Gene expression pattern

Dopamine transporter expression distinguishes dopaminergic neuronsfrom other catecholaminergic neurons in the developing zebra®sh embryo

Jochen Holzschuh, Soojin Ryu, Fritz Aberger1, Wolfgang Driever*

Department of Developmental Biology, Institute Biology 1, University of Freiburg, Hauptstrasse 1, D-79104 Freiburg, Germany

Received 4 October 2000; received in revised form 1 December 2000; accepted 1 December 2000

Abstract

To characterize the formation of the dopaminergic system in the developing zebra®sh CNS, we cloned cDNAs encoding tyrosine

hydroxylase (th), an enzyme in dopamine synthesis, and the dopamine transporter (dat), a membrane transport protein which terminates

dopamine action by re-uptake. Dopaminergic neurons are ®rst detected between 18 and 19 h post-fertilization in a cluster of cells in the

ventral diencephalon. Subsequently, th and dat detection identi®es dopaminergic neurons in the olfactory bulb, the pretectum, the retina and

the locus coeruleus. Neurons expressing th but not dat are adrenergic or noradrenergic, and are found in the locus coeruleus, the medulla, the

likely analog of the carotid body, and precursors of the enteric and sympathetic nervous system. q 2001 Elsevier Science Ireland Ltd. All

rights reserved.

Keywords: Zebra®sh; Dopaminergic system; Catecholaminergic system; Tyrosine hydroxylase; Dopamine transporter; Diencephalon; Olfactory bulb; Pretec-

tum; Retina; Locus coeruleus; Medulla; Carotid body; Parkinson's disease

1. Results and discussion

1.1. Cloning of tyrosine hydroxylase and the dopamine

transporter from zebra®sh

The zebra®sh Danio rerio provides an excellent system to

study development of the dopaminergic system, as it is

accessible to genetic analyses while allowing visual access

to single neurons in living embryos. The catecholaminergic

system (CA) consists of dopaminergic (DA), noradrenergic

(NA) and adrenergic neurons. Tyrosine hydroxylase (TH)

catalyses the initial step in the catecholamine biosynthetic

pathway, the conversion of tyrosine to l-DOPA, and is

expressed by all catecholaminergic neurons. To distinguish

DA neurons from other CA neurons we cloned the dopa-

mine transporter (dat), which in vertebrates is exclusively

expressed in dopaminergic neurons (Shimada et al., 1992;

Augood et al., 1993; Cerruti et al., 1993; Ciliax et al., 1995;

Hersch et al., 1997). Previously, zebra®sh neurons expres-

sing TH but not dopamine b-hydroxylase (DbH, converts

dopamine to noradrenaline) were considered dopaminergic

(Guo et al., 1999). By assaying the expression of dat we can

identify dopaminergic neurons more directly.

We isolated, by reverse transcriptase-polymerase chain

reaction (RT-PCR), a 851 bp cDNA fragment for th, and a

704 bp fragment for dat (Guo et al., 1999). We derived the

full-length open reading frame (ORF) for dat by using the

RT-PCR fragment, genomic sequences, and rapid ampli®-

cation of cDNA ends (3 0-RACE). The identity of our dat

ORF as the zebra®sh dat gene is con®rmed by high sequence

homology to other dat genes (Fig. 1) and its speci®c expres-

sion in cells expected to contribute to the dopaminergic

system.

1.2. Expression of th and dat during zebra®sh development

Expression of th mRNA has previously been reported for

20-somite stage zebra®sh embryos (Guo et al., 1999). We

have assayed th and dat expression throughout zebra®sh

embryogenesis up to 96 h post-fertilization (hpf). For

early embryonic stages, we use general anatomical terms

to describe the expression patterns, as teleost neuroanato-

mical structures are not fully differentiated yet (Wullimann

et al., 1996). Expression of dat or th was not detected before

the 16-somite stage (18 hpf). We discuss the catecholami-

nergic clusters in the order in which they appear.

(1) Ventral diencephalon. 18±20 hpf: 1±3 neurons begin

to express dat and th in the ventral diencephalon, bilaterally

Mechanisms of Development 101 (2001) 237±243

0925-4773/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved.

