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First published online 8 October 2003
doi: 10.1242/dev.00816

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Noradrenergic neurons in the zebrafish hindbrain are induced by retinoic acid and require tfap2a for expression of the neurotransmitter phenotype

Jochen Holzschuh1,*, Alejandro Barrallo-Gimeno2,{dagger}, Anne-Kathrin Ettl1, Katrin Dürr1, Ela W. Knapik2 and Wolfgang Driever1,{ddagger}

1 Developmental Biology, Institute Biology 1, University of Freiburg, Hauptstrasse 1, D-79104 Freiburg, Germany
2 GSF, Institute for Mammalian Genetics, Ingolstadter Landstrasse 1, D-85764 Neuherberg, Germany



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  		Fig. 1. The phenotype of tfap2a mutant embryos. (A) Live wild-type and (B) tfap2a mutant embryos at 4 dpf. Jaw defects, a slightly smaller head and reduced pigmentation in the tail characterize mob mutant embryos. (C-J) Detection of th expression in zebrafish embryos at 54 hpf by whole-mount in situ hybridization. (C,E) th expression is normal in the forebrain of tfap2a mutant embryos. (D,F,G,I) At 54 hpf, no th expression is detectable (white arrows) in the hindbrain of tfap2a mutant embryos. (H,J) th expression cannot be detected in tfap2a mutant embryos in the region where sympathetic ganglia form in wild-type. (K-P) Detection of dbh expression in zebrafish embryos at 4 dpf by whole-mount in situ hybridization. (K,M,N) At 4 dpf, dbh expression can be detected in the wild type in the locus coeruleus, the medulla oblongata/area postrema, sympathetic neurons as well as a group of arch associated neurons that may later contribute to the carotid body. (L,O,P) In tfap2a/mobm819mutant embryos, the arch associated NA neurons develop normally, but cells of the locus coeruleus and sympathicus do not express dbh. A small number of cells starts to express dbh in the medulla oblongata/area postrema: both the number of cells and the expression level of dbh are severely reduced when compared with wild type. (Q-T) Immunohistochemistry with anti-serotonin antibodies reveals that the development of serotonergic neurons in the forebrain (Q,S) and hindbrain (R,T) is not affected in tfap2a mutant embryos. (A-T) anterior towards the left; (A-C,E,K,L) lateral views; (D,F-J,M-T) dorsal views. (A,C,D,G,H,K,M,N,Q,R) Wild-type; (B,E,F,I,J,L,O,P,S,T) tfap2a/mobm819 mutant embryo. AAC, arch associated cluster (carotid body); DDC, ventral diencephalic dopaminergic cluster; LC, locus coeruleus; MC, medulla oblongata noradrenergic cluster; ObC, olfactory bulb dopaminergic cluster; s, somite; symp, sympathetic neurons.

 


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  		Fig. 4. Early development of the visceral sensory ganglia is affected in tfap2a mutant embryos. (A,B) In situ hybridization at 24 hpf reveals a strong reduction in dlx2-expressing neural crest cells in branchial arches of tfap2a mutant embryos. (C,D) By 24 hpf, ret1 expression in the branchial arches is reduced in tfap2a mutant embryos. (E,F) At 4 dpf, ret1 expression reveals a complete loss of epibranchial placode-derived sensory ganglia in tfap2a mutant embryos. (G,H) Expression of phox2a, which marks placodal-derived neurons, is also missing in tfap2a mutant embryos. (I,J) Alcian Blue staining of cartilage combined with anti-acetylated-tubulin immunohistochemistry in 4 dpf embryos. The axon bundles of the cranial nerves are missing in tfap2a mutant embryos. (A-H) Lateral views, anterior towards the left. (I,J) Ventral views, anterior towards the left. (A,C,E,G,I) wild-type; (B,D,F,H,J) tfap2a mutant embryos. allg, anterior lateral line ganglion; b, branchial stream, cb, ceratobranchials; ch; ceratohyal; fs, facial sensory ganglion; gs, glossopharyngeal sensory ganglion; h, hyoid stream; m, mandibular stream; pllg; posterior lateral line ganglion; vs, vagal sensory ganglia; V, axons of the facial cranial nerve; VII, axons of the glossopharyngeal cranial nerve; X axons of the vagus cranial nerve. Scale bars: 50 µm.

 


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  		Fig. 2. Genomic organization of the zebrafishtfap2a{alpha} locus. (A) Genetic map of linkage group 24 (LG24) showing the position of mont blanc/tfap2a in relation to some SSLP markers. (B) The genomic organization of the zebrafish tfap2a gene; exons Ia, Ib and Ic represent alternative first exons for isoforms 1, 2 and 3. (C) Identification of the mobm819 mutation revealed an A to T transition (red) at the beginning of exon V, which introduces a stop codon (black box) and a DraII restriction site (green with red T). (D) RFLP was used for linkage analysis. Intron primers P1 and P2 give rise to a 360 bp PCR product. Amplified mutant DNA can be cleaved by DraII into 123 bp and 237 bp fragments. (E) The stop codon causes a truncation of Tfap2a at amino acid 264. The truncated protein lacks the dimerization and DNA binding domain (DDB, indicated in green). 1a, 1b and 1c are alternative first exons; M, 100 bp marker; TA, transactivation domain; und., undigested PCR product.

