Hedgehog and Fgf signaling pathways regulate the development of tphR-expressing serotonergic raphe neurons in zebrafish embryos

Abstract

Serotonin (5HT) plays major roles in the physiological regulation of many behavioral processes, including sleep, feeding, and mood, but the genetic mechanisms by which serotonergic neurons arise during development are poorly understood. In the present study, we have investigated the development of serotonergic neurons in the zebrafish. Neurons exhibiting 5HT-immunoreactivity (5HT-IR) are detected from 45 h postfertilization (hpf) in the ventral hindbrain raphe, the hypothalamus, pineal organ, and pretectal area. Tryptophan hydroxylases encode rate-limiting enzymes that function in the synthesis of 5HT. As part of this study, we cloned and analyzed a novel zebrafish tph gene named tphR. Unlike two other zebrafish tph genes (tphD1 and tphD2), tphR is expressed in serotonergic raphe neurons, similar to tph genes in mammalian species. tphR is also expressed in the pineal organ where it is likely to be involved in the pathway leading to synthesis of melatonin. To better understand the signaling pathways involved in the induction of the serotonergic phenotype, we analyzed tphR expression and 5HT-IR in embryos in which either Hh or Fgf signals are abrogated. Hindbrain 5HT neurons are severely reduced in mutants lacking activity of either Ace/Fgf8 or the transcription factor Noi/Pax2.1, which regulates expression of ace/fgf8, and probably other genes encoding signaling proteins. Similarly, serotonergic raphe neurons are absent in embryos lacking Hh activity confirming a conserved role for Hh signals in the induction of these cells. Conversely, over-activation of the Hh pathway increases the number of serotonergic neurons. As in mammals, our results are consistent with the transcription factors Nk2.2 and Gata3 acting downstream of Hh activity in the development of serotonergic raphe neurons. Our results show that the pathways involved in induction of hindbrain serotonergic neurons are likely to be conserved in all vertebrates and help establish the zebrafish as a model system to study this important neuronal class.

Figure 1

tphR is a novel tryptophan hydroxylase encoding gene. Amino acid sequence alignment (A) and phylogenetic tree (B) of TphR with tryptophan hydroxylase sequences of other species recorded in Genbank. Human (X52836), mouse (J04758), rabbit (L29305), chicken (U26428), Xenopus (L20679), zebrafish tphR (AB125219), zebrafish tphD1 (AF548566), zebrafish tphD2 (EST fl56d09.y1), Drosophila (PAH/TPH: M81833), and C. elegans (AF135186). The different colors in (A) highlight conserved amino acids.

Figure 2

tphR is expressed in serotonergic raphe neurons. Lateral (A,C,E,G) and dorsal (B,D,F,H) views of brains of embryos labeled to detect the expression of genes or proteins (indicated bottom right). Typeface color matches reaction product color in double-labeled preparations. GFP-IR reveals cranial motor neurons expressing an Islet-1:GFP transgene (G,H). Age is indicated bottom left. Black arrows and arrowheads point to the anterior and posterior clusters of serotonergic raphe neurons. Asterisks indicate the pineal organ. Abbreviations: a, i, p, anterior, intermediate, and posterior regions of the paraventricular organ; IV, trochlear motor nucleus; V, trigeminal motor nucleus.

Figure 3

tphR is expressed in the pineal organ. Dorsal (A,C,E,G) and lateral (B,D,F,H) views of the epithalamus showing expression of tphR, Opsin-IR, tphD1, and 5HT-IR in the pineal organ. Age is indicated bottom left. The arrowhead indicates pineal organ cells and the arrow pineal stalk cells in (G) and (H). The asterisk indicates a 5HT-IR cell in the skin.

Figure 4

Ace (Fgf8) and Noi (Pax2.1) both promote development of serotonergic raphe neurons. Dorsal views of wild-type (wt), ace^−/− (ace), and noi^−/− (noi) embryos labeled to show tphR expression and 5HT immunoreactivity. Arrows point to serotonergic raphe neurons.

Figure 5

Inhibition of Fgf receptor activity leads to loss of serotonergic raphe neurons. Dorsal views of tphR expression and 5HT-IR in the brains of wild-type embryos treated with 9 μM SU5402 from 24 hpf to the stage indicated bottom left. Arrows point to serotonergic raphe neurons.

Figure 6

Rhombomeric patterning is still evident in embryo lacking Ace (Fgf8) and Noi (Pax2.1) function. Lateral (A–D) and dorsal (E–H) views of ace^−/− (ace), noi^−/− (noi), and sibling embryos (WT) labeled to show ephA4 expression (A–D) and cranial motorneurons (E–H) revealed by expression of Islet1. Age is indicated bottom left. Arrowheads indicate ephA4 expression in r1, r3, and r5 in (A–D). The asterisks indicate a fused nucleus that replaces nuclei III and IV in ace^−/− and noi^−/− embryos. Roman numerals refer to the identity of the cranial motor nuclei and OLe are otic and lateral line efferent neurons.

Figure 7

Hh signaling is required for the development of serotonergic raphe neurons. Dorsal views of brains of wild-type and mutant (indicated top right) embryos or wild-type embryos treated with 100 μM cyclopamine (CCP) from 24 hpf to the stage indicated bottom left labeled to detect the expression of genes or proteins (indicated bottom right). Black arrows point to serotonergic raphe neurons (or the position where they should be present) in (A–J), and the arrowhead points to anterior clusters of serotonergic neurons in the hypothalamus in (H).

Figure 8

Requirement of Hh signals for gata3 expression in the raphe region. Dorsal (A,B) and lateral (C–F) views of gata3 expression in the brains of smu^−/− (smu), syu^−/− (syu), and sibling (wt) embryos at 48 hpf. Arrows indicate gata3 expression in the raphe region.

Figure 9

Exogenous Shh increases tphR expression. Lateral (A,C) and dorsal (B,D,E,F) views of tphR and nk2.2 expression in the brains of 48 h wild-type and shh-injected embryos. Arrows in (A–D) indicate serotonergic raphe neurons and indicate the width of the medial expression domain of nk2.2 in (E) and (F).