PII: S0925-4773(01)00287-8

www.elsevier.com/locate/modo

* Corresponding author. Tel.: 149-761-203-2587; fax: 149-761-203-

2597.

E-mail address: driever@ruf.uni-freiburg.de (W. Driever).1 Present address: Institute of Genetics, University of Salzburg, Hellbrun-

nerstrasse 34, 5020 Salzburg, Austria.

(Figs. 2A and 3A). By 3 days post-fertilization (dpf) the

number of th-expressing neurons increases to 20±30 (Fig.

2I).

(2) Locus coeruleus. By 24 hpf, cells in the hindbrain at

the position corresponding to the developing locus coeru-

leus (LC) express th but not dat (Figs. 2E and 3A). Surpris-

ingly, from 60 hpf onward, one or two neurons express dat

in the region corresponding to the LC (Fig. 3F). The number

of th-expressing neurons in the LC does not increase signif-

icantly during embryonic development, with a maximum of

seven cells through adulthood (Ma, 1994a,b).

(3) Medulla oblongata. At about 36 hpf, neurons of the

medulla oblongata in the hindbrain start to express th (MC,

Fig. 2I,L).

(4) Olfactory bulb. Dopaminergic neurons expressing th

and dat are detected in the olfactory bulbs from 48 hpf

onward (ObC, Fig. 2I; ObD, Fig. 3B,D,E).

(5) Pretectum. By 60 hpf, dat and th are expressed in

neurons in the pretectum (PtC, Fig 2I; PtD, Fig 3E).

(6) Retina. By 60 hpf, a restricted number of amacrine

cells in the inner nuclear layer of the retina starts to express

dat and th (Figs. 2J and 5B).

(7) Area of optic nerve. By 48 hpf, dat expression occurs

in cells surrounding the optic nerve (Fig. 3G). The dat-posi-

tive cells extend from the head of the optic nerve, in the

ganglion cell layer, to the junction of the optic nerve with

the optic tract (Fig. 3H). This distribution and the epithelial

characteristics coincide with those of the reticular astrocytes

of the optic nerves of other teleost ®sh (Maggs and Scholes,

1990; MacDonald et al., 1997).

(8) Arch-associated neurons: carotid body. After 20 hpf,

two bilateral clusters of cells located outside the CNS lateral

to rhombomere 1 begin expressing th (Figs. 2C and 3A).

These cells are associated with the arch primordia. These

clusters then migrate anteriorly and by 3 dpf fuse at the

midline slightly rostral and dorsal to the heart (Fig. 2K,L).

(9) PNS. By 48 hpf, neurons of the sympathetic nervous

system located bilaterally dorsal to the forming gut express

th (Fig. 2H) but not dat (Fig. 3C). Later (55 hpf), some

neurons of the enteric nervous system, located at the

midline anterior to the swim bladder, start to express th

(Figs. 2L and 4B). In contrast, the th-expressing sympa-

thetic neurons are located ventrolateral to the cranial noto-

chord (Fig. 4A,C).

In contrast to mouse pancreatic precursor cells that

express tyrosine hydroxylase (Teitelman et al., 1993), th

expression was not detected in the zebra®sh pancreas at

any developmental stage.

J. Holzschuh et al. / Mechanisms of Development 101 (2001) 237±243238

Fig. 1. The amino acid sequence from the zebra®sh (zfh) dopamine transporter (DAT) is compared with those of human (hum) DAT (Giros et al., 1992), human

noradrenaline transporter (hum-NAT) (Porzgen et al., 1995) and human serotonin transporter (hum-5HT) (Ramamoorthy et al., 1993). Gaps have been added to

optimize the alignment. These four transporters share 39% sequence identity (asterisks). zfh-DAT is highly homologous to hum-DAT (identical sequences are

underlined). The predicted zebra®sh DAT protein has 76% amino acid identity and 9% similarity to human DAT (hum-DAT; Giros et al., 1992), 63% identity

and 14% similarity to human noradrenaline transporter (hum-NAT; Porzgen et al., 1995), and 45% identity and 20% similarity to the human serotonin

transporter (hum-5HT; Ramamoorthy et al., 1993). Thirteen percent of the aligned amino acids of zfh-DAT are shared with hum-DAT (red shading), but not

with hum-NAT or hum-5-HT.