 


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  		Fig. 3. Expression of tfap2a during development of zebrafish embryos. (A-F) tfap2a is expressed widely at 24 hpf in (A) spinal cord neurons, the pronephric duct and widely in the epidermis; in (B) neural crest cells in the arch primordia; in (C) migrating posterior lateral line primordium; in (D) epidermal cells surrounding the olfactory vesicle; in (E) segmentally arranged cell groups in the lateral hindbrain; and in (F) bilateral clusters of cells in the tegmentum. (G,H) Expression of tfap2a is increased in the central nervous system at 48 hpf. (G) tfap2a is expressed in the telencephalon, tectum, pretectum and tegmentum. (H) Two laterally stripes of hindbrain neurons express tfap2a. The white arrowhead indicates the lateral and the black arrowhead indicates the more medial stripe of the tfap2a expression domain. In the isthmus, expression of tfap2a is not detected, while strong expression is established in the adjacent optic tectum and cerebellum. (I) The cells in the inner nuclear layer of the retina express tfap2a. (J) The lateral line organs express tfap2a. (K,L) At 72 hpf, a few cells adjacent to the forming gut tube express tfap2a. which are probably neurons of the sympathetic ganglia. (L,M) Higher magnification of the area adjacent to the gut tube where sympathetic ganglia form (72 hpf and 96 hpf respectively; arrowheads indicate tfap2a-expressing cells). (A-M) Anterior towards the left; (A-D,G,I,J) lateral views; (F,H,K-M) dorsal views. ap, arch primordia; ce, cerebellum; ep, epidermal cells; epi, epiphysis; hb, hindbrain; i, isthmus; inl, inner nuclear layer of the retina; llp, lateral line primordia; nm, neuromast cells; ot, otic vesicle; ov, olfactory vesicle; pnd, pronephric duct; sc, spinal cord; symp, cells of sympathetic ganglia; teg, tegmentum; tec, tectum.

 


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  		Fig. 5. tfap2a and phox2a expression and NA neuron specification in the LC. (A) Lateral view and (B) dorsal view of a wild-type embryo at 33 hpf showing expression of tfap2a (red) and phox2a (blue) in the hindbrain. phox2a and tfap2a are expressed in a segmental pattern reflecting rhombomeres 1-7 (r1-r7). In the rhombomeres, small groups of neurons in the ventral medial expression domain of tfap2a co-express phox2a (black arrowheads; white arrowheads indicate the lateral expression of tfap2a). In the spinal cord, both genes are expressed widely. (C) Dorsal view of a wild-type embryo at 33 hpf. In the locus coeruleus, tfap2a (red) and phox2a (blue) are co-expressed. (D) Dorsal view of a wild-type embryo at 33 hpf. Double staining for th (blue) and tfap2a (red) confirmed the co-localization of th and phox2a in the LC. (E) Lateral view of a wild-type embryo at 38 hpf. In the ventral part of r6-r7, most of the cells co-express tfap2a (red) and phox2a (blue), while in the dorsal part, only tfap2a is expressed. (F) Dorsal view of the same embryo. Co-expression of tfap2a and phox2a continues in the medial line of tfap2a expression (black arrowheads; white arrowheads indicate the lateral expression of tfap2a). (G-J) Transversal sections of embryos at 33 hpf showing expression of tfap2a and phox2a at the level of the LC (G,I; left and right halves show left and right LC from different sections) and rhombomere 6-7 and the medulla (H,J). In I and J, the same sections as in G and H are shown, but the Fast Red dye used to detect tfap2a expression is visualized by fluorescence microscopy. The phox2a expression domains in LC and medulla are part of the tfap2a expression domain, albeit tfap2a is expressed at relatively low levels in the region of phox2a-expressing cells. (K) Lateral view of a wild-type embryo at 48 hpf. phox2a is expressed in the LC and anterior lateral line ganglia (allg) and sensory ganglia (sg). (L) Lateral view of a tfap2a mutant embryo at 48 hpf. The LC develops in the mutant embryo, as revealed by the expression of phox2a in this region. The white arrow highlights the absence of phox2a expression at the position of the sensory ganglia. (M) Dorsal view of the hindbrain of a wild-type embryo at 72 hpf showing th expression in the posterior hindbrain. (N) Dorsal view of a soulless/phox2a mutant embryo, with th expression in the posterior hindbrain that is not distinguishable from wild-type. (A-F,K-N), anterior towards the left; (G-J), transverse sections. LC, locus coeruleus; MO, medulla oblongata; sc, spinal cord; sg, sensory ganglia; tec, tectum; 1-7, rhombomeres 1-7.

