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. 1999 Dec 1;19(23):10348-56.
doi: 10.1523/JNEUROSCI.19-23-10348.1999.

The ETS domain factor Pet-1 is an early and precise marker of central serotonin neurons and interacts with a conserved element in serotonergic genes

T Hendricks  1 , N Francis, D Fyodorov, E S Deneris
Affiliations

The ETS domain factor Pet-1 is an early and precise marker of central serotonin neurons and interacts with a conserved element in serotonergic genes

T Hendricks et al. J Neurosci. .

Abstract

Serotonin (5-HT) plays a crucial neuromodulatory role in numerous physiological and behavioral functions, and dysfunction of the serotonergic system has been implicated in several psychiatric disorders. Despite the widespread importance of the central serotonergic neurotransmitter system, little is known about the molecular mechanisms controlling the development of 5-HT neurons. We previously identified an ETS domain transcription factor, Pet-1, that is expressed in a small number of tissues, including the brain. Here, we show that expression of Pet-1 RNA in the brain is restricted to, and marks, the entire rostrocaudal extent of rat serotonergic hindbrain raphe nuclei. Remarkably, Pet-1 RNA colocalizes with tryptophan hydroxylase-positive neurons in raphe nuclei but not with their nonserotonergic neuron or non-neuronal neighbors. Pet-1 RNA is limited to two domains in the developing hindbrain, which precedes the appearance of 5-HT in each domain by approximately a half day. Conserved Pet-1 binding sites are present in or near the promoter regions of the human and mouse 5-HT1a receptor, serotonin transporter, tryptophan hydroxylase, and aromatic L-amino acid decarboxylase genes whose expression is characteristic of the serotonergic neuron phenotype. These sites are capable of supporting transcriptional activation through interactions with the Pet-1 ETS domain and can function as enhancers. Together, our findings establish Pet-1 as an early and precise marker of 5-HT neurons and suggest that it functions specifically in the differentiation and maintenance of these neurons.