J. Holzschuh et al. / Mechanisms of Development 101 (2001) 237±243 239

Fig. 2. Tyrosine hydroxylase (th) expression during zebra®sh embryogenesis. Lateral views (except F,K: ventral views, and H: dorsal view) of whole-mount

embryos following in situ hybridization for th, rostral is to the left. (A) Eighteen hpf embryo (yolk removed). (B,C) Twenty hpf embryo at medial (B) and

lateral (C) focal planes. (D,E) Twenty-four hpf old embryos at the focal planes of the diencephalic (D) and locus coeruleus th-expressing cell clusters. (F±H)

Forty-eight hpf embryos in ventral, lateral and dorsal views to show th expression in the CNS, in arch-associated cells (AAN), and in presumptive precursors of

the sympathetic nervous system. (I) Seventy-two hpf embryo at a medial focal plane. th-Expressing cell clusters in the CNS are labeled. In some embryos a few

th-expressing cells can be found outside of the catecholaminergic cell cluster (asterisks). (J±L) Ninety-six hpf embryos: lateral view focusing on the amacrine

cell layer of the retina (J); ventral view showing the arch-associated neurons that have converged to the midline just rostral to the heart (K); and lateral view of

central and peripheral cell clusters at a medial focal plane (L). AAN, arch-associated neurons; AC catecholaminergic amacrine cells; DC, diencephalic

catecholaminergic cluster; ent, enteric neurons; hpf, hours post-fertilization; LC, locus coeruleus; MC, medulla catecholaminergic cluster; ObC, catechola-

minergic cluster in olfactory bulbs; PtC, pretectal catecholaminergic cluster; sym, sympathetic neurons.

J. Holzschuh et al. / Mechanisms of Development 101 (2001) 237±243240

Fig. 3. Expression of the dopamine transporter (dat) during zebra®sh

embryogenesis. (A±H) Whole-mount in situ hybridization detecting dat

expression. Rostral is to the left (A±G): (A,B,E) lateral views; (C,F) dorsal

views; (D,G) ventral views; (H) transversal histological section. (A) In the

24 hpf embryo dat is only expressed in the diencephalon but not in the area

where arch-associated neurons form (white arrow). (B±D) By 48 hpf dopa-

minergic neurons have formed in the olfactory bulb and in the diencepha-

lon, whereas no dat expression can be detected in the areas where the locus

coeruleus, the sympathetic neurons and the arch-associated neurons form

(white arrows). (E,F) By 72 hpf dat expression begins in a subset of neurons

in the pretectum, while expression in the diencephalon and olfactory bulbs

is maintained. (F) Bilateral clusters of neurons in the hindbrain posterior to

the midbrain-hindbrain boundary (arrow heads) express dat. The more

lateral ones are located in the area of the forming locus coeruleus. (G)

By 48 hpf, reticular astrocytes found in parallel to the optic nerve start to

express dat. (H) Transverse section through the eye at 72 hpf, counter-

stained with nuclear red dye (red), shows reticular astrocytes expressing

dat surrounding the optic nerve. A subset of amacrine neurons also

expresses dat. AD, dopaminergic amacrine neurons; DD, diencephalic

dopaminergic cluster; gcl, ganglion cell layer; hpf, hours post-fertilization;

inl, inner nuclear layer; ipl, inner plexiform layer; ObD, olfactory bulb

dopaminergic cluster; on, optic nerve; onl, outer nuclear layer; opl, outer

plexiform layer; pcl, photoreceptor cell layer; PtD, pretectal dopaminergic

cluster; ra, reticular astrocytes.

Fig. 4. Catecholaminergic neurons outside of the CNS. Transverse section

through a zebra®sh embryo (3 dpf) in which th expression has been detected

by whole-mount in situ hybridization (blue), counterstained with nuclear

red dye (red). (A) Section at the level of the ®rst somite. Sympathetic

neurons expressing th lie ventral to the notochord. (B) Section at the

level of the third somite. th-Expressing enteric neurons are located ventral

to the somites and dorsal to the intestine. (C) Sagittal section (dorsal at top

and anterior to the left) through an embryo at 4 dpf. The enteric ganglia is

now more than one cell layer thick and embedded in gastrointestinal tissue.

