, (C-E") lateral views, anterior towards the left. Scale bars: in C, 100 µm for A,C; in B, 100 µm; in D",E",G", 50 µm for D-G". Images in B,D-G" are z-projections of multiple adjacent focal planes, depths (B,D) 12 µm, (E) 30 µm and (F,G) 20 µm. d, diencephalon; m, mesencephalon; PO, preoptic region.

 

Figure 3
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  		Fig. 3. Similar reduction of DA cells in embryos injected with arnt2 or sim1a antisense morpholinos. (A-H) Expression of th at 4 dpf in uninjected wild-type embryos (A,E), arnt2m1055 mutants (B,F), embryos injected with 2.8 ng arnt2 splice morpholino (C,G) and embryos injected with 1.1 ng sim1a splice morpholino (D,H). (A-D) Affected groups are marked by a bracket. (E-H) Magnification of ventral diencephalic groups 1-6. (A-D) Lateral views; (E-H) dorsal views. Anterior towards the left. Scale bar in H: 100 µm for A-D, 50 µm for E-H. Images in E-H represent z-projections from several adjacent focal planes. AAC, arch associated cluster; LC, locus coeruleus; MO, medulla oblongata; OB, olfactory bulb; PO, preoptic region; Pr, pretectum; PT, posterior tuberculum; SP, subpallium; sym, sympathetic CA neurons; VT, ventral thalamic cluster.

 

Figure 4
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  		Fig. 4. Reduction of neurohormone-producing cells in the hypothalamus of arnt2m1055 mutants and sim1a morphants. (A-P) Expression of crh (A-C), trh (D-F), itnp (G-I), vsnp (K-M) and sst1 (N-P) at 76 hpf in wild-type embryos (A,D,G,K,N), arnt2m1055 mutants (B,E,H,L,O) and embryos injected with 1 ng sim1a morpholino (C,F,I,M,P). (A-C) crh neurons in the PO (lower arrowheads) and in the posterior tuberculum and hypothalamus (upper arrowheads) are reduced. (D-F) trh-expressing cells in the PO (arrowheads) are strongly reduced. (G-I) itnp-expressing cells in the PO (arrowheads) are strongly reduced or absent. In sim1a morphants, itnp-expressing cells are detected at ectopic locations within the diencephalon (arrow). (K-M) vsnp-expressing cells in the PO (arrowheads) are strongly reduced or absent. (N-P) A group of sst1-expressing cells in the PO (arrowheads) is strongly reduced or absent, whereas all other sst1-positive domains, including a second group in the PO (arrow) are not affected. (Q,R) Camera lucida drawing showing the relative position of all analyzed neuronal groups reduced in arnt2 mutants or sim1a morphants in lateral (Q) or dorsal (R) views; non-affected neuronal groups are not included. (A-P) Whole-mount in situ hybridization, lateral views; anterior is towards the left. Scale bar in P: 50 µm for A-P. Images represent z-projections from several consecutive focal planes. ac, anterior commissure; d, diencephalon; h, hindbrain; H, hypothalamus; m, mesencephalon; PO, preoptic region; poc, post optic commissure; PT, posterior tuberculum.

 

Figure 5
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  		Fig. 5. Mutually independent expression of sim1 and otp genes in DA neurons. (A-B") Detection of otpa (green) and sim1b (red) expression by fluorescent whole-mount in situ hybridization combined with immunohistochemistry for TH (white) at 1 dpf. (A) THir cells are located within an otpa- and sim1b-expressing domain in the diencephalon (z-projection, 16 µm). (B-B") Higher magnification of area framed in A (z-projection 10 µm). All THir cells co-express both otpa and sim1b (arrows in B,B'). (C-F) Expression analysis of sim1a (C,D) and sim1b (E,F) in wild-type embryos (C,E) and in otpam866 homozygous mutants injected with 2 ng otpb morpholino (D,F) at 2 dpf. (G-K) Expression analysis of otpa (G,H) and otpb (I-K) in wild-type (G,I) and in arnt2m1055 (H,K) embryos at 2 dpf. (A-B") Dorsal, (C-K) lateral views. Anterior towards the left. Scale bars: in B", 100 µm for A, 50 µm for B-B"; in K, 100 µm for C-K. d, diencephalon; h, hindbrain; H, hypothalamus; m, mesencephalon; Pit, pituitary; PO, preoptic region; PT, posterior tuberculum.

 

Figure 6
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  		Fig. 6. Combined overexpression of Otpa and Sim1a leads to supernumerary DA and CRH cells in the ventral diencephalon. (A-H) Whole-mount in situ hybridization. Expression of th at 24 hpf (A,B), 32 hpf (C,D) and 48 hpf (E,F), and crh at 32 hpf (G,H) in wild-type controls (A,C,E,G) compared with transgenic embryos expressing otpa from a hsp70 promoter, and injected with sim1a mRNA (Otp/Sim1OE; B,D,F,H). Only the number of group 2 cells is strongly increased in Otp/Sim1OE embryos, whereas groups 4/6 are not affected (A-F). The number of CRH cells is increased in Otp/Sim1OE embryos (G,H). Lateral views, anterior towards the left. Scale bar: 50 µm for A,B; 62.5 µm for C,D,G,H; 80 µm for E,F.

 

Figure 7
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  		Fig. 7. Quantification of DA and CRH cell numbers in Otpa- and Sim1a-overexpressing embryos. (A-D) Bar charts illustrating the average number of th- (A,B), dat- (C) and crh- (D) positive neurons in hsOtpa transgenic embryos injected with sim1a mRNA (Otp/Sim1OE, black bars), uninjected hsOtpa transgenic embryos (OtpOE, dark-gray bars), wild-type embryos injected with sim1a mRNA (Sim1OE, light-gray bars) and uninjected wild-type embryos (control, white charts). Error bars indicate standard deviation of the mean. Significance was evaluated by paired Student's t-test. Asterisks indicate significant differences compared with the same stage control (P<0.001). The developmental stage as well as the number of embryos analyzed are depicted.

 

Figure 8
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  		Fig. 8. Model summarizing the roles of Otp, Sim1 and Arnt2 during development of DA and neurosecretory neurons in the hypothalamus. Top to bottom schematic time axis from early patterning through neuronal specification and differentiation. Left to right DA and neurosecretory cell groups of the hypothalamus (for abbreviations, see text). The model is based on analysis in zebrafish. Tentative assignments of homologous mammalian groups are indicated using the mammalian nomenclature. The zebrafish diencephalic group 1 (mammalian A13) is located in the prethalamus and, thus, not included in this scheme.

 

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© The Company of Biologists Ltd 2009