 


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  		Fig. 6. Development of the peripheral nervous system in tfap2a. (A-F) Detection of PNS and CNS components by whole-mount immunohistochemistry with anti-Hu antibodies. (A,B) Dorsal view of the anterior intestine at 72 hpf in (A) wild-type and (B) tfap2a mutant embryos showing sympathetic (black arrows) and enteric neurons (white arrows). (C,D) Dorsal view of the posterior gut tube at 48 hpf. (D) In tfap2a mutant embryos no enteric neurons can be detected in the posterior gut tube. (E,F) Lateral view of the trunk region of (E) wild-type and (F) tfap2a mutant embryos 48 hpf: Rohan Beard sensory neurons (white arrows) and dorsal root ganglia (black arrows) can be detected in mutants, but the number of neurons is reduced in each group. (G,H) Whole-mount in situ hybridization with isl1 riboprobes revealing presence of sympathetic neurons (black arrows) in (G) wild-type and (H) tfap2a mutant embryos. (I,J) Visualization of sympathetic neurons (black arrows) in (I) wild-type and (J) tfap2a mutant embryos by whole-mount in situ hybridization with phox2a riboprobes. (A-J) anterior towards the left. no, notocord; sc, spinal cord.

 


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  		Fig. 7. Retinoic acid (RA) is required and sufficient to induce formation of th-expressing NA neurons in the posterior hindbrain. (A-P) Analysis of th expression in 72 hpf embryos by whole-mount in situ hybridization. In the diencephalon (DC) no differences of th expression are detected in (B) RA treated wild-type embryos, (C) nls/raldh2 mutants and (D) tfap2a mutant embryos exposed to RA. (E-H) th expression is normal in the locus coeruleus (LC) of (F) RA-treated embryos, (G) nls/raldh2 and (H) remains absent in tfap2a mutant embryos exposed to RA. (J) In the medulla oblongata and area postrema (MO), RA treatment induces th expression. (K) nls/raldh2 mutant embryos express th in one or two cells of the MO only. (L) RA fails to induce th expression in the hindbrain of tfap2a mutant embryos. (M,N) A dramatic increase of enteric neurons expressing th in the gut region occurs after RA treatment. (O) Embryos mutant for nls/raldh2 have a reduced number of th-expressing cells in the sympathetic ganglia (Symp). (P) RA is not able to induce th expression in the gut region of tfap2a mutant embryos. (A-P) Dorsal views, anterior towards the left.

 


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  		Fig. 9. Retinoic acid-mediated induction of th-expressing cells requires protein synthesis and coincides with increased tfap2a expression. (A-D) Cycloheximide (CHX; B), retinoic acid (RA; C) or a combination of both treatments (D) does not alter the expression of th in the locus coeruleus (LC) compared with untreated wild-type embryos (A). (E-H) th expression at 33 hpf in the hindbrain of wild-type embryos (H) and embryos exposed to (F) cycloheximide, (G) retinoic acid and (H) a combination of cycloheximide and RA. CHX blocks the RA induced expression of th in medulla at 33 hpf. (H) Brown cells are pigment cells and not NA neurons, which would stain blue from whole-mount in situ hybridization procedure. (I-L) Expression of th in the medulla oblongata of wild-type embryos at 72 hpf. (J) Cycloheximide-treated embryos, (K) retinoic acid-treated embryos, (L) embryos treated with both cycloheximide and RA. (M-P) Expression of th in the region in which sympathetic neurons form in wild-type embryos (M) at 33 hpf. Embryos exposed to cycloheximide (N), retinoic acid (O) and a combination of both (P). CHX blocks the RA-induced expression of th in sympathetic ganglia. (Q,R) Expression of tfap2a in wild-type embryos (Q) and embryos treated with RA between 24 and 33 hpf (R). RA induces the expression of tfap2a in the hindbrain. (S,T) Expression of tfap2a (red) and phox2a (blue) in the hindbrain of wild-type embryos (S) and nls mutant embryos (T). The expression of tfap2a (red) and phox2a (blue) is reduced in the posterior hindbrain (r6,7) of neckless/raldh2-deficient mutants. (A-R) dorsal views, anterior towards the left; (S,T) lateral views, anterior towards the left. ce, cerebellum; hb, hindbrain; sc, spinal cord; tec, tectum; 1-7, rhombomeres r1-7; sympat, region in which sympathetic neurons form.

 


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  		Fig. 8. Retinoic acid treatment between 24 and 33 hpf does not affect the anterioposterior pattern of the hindbrain. (A-H) Analysis of the hoxa2, hoxb2, hoxb3 and hoxd3 expression patterns did not reveal any changes in anterioposterior patterning of the hindbrain when embryos were exposed to RA between 24 and 33 hpf. (I,J) In situ hybridization for epha4 expression did not reveal any anterioposterior shifts in the hindbrain rhombomere pattern of 33hpf embryos after RA treatment. (K,L) At 48 hpf, embryos treated with retinoic acid show a loss of the lateral expression domains of epha4 in rhombomere 3 and 5. cont, control embryos exposed to DMSO; r, rhombomere; RA, retinoic acid. (A-L) Dorsal views of flat-mount preparations, anterior towards the left.

 

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© The Company of Biologists Ltd 2003