(D) Sagittal section (dorsal at top and anterior to the left) through a zebra-

®sh embryo at 3 dpf. The th-expressing cells of the arch-associated neurons

are located dorsal to the developing heart. AAN, arch-associated neurons;

AP area postrema of the medulla oblongata; dpf, days post-fertilization; ent,

enteric ganglia; HB, hindbrain; in, intestine; no, notochord; sc, spinal cord;

so, somite; sym, sympathetic ganglia; tel, telencephalon; th, tyrosine hydro-

xylase; y, yolk.

1.3. dat expression identi®es dopaminergic neurons

To determine in which cells th and dat are coexpressed,

we performed in situ hybridization for dat followed by anti-

TH immunohistochemistry (Fig. 5). By two dpf, TH and dat

are coexpressed in cell clusters in the olfactory bulbs, the

pretectum, and in the thalamus and the hypothalamus (Fig.

5A,C). By two dpf, catecholaminergic neuronal clusters

J. Holzschuh et al. / Mechanisms of Development 101 (2001) 237±243 241

Fig. 5. Identi®cation of the relative positions of dopaminergic and adrenergic/noradrenergic neurons in the larval zebra®sh brain. (A±C,E) Lateral views,

anterior is to the left, dorsal to the top; (D) dorsal view, anterior is to the left. (A±D) Catecholaminergic neurons in the developing brain. dat-Expressing cells

are labeled in blue by whole-mount in situ hybridization, TH-containing cells are labeled in brown by whole-mount immunohistochemistry. Dark brown cells

are double-labeled cells containing dat and TH. (A) Forty-eight hpf embryo. (B) At 96 hpf dopaminergic neurons expressing both dat and TH can be identi®ed

in the amacrine layer of the retina. (C,D) By 96 hpf, all clusters of catecholaminergic neurons described in adult zebra®sh can be found. (E) A diagram

representing the catecholaminergic cell groups of the zebra®sh embryonic brain. AC, dopaminergic amacrine cells; dat, dopamine transporter; HB, hindbrain;

hpf; hours post-fertilization; NC; notochord; for further abbreviations see panel (E).

expressing only TH but not dat are present in the thalamus,

the hypothalamus, the locus coeruleus and the medulla

oblongata.

At four dpf TH and dat are co-expressed in some of the

th-expressing cells in bilateral cell clusters corresponding to

the position of the locus coeruleus (Figs. 3F and 5D). This

demonstrates that a subset of neurons in the locus coeruleus

is dopaminergic. Thus the hypothesis that this region

contains exclusively adrenergic and noradrenergic neurons

in teleosts (Meek, 1994) cannot be sustained.

At four dpf, bilateral dat-expressing cell clusters in the

anterior hindbrain had no anti-TH immunoreactivity (Fig.

5D,F). In adult zebra®sh, cells in the corresponding region

of the hindbrain are immunoreactive for both DAT and TH

(Ma, 1997). Similarly, a cluster in the most rostroventral

part of the ventral thalamus expresses dat only. It is possible

that these two cell clusters may start to express th only later

in development. Immunohistochemical studies in other tele-

ost species also have shown DA and/or TH immunoreactive

neurons within the most ventral part of the hypothalamus

(Hornby et al. 1987; Meek, 1994).

Six main groups of catecholaminergic neurons have been

described in the vertebrate CNS: (1) a caudal rhombence-

phalic group (medulla); (2) a rostral rhombencephalic group

(locus coeruleus); (3) a mesencephalic group; (4) a dience-

phalic group; (5) an olfactory bulb group; (6) a retinal group

(reviewed in Smeets and Reiner, 1994). With the exception

of the mesencephalic group, each of these groups is present

in the developing zebra®sh brain (see scheme in Fig. 5E).

dat or th expression, or TH immunoreactivity does not

appear in the mesencephalon at any developmental stage

in the zebra®sh, similar to other teleost species (EkstroÈm

et al., 1992). Rink and Wullimann (2000) recently argued

that adult zebra®sh basal diencephalic DA clusters may be

functionally homologous to basal mesencephalic DA clus-

ters in higher vertebrates.

