Expression and function of nr4a2, lmx1b, and pitx3in zebrafish dopaminergic and noradrenergic neuronal development

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Abstract
Dopaminergic neurons form in diverse areas of the vertebrate di- and mesencephalon to constitute several major neuromodulatory systems. While much is known about mammalian mesencephalic dopaminergic neuron development, little is known about the specification of the diencephalic dopaminergic groups. The transcription factors Pitx3 and Lmx1b play an important role in mammalian mesencephalic dopaminergic specification, and Nurr1/Nr4a2 has been shown to contribute to specification of the dopaminergic neurotransmitter phenotype. We use zebrafish to analyze potentially evolutionarily conserved roles of these transcription factors in a vertebrate brain that lacks a mesencephalic dopaminergic system, but has an ascending dopaminergic system in the ventral diencephalon. We use a combination of fluorescent in situ hybridization and immunohistochemistry to determine whether nr4a2, lmx1b, and pitx3 genes are expressed in mature dopaminergic neurons or in potential precursor populations. We identify a second nr4a2 paralogue, nr4a2a, and find it co-expressed with Tyrosine hydroxylase in preoptic, pretectal and retinal amacrine dopaminergic neurons, while nr4a2b is only expressed in preoptic and retinal dopaminergic neurons. Both zebrafish nr4a2 paralogues are not expressed in ventral diencephalic dopaminergic neurons with ascending projections. Combined morpholino antisense oligo mediated knock-down of both nr4a2a and nr4a2b transcripts reveals that all zebrafish dopaminergic neurons expressing nr4a2a depend on Nr4a2 activity for tyrosine hydroxylase and dopamine transporter expression. Zebrafish lmx1b.1 is expressed in noradrenergic neurons of the locus coeruleus and medulla oblongata, but knock-down reveals that it is specifically required for tyrosine hydroxylase expression only in the medulla oblongata area postrema noradrenergic neurons. Both lmx1b genes and pitx3 are not expressed in dopaminergic neurons, but in a diencephalic territory that might contain precursor cells for ventral diencephalic dopaminergic neurons. Upon morpholino knock-down of both lmx1b paralogues, the number of neurons in diencephalic dopaminergic clusters with ascending projections appears specifically reduced. Thus lmx1b paralogues may contribute to the generation of diencephalic dopaminergic precursors. Conversely, knock-down of pitx3 does not specifically affect any diencephalic DA cluster. Our data indicate a conserved evolutionary role of Nr4a2 proteins in specification of the neurotransmitter phenotype, albeit it appears to be only one of several regulatory modules of dopaminergic differentiation, as most ventral diencephalic dopaminergic neurons do not express nr4a2 genes in zebrafish. For zebrafish lmx1b genes, which are not expressed in mature dopaminergic neurons, our data suggest a role in diencephalic precursor populations contributing to the ascending dopaminergic systems. A di-mesencephalic longitudinal domain of lmx1b expression may be the basis for the expansion and posterior shift of ventral di-/mesencephalic dopaminergic populations with ascending projections during evolution.