Expression and function of nr4a2, lmx1b, and pitx3 in zebrafish dopaminergic and noradrenergic neuronal development.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

Differential roles of transcriptional mediator complex subunits Crsp34/Med27, Crsp150/Med14 and Trap100/Med24 during zebrafish retinal development.

The transcriptional mediator complex has emerged as an important component of transcriptional regulation, yet it is largely unknown whether its subunits have differential functions in development. We demonstrate that the zebrafish mutation m885 disrupts a subunit of the mediator complex, Crsp34/Med27. To explore the role of the mediator in the control of retinal differentiation, we employed two additional mutations disrupting the mediator subunits Trap100/Med24 and Crsp150/Med14. Our analysis shows that loss of Crsp34/Med27 decreases amacrine cell number, but increases the number of rod photoreceptor cells. In contrast, loss of Trap100/Med24 decreases rod photoreceptor cells. Loss of Crsp150/Med14, on the other hand, only slightly reduces dopaminergic amacrine cells, which are absent from both crsp34(m885) and trap100(lessen) mutant embryos. Our data provide evidence for differential requirements for Crsp34/Med27 in developmental processes. In addition, our data point to divergent functions of the mediator subunits Crsp34/Med27, Trap100/Med24, and Crsp150/Med14 and, thus, suggest that subunit composition of the mediator contributes to the control of differentiation in the vertebrate CNS.

Presence of pituitary adenylate cyclase-activating polypeptide (PACAP) defines a subpopulation of hypothalamic POMC neurons.

Several lines of evidence support a role for pituitary adenylate cyclase-activating polypeptide (PACAP) in the regulation of energy balance. In the present study, we have used fluorescent in situ hybridization and immunohistochemistry to investigate in detail the cellular localization and chemical content of PACAP mRNA- and peptide-containing neuronal cell bodies in the mediobasal hypothalamus of the rat. PACAP mRNA-containing cell bodies were demonstrated in high numbers in the ventromedial hypothalamic nucleus (VMH) and in lower numbers in the arcuate nucleus (Arc). In colchicine-treated rats, PACAP immunoreactivity was demonstrated in many cell bodies of the VMH and several cell bodies of the ARC. Double-labeling revealed that PACAP immunoreactivity was present in approximately 20% of pro-opiomelanocortin (POMC) neurons in the ventrolateral Arc as shown by presence of alpha-melanocyte-stimulating hormone (alpha-MSH), but not in agouti-related peptide (AgRP)-containing neurons in the ventromedial aspect of the Arc. PACAP immunoreactivity was also colocalized with the vesicular acetylcholine transporter (VAChT; a marker for cholinergic neurons) in Arc POMC neurons. Brainstem POMC neurons in the commissural part of the solitary tract nucleus were devoid of PACAP immunoreactivity. However, several VAChT-positive neurons in the dorsal motor nucleus of the vagus nerve were also PACAP immunoreactive, whereas VAChT-positive neurons of the motor nucleus of the hypoglossal nerve were PACAP-negative. The results show presence of PACAP with alpha-MSH in a subpopulation of hypothalamic POMC neurons and point further to the neurochemical heterogeneity of hypothalamic, but not brainstem, POMC neurons.

Hypothalamic proopiomelanocortin (POMC) neurons have a cholinergic phenotype.

Neuronal networks originating in the hypothalamic arcuate nucleus play fundamental roles in the control of energy balance. Neuropeptide Y (NPY)-producing neurons in the arcuate nucleus stimulate food intake, whereas arcuate nucleus neurons that release the proopiomelanocortin (POMC)-derived peptide alpha-melanocyte-stimulating hormone (alpha-MSH) potently reduce food intake. Relatively little attention has been focused on classical neurotransmitters in regulation of food intake. Here, we have investigated the potential presence of acetylcholine (ACh) in NPY- and POMC-containing neuronal populations of the arcuate nucleus. Antisera to proteins required for cholinergic neurotransmission, including choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT), were employed in double-labeling immunohistochemical experiments. In colchicine-treated rats, ChAT- and VAChT-immunopositive cell bodies were located in the ventral aspect of the arcuate nucleus. ChAT and VAChT immunoreactivities were demonstrated in alpha-MSH- and cocaine- and amphetamine-regulated transcript (CART)-containing cell bodies of the arcuate nucleus, whereas cell bodies containing NPY or agouti-related peptide (AGRP) were distinct from VAChT-immunoreactive neuronal perikarya. VAChT immunoreactivity was also present in a large number of alpha-MSH-containing nerve fiber varicosities throughout the central nervous system. In the commissural part of the nucleus tractus solitarius, no alpha-MSH-containing cell bodies were found to have ChAT or VAChT immunoreactivity. The presence of markers for cholinergic neurotransmission in a subpopulation of hypothalamic POMC/CART neurons suggests co-release of ACh with peptides derived from the POMC precursor and CART. The results indicate a role for ACh in control of energy balance, mediating the effects of peripheral hormones such as leptin and insulin.

A mutation in the zebrafish Na,K-ATPase subunit atp1a1a.1 provides genetic evidence that the sodium potassium pump contributes to left-right asymmetry downstream or in parallel to nodal flow.

While there is a good conceptual framework of dorsoventral and anterioposterior axes formation in most vertebrate groups, understanding of left-right axis initiation is fragmentary. Diverse mechanisms have been implied to contribute to the earliest steps of left-right asymmetry, including small molecule signals, gap junctional communication, membrane potential, and directional flow of extracellular liquid generated by monocilia in the node region. Here we demonstrate that a mutation in the zebrafish Na,K-ATPase subunit atp1a1a causes left-right defects including isomerism of internal organs at the anatomical level. The normally left-sided Nodal signal spaw as well as its inhibitor lefty are expressed bilaterally, while pitx2 may appear random or bilateral. Monocilia movement and fluid circulation in Kupffer's vesicle are normal in atp1a1a(m883) mutant embryos. Therefore, the Na,K-ATPase is required downstream or in parallel to monocilia function during initiation of left-right asymmetry in zebrafish.

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

Tfap2a is a transcriptional activator expressed in many different cell types, including neurons, neural crest derivatives and epidermis. We show that mutations at the zebrafish locus previously called mont blanc (mob) or lockjaw (low) encode tfap2a. The mutant phenotype reveals that tfap2a is essential for the development of hindbrain noradrenergic (NA) neurons of the locus coeruleus, medulla and area postrema, as well as for sympathetic NA neurons, epibranchial placode derived visceral sensory ganglia, and craniofacial and trunk crest derivatives. We focus our analysis on the role of tfap2a NA differentiation in the CNS. In the locus coeruleus, Phox2a and Tfap2a are co-expressed and are both required for NA development. By contrast, in the medulla Phox2a and Tfap2a are expressed in adjacent overlapping domains, but only tfap2a activity is required for NA differentiation, as NA neurons develop normally in soulless/phox2a mutant medulla. phox2a and tfap2a do not appear to affect each others expression. Our studies show that two distinct inductive mechanisms control NA development in the zebrafish hindbrain. For the posterior hindbrain, we identify retinoic acid as an important signal to induce NA differentiation in the medulla oblongata and area postrema, where it expands the tfap2a expression domain and thus acts upstream of tfap2a. By contrast, previous work revealed Fgf8 to be involved in specification of NA neurons in the locus coeruleus. Thus, although the inductive signals may be distinct, hindbrain NA neurons of the locus coeruleus and the posterior groups both require Tfap2a to establish their noradrenergic identity.