2. Methods

2.1. Cloning and sequencing

Zebra®sh th and dat fragments were ampli®ed by RT±

PCR from RNA of 3dpf embryos using degenerate primers:

DAT forward: 5 0-GC(A/C/G/T)-GG (A/C/G/T)-ATG-CC

(A/C/G/T)-(C/T)T(A/C/G/T)-TA(C/T)-ATG-GA-3 0; DAT

reverse: 5 0-TA(A/G)-CA(A/G)-TT(A/G)-TT(A/C/G/T)-

GT(A/G)-AA(C/T)-TT(A/G)-TT(A/G)-TA-3 0. The 704 bp

dat RT±PCR product was used to screen a zebra®sh PAC

®lter library (RZPD, Berlin). dat 5 0 sequence was obtained

from genomic sequences. dat 3 0 end sequence was obtained

using SMART-RACE kit (Clontech). The dat sequence has

been submitted to GenBank (AF318177).

2.2. In situ hybridization and immunohistochemistry

Embryos of AB strain zebra®sh were staged according to

Kimmel et al. (1995). Embryos were raised at 28.58C with

0.2 mM phenylthiourea (Sigma) in egg water to prevent

pigmentation (Wester®eld, 1995). For some stages, expres-

sion of th was compared between phenylthiourea treated

wild-type and non-pigmented albino homozygous embryos.

No differences in th expression were detected (data not

shown). Whole-mount in situ hybridization (Hauptmann

and Gerster, 1994) and immunohistochemistry (Solnica-

Krezel and Driever, 1994) were as described. Primary anti-

body: rat anti-TH (Chemicon, Hofheim, Germany).

Acknowledgements

We thank Annette Bodenhausen and Roswitha Koppa for

excellent technical assistance, and Gerlinde Wussler for

animal care. We are grateful to Karen Lunde for critical

reading of the manuscript. S.R. was supported by a Alex-

ander von Humboldt Fellowship, and F.A. by a Schroedin-

ger Fellowship and a Long Term Fellowship from EMBO.

This study was supported by DeveloGen AG, GoÈttingen,

Germany, a Landesschwerpunktprogramm Baden-WuÈrt-

temberg, and the Center for Applied Biosciences (ZAB),

University of Freiburg.

References

Augood, S.J., Westmore, K., McKenna, P.J., Emson, P.C., 1993. Coexpres-

sion of dopamine transporter mRNA and tyrosine hydroxylase mRNA

in ventral mesencephalic neurons. Brain Res. Mol. Brain Res. 20, 328±

334.

Cerruti, C., Walther, D.M., Kuhar, M.J., Uhl, G.R., 1993. Dopamine trans-

porter mRNA expression is intense in rat midbrain neurons and modest

outside the midbrain. Brain Res. Mol. Brain Res. 18, 181±186.

Ciliax, B.J., Heilman, C., Demchyshyn, L.L., Pristupa, Z.B., Ince, E.,

Hersch, S.M., Niznik, H.B., Levey, A.I., 1995. The dopamine transpor-

ter: Immunochemical characterization and localization in the brain. J.

Neurosci. 15, 1714±1723.

EkstroÈm, P., Honkanen, T., Borg, B., 1992. Development of tyrosine hydro-

xylase-, dopamine-, and dopamine-b-hydroxylase-immunoreactive

neurons in a teleost, the three-spined stickleback. J. Chem. Neuroanat.

5, 481±501.

Giros, B.:., el Mestikawy, S., Godinot, N., Zheng, K., Han, H., Yang-Feng,

T., Caron, M.G., 1992. Cloning, pharmacological characterization, and

chromosome assignment of the human dopamine transporter. Mol.

Pharmacol. 42, 383±390.

Guo, S., Wilson, W.S., Cooke, S., Chitnis, A.B., Driever, W., Rosenthal, A.,

1999. Mutations in zebra®sh unmask shared regulatory pathways

controlling the development of catecholaminergic neurons. Dev. Biol.

208, 473±487.

Hauptmann, G., Gerster, T., 1994. Two-color whole-mount in situ hybri-

dization to vertebrate and Drosophila embryos. Trends Genet. 10, 266±

269.

Hersch, S.M., Yi, H., Heilman, C.J., Edwards, R.H., Levley, A.I., 1997.

Subcellular localization and molecular topology of dopamine transpor-

ter in the striatum and substantia nigra. J. Comp. Neurol. 388, 211±227.

