Zebrafish tyrosine hydroxylase 2 gene encodes tryptophan hydroxylase - PubMed (original) (raw)

Zebrafish tyrosine hydroxylase 2 gene encodes tryptophan hydroxylase

Guiqi Ren et al. J Biol Chem. 2013.

Abstract

The primary pathological hallmark of Parkinson disease (PD) is the profound loss of dopaminergic neurons in the substantia nigra pars compacta. To facilitate the understanding of the underling mechanism of PD, several zebrafish PD models have been generated to recapitulate the characteristics of dopaminergic (DA) neuron loss. In zebrafish studies, tyrosine hydroxylase 1 (th1) has been frequently used as a molecular marker of DA neurons. However, th1 also labels norepinephrine and epinephrine neurons. Recently, a homologue of th1, named tyrosine hydroxylase 2 (th2), was identified based on the sequence homology and subsequently used as a novel marker of DA neurons. In this study, we present evidence that th2 co-localizes with serotonin in the ventral diencephalon and caudal hypothalamus in zebrafish embryos. In addition, knockdown of th2 reduces the level of serotonin in the corresponding th2-positive neurons. This phenotype can be rescued by both zebrafish th2 and mouse tryptophan hydroxylase 1 (Tph1) mRNA as well as by 5-hydroxytryptophan, the product of tryptophan hydroxylase. Moreover, the purified Th2 protein has tryptophan hydroxylase activity comparable with that of the mouse TPH1 protein in vitro. Based on these in vivo and in vitro results, we conclude that th2 is a gene encoding for tryptophan hydroxylase and should be used as a marker gene of serotonergic neurons.

Keywords: Dopamine; Neurons; Parkinson Disease; Serotonin; Tryptophan Hydroxylase; Tyrosine Hydroxylase 2; Zebrafish.

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Figures

FIGURE 1.

FIGURE 1.

Temporal and spatial localization analysis of th2 and 5-HT shows that they co-localize in the ventral diencephalon and caudal hypothalamus at 72 hpf. A–F, 24-hpf embryo, lateral view, anterior to the left. A–C show that th2 expresses at close proximity with GFP of ETvmat2:GFP in the diencephalon. _C_′ shows a magnification of the white box in C; D–F indicate that 5-HT also localizes at close proximity with the GFP in the diencephalon. _F_′ shows a magnification of white box in F. G–L, 48-hpf embryo, dorsal view, anterior to the top. G–I show that th2 expresses in three clusters of neurons but not with the GFP in the diencephalon; J–L show that 5-HT localizes in the epiphysis, and a weak level of 5-HT can be detected in the raphe nucleus. M–R, 60-hpf embryo, dorsal view, anterior to the top. M–N show that th2 co-expresses with the GFP in the neural cluster of paraventricular organ (Pa in M–O). P–R show that 5-HT also co-localizes with the GFP in the neural cluster of the paraventricular organ (Pa in P–R). S–X, 72-hpf embryo, dorsal view, anterior to the top. S–U, GFP immunochemistry and th2 fluorescent in situ hybridization show that there are four clusters of _th2_-positive neurons, 3b, 8b, 9b, and 10b in T; three of them co-localize with GFP in neural cluster of paraventricular organ, intermediate hypothalamus neural cluster (Hi), and caudal hypothalamus neural cluster (Hc) (S and X); V–X, GFP and 5-HT immunochemistry shows that 5-HT co-localizes with the same GFP-immunoreactive neurons of paraventricular organ, intermediate hypothalamus, and caudal hypothalamus. Di, neural cluster of diencephalon; Ep, neural cluster of epiphysis; Ra, neural cluster of raphe nucleus. Scale bar, 200 μm (A–F), 100 μm (_C_′ and _F_′), and 100 μm (G–X).

FIGURE 2.

FIGURE 2.

Verification of splicing MO and ATG MO. A, RT-PCR shows altered mRNA product (b and c) in the 12-ng splicing MO group. Bottom, schematic representation of the sequencing result shows that there is intron 2-3 insertion in b; there is intron 2-3 and intron 3-4 insertion in c. Bottom, schematic representation shows that both of the altered mRNA products will generate the same very short truncated protein product. B, strong GFP appeared in embryos injected with ATG MO target site fusion EGFP but not in embryos co-injected with ATG MO target site fusion EGFP and 1 ng of ATG MO. A lateral view is shown, anterior to the top. Scale bar, 250 μm.