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Figures

Fig. 1.
Fig. 1.
Pet-1 RNA is expressed in the B1–B9 groups of central serotonergic neuron clusters. The data are presented as x-ray film autoradiography of [35S]-labeled probe in coronal sections of adult rat brain. Analyses of several rat brains from olfactory bulbs to spinal cord did not reveal other sites of Pet-1 RNA expression in the adult rat brain. B1, Raphe pallidus and caudal ventrolateral medulla; B2, raphe obscurus; B3, raphe magnus, rostral ventrolateral medulla, and lateral paragigantocellular reticular nucleus;B4, central gray of the medulla oblongata;B5, pontine median raphe nucleus; B6, pontine dorsal raphe nucleus; B7, midbrain dorsal raphe nucleus; B8, midbrain median raphe nucleus;B9, medial lemniscus.
Fig. 2.
Fig. 2.
Pet-1 expression in brain is restricted to serotonergic neurons. A, B, DIG-Pet-1 antisense RNA probes were used to the compare the distribution of Pet-1 RNA (A) with that of 5-HT immunoreactivity (B) in adjacent 20 μm coronal sections through adult dorsal and median raphe. C, Double-label analysis at the level of the ventral field of the dorsal raphe using DIG-Pet-1 RNA probe and a monoclonal antibody raised against rabbit TPH.D, Higher magnification photomicrograph of Pet-1-positive and TPH-positive neurons. Dark bluereaction product represents Pet-1 RNA, and brownreaction product represents TPH immunoreactivity. MnR, Median raphe. Asterisk, Isolated double-labeled neuron in the reticular formation. Magnifications: A,B, 30×; C, 220×; D, 675×.
Fig. 3.
Fig. 3.
Pet-1 is expressed in the developing hindbrain.A, In situ hybridization for Pet-1 with digoxigenin-labeled Pet-1 RNA probe at E14.0. Hybridization of a sagittal section close to the midline indicates two domains of Pet-1 expression with one just caudal to the mesencephalic flexure (arrow) and the other caudal to the pontine flexure (asterisk). B, C, Hybridization of a transverse section of neural tube at E14.0 shows Pet-1 expression in a bilateral cluster (top), which is located adjacent to the floor pate and near the outer surface of the ventricular zone (bottom). FP, Floor plate. Scale bar:A, 1.5 mm; B, 300 μm; C, 37.5 μm.
Fig. 4.
Fig. 4.
Pet-1 gene expression in the developing hindbrain precedes the appearance of 5-HT-positive neurons. A, Pet-1 expression at E12.75 in scattered cells (arrows) caudal to the mesencephalic flexure. B, At E13.0 on sagittal sections, Pet-1 expression is seen as a single longitudinal band caudal to the mesencephalic flexure (arrow).C, At higher magnification, the Pet-1-positive cells shown in B can be seen near the outer surface of the ventricular zone. D, 5-HT immunohistochemistry reveals the first appearance of 5-HT-positive neurons at E13.0 caudal to the mesencephalic flexure; inset shows the morphology of the two cells from this field (dashed lines indicate boundaries of the neural tube). E, At E13.5, a caudal domain of Pet-1 expression (arrow) appears caudal to the pontine flexure. F, An additional sagittal section showing the extent of Pet-1 expression in the caudal domain at E13.5.G, At E13.5, 5-HT-positive neurons form a longitudinal band caudal to the mesencephalic flexure, which comprises the rostral 5-HT cluster, but the caudal 5-HT cluster is not yet evident caudal to the pontine flexure (arrow). H, At E14.0, 5-HT-positive neurons form an extensive rostral cluster, and the first 5-HT-positive neurons of the caudal group appear below the pontine flexure (arrow). Asterisks, Pontine flexure. Scale bar: A, C,D, 37.5 μm; B, E–H, 225 μm; inset in D, 15 μm.
Fig. 5.
Fig. 5.
Identification of Pet-1 binding sites in serotonergic genes. Mobility shift assays were performed with bacterially expressed Pet-1 protein and oligonucleotides composed of sequences obtained from each of the indicated genes. The potential Pet-1 binding site present in each of these probes is shown in Table 1. Analysis of each binding site included incubation with indicated probe and Pet-1 protein (lanes 1, 4,7, 10, 13,16, 19, 22,25), competition of probe and Pet-1 protein interaction with 200–400 m excess of unlabeled PEA3 oligonucleotides (lanes 2, 5, 8,11, 14, 17,20, 23, 26), and competitions of probe and Pet-1 protein interaction with unlabeled oligonucleotides in which the 5′-GGA core of the PEA3 binding site was changed to 5′-TAC (lanes 3, 6,9, 12, 15,18, 21, 24,27). Asterisk, Complex formed with probe and a Pet-1 protein degradation product.
Fig. 6.
Fig. 6.
Functional analysis of Pet-1 binding sites.A, Four copies of the mouse −2024/−2014 5-HTT Pet-1 binding site and two copies of the human −1172/−1162; −1154/−1144 tandem Pet-1 binding site were each cloned upstream of the adenovirus 2 MLP to prepare 4xm5HTT-luc and 2xh5HTT-luc, respectively. These reporters were transfected into dissociated retinal cultures along with CMV-Pet-1-VP16 or CGS empty vector. Bars represent the ratio of Pet-1-VP16 responses of luciferase reporters containing 5-HTT binding sites over the activity of these reporters in the absence of Pet-1-VP16. This ratio included normalization to the nonspecific response of the MLP promoter by Pet-1-VP16 relative to MLP reporter alone. Error bars indicate ±SEM. B, PC12 cells were transfected with reporters carrying either MLP, four copies of the 5-HT1a receptor Pet-1 binding site placed immediately upstream of MLP (4x5HT1a-luc), or four copies of the 5-HT1a receptor site upstream of MLP except that each copy had TAC residues in place of the GGA core (mut4x5HT1a-luc). Data are the average of four separate transfections for each reporter and represent the mean ± SD relative light units.
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