Hornby, P.J., Piekut, D.T., Demski, L.S., 1987. Localization of immunor-

eactive tyrosine hydroxylase in the gold®sh brain. J. Comp. Neurol.

261, 1±14.

Kimmel, C.B., Ballard, W.W., Kimmel, S.R., Ullmann, B., Schilling, T.F.,

J. Holzschuh et al. / Mechanisms of Development 101 (2001) 237±243242

1995. Stages of embryonic development of zebra®sh. Dev. Dyn. 203,

253±310.

Ma, P.M., 1994a. Catecholaminergic systems in the zebra®sh. I. Number,

morphology, and histochemical characteristics of neurons in the locus

coeruleus. J. Comp. Neurol. 344, 242±255.

Ma, P.M., 1994b. Catecholaminergic systems in the zebra®sh. II. Projection

pathways and pattern of termination of the locus coeruleus. J. Comp.

Neurol. 344, 256±269.

Ma, P.M., 1997. Catecholaminergic systems in the zebra®sh. III. Organiza-

tion and projection pattern of medullary dopaminergic and noradrener-

gic neurons. J. Comp. Neurol. 381, 411±427.

Macdonald, R., Scholes, J., StraÈhle, U., Brennan, C., Holder, N., Brand, M.,

Wilson, S.W., 1997. The Pax protein Noi is required for commissural

axon pathway formation in the rostral forebrain. Development 124,

2397±2408.

Maggs, A., Scholes, J., 1990. Reticular astrocytes in the ®sh optic nerve:

macroglia with epithelial characteristics form an axially repeated lace-

work pattern, to which nodes of Ranvier are apposed. J. Neurosci. 15,

3716±3729.

Meek, J., 1994. Catecholamines in the brains of Osteichthyes. In: Smeets,

W.J.A.J., Reiner, A. (Eds.). Phylogeny and Development of Catecho-

lamine Systems in the CNS of Vertebrates, Cambridge University Press,

Cambridge, UK, pp. 49±76.

Porzgen, P., Bonisch, H., Bruss, M., 1995. Molecular cloning and organiza-

tion of the coding region of the human norepinephrine transporter gene.

Biochem. Biophys. Res. Commun. 215, 1145±1150.

Ramamoorthy, S., Bauman, A.L., Moore, K.R., Hong, H., Yang-Feng, T.L.,

Chang, A.S., Ganapathy, V., Blakely, R.D., 1993. Antidepressant and

cocaine sensitive human serotonin transporter: Molecular cloning,

expression, and chromosomal localization. Proc. Natl. Acad. Sci.

USA 90, 2542±2546.

Rink, E., Wullimann, M.F., 2000. The teleostean (zebra®sh) dopamine

system ascending to the subpallium (striatum) is located in the basal

diencephalon (posterior tuberculum). Brain Res. 1, 1±15.

Shimada, S., Kitayama, S., Walther, D., Uhl, G., 1992. Dopamine trans-

porter mRNA: dense expression in ventral midbrain neurons. Brain.

Res. Mol. Brain Res. 13 (4), 359±362.

Smeets, W.J.A.J., Reiner, A. (Eds.), 1994. Phylogeny and Development of

Catecholamine Systems in the CNS of Vertebrates Cambridge Univer-

sity Press, Cambridge.

Solnica-Krezel, L., Driever, W., 1994. Microtubule arrays of the zebra®sh

yolk cell: organization and function during epiboly. Development 120,

2443±2455.

Teitelman, G., Alpert, S., Polak, J.M., Martinez, A., Hanahan, D., 1993.

Precursor cells of mouse endocrine pancreas coexpress insulin, gluca-

gon and neuronal proteins tyrosine hydroxylase and neuropeptide Y, but

not pancreatic polypeptide. Development 118, 1031±1039.

Wester®eld, M., 1995. The Zebra®sh Book. A Guide for the Laboratory Use

of Zebra®sh (Danio rerio). 3rd Edition. University of Oregon Press,

Eugene, OR.

Wullimann, M.F., Rupp, B., Reichert, H., 1996. Neuroanatomy of the

Zebra®sh Brain, BirkhaÈuser, Basel.

J. Holzschuh et al. / Mechanisms of Development 101 (2001) 237±243 243