FIGURE 3.

FIGURE 3.

Knockdown of p53 does not cause decrease of 5-HT in 8b, 9b, and 10b neurons. A dorsal view is shown, anterior to the top. A and B, _Z_-stack (70-μm) images of 5-HT immunoreactivity of 72-hpf embryos. A, control; B, p53 MO 4 ng. C, statistical result shows that the 5-HT level is not significantly different between control and the p53 morphant group (p = 0.85). The y axis shows relative 5-HT level. n = 20 in each group; error bars, S.E. Scale bar, 100 μm.

FIGURE 4.

FIGURE 4.

Knockdown of th2 causes reduction of 5-HT in the corresponding neurons, and this reduction can be rescued by both mRNA of th2 and mouse Tph1 as well as 5-HTP. A dorsal view is shown, anterior to the top. A–E, G, and H, _z_-stack (70 μm) images of 5-HT immunoreactivity of 72 hpf embryos. The white circles indicate the regions of interest used for statistical analysis. F, statistical result of the mRNA and chemical rescue experiment in both morphant groups. The y axis shows relative 5-HT level. Both MOs generate significant reduction of the 5-HT level compared with control (p < 0.05). mRNA of th2 and mouse Tph1 and 5-HTP show significant rescue of 5-HT compared with both morphant groups (p < 0.05). In contrast, mRNA of th1 and

l

-DOPA do not have a significant rescue effect in both morphant groups (p > 0.05). n = 20 in each group; error bar, S.E; scale bar, 100 μm. White circles indicate regions of interest. H, hypothalamic region immediately anterior to the raphe nucleus (arrow); Ra, raphe nucleus.

FIGURE 5.

FIGURE 5.

Maximum intensity section of 5-HT in diencephalon and GFP immunoreactivity at 72 hpf shows that the decrease of 5-HT is not caused by cell loss. A dorsal view is shown, anterior to the top. A–C, control embryo; D–F, ATG MO (4 ng) + p53 MO (4 ng) embryo. The intensity of 5-HT in the morphant reduced sharply (E) compared with that of control (B), whereas the intensity of GFP in both groups did not change (A and D). Scale bar, 100 μm.

FIGURE 6.

FIGURE 6.

Th2 has tryptophan hydroxylase activity, not tyrosine hydroxylase activity, in vitro. A, Coomassie Brilliant Blue staining after protein purification shows that Th1, Th2, and mouse TPH1 express successfully (compare with the GST lane). Top, Th1, Th2, and mouse TPH1 tagged with GST. Bottom, GST expression of each group near a molecular mass of 25 kDa. B, tryptophan hydroxylase activity of GST, Th1, Th2, and mouse TPH1. There are no differences between the GST and Th1 groups (p = 0.998) or between the Th2 and mouse TPH1 groups (p = 0.554). In contrast, the activities of Th2 and mouse TPH1 are more than 1 order magnitude higher than that of GST (p = 0.002 and 0.000, respectively). C, Coomassie Brilliant Blue staining after protein purification shows that Th1, Th2, and mouse TH were expressed successfully (compare with the GST lane). Top, Th1, Th2, and mouse TH tagged with GST. Bottom, GST expression of each group near a molecular mass of 25 kDa. D, tyrosine hydroxylase activity of GST, Th1, Th2, and mouse TH. The tyrosine activity of Th1 and mouse TH is 65 ± 4.9 and 61 ± 5.6 p

m l

-DOPA/min/mg, respectively. There are no differences between the GST and Th2 groups (p = 0.954) or between the Th2 and mouse TH groups (p = 0.464). Error bars, S.E.

FIGURE 7.

FIGURE 7.

Similarity dendrogram of zebrafish th1, th2, pah, and tph cluster genes using MEGA program. Based on sequence homology, th1 and th2 clustered in one group, whereas the distance between th1 and tph cluster genes is similar to that of th2. pah is used as an outside group.

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