Presentation_1_Tachykinin-3 Genes and Peptides Characterized in a Basal Teleost, the European Eel: Evolutionary Perspective and Pituitary Role.PDF

<p>In mammals, neurokinin B (NKB) is a short peptide encoded by the gene tac3. It is involved in the brain control of reproduction by stimulating gonadotropin-releasing hormone (GnRH) neurons, mainly via kisspeptin. We investigated tac3 genes and peptides in a basal teleost, the European eel, which shows an atypical blockade of the sexual maturation at a prepubertal stage. Two tac3 paralogous genes (tac3a and tac3b) were identified in the eel genome, each encoding two peptides (NKBa or b and NKB-related peptide NKB-RPa or b). Amino acid sequence of eel NKBa is identical to human NKB, and the three others are novel peptide sequences. The four eel peptides present the characteristic C-terminal tachykinin sequence, as well as a similar alpha helix 3D structure. Tac3 genes were identified in silico in 52 species of vertebrates, and a phylogeny analysis was performed on the predicted TAC3 pre-pro-peptide sequences. A synteny analysis was also done to further assess the evolutionary history of tac3 genes. Duplicated tac3 genes in teleosts likely result from the teleost-specific whole genome duplication (3R). Among teleosts, TAC3b precursor sequences are more divergent than TAC3a, and a loss of tac3b gene would have even occurred in some teleost lineages. NKB-RP peptide, encoded beside NKB by tac3 gene in actinopterygians and basal sarcopterygians, would have been lost in ancestral amniotes. Tissue distribution of eel tac3a and tac3b mRNAs showed major expression of both transcripts in the brain especially in the diencephalon, as analyzed by specific qPCRs. Human NKB has been tested in vitro on primary culture of eel pituitary cells. Human NKB dose-dependently inhibited the expression of lhβ, while having no effect on other glycoprotein hormone subunits (fshβ, tshβ, and gpα) nor on gh. Human NKB also dose-dependently inhibited the expression of GnRH receptor (gnrh-r2). The four eel peptides have been synthesized and also tested in vitro. They all inhibited the expression of both lhβ and of gnrh-r2. This reveals a potential dual inhibitory role of the four peptides encoded by the two tac3 genes in eel reproduction, exerted at the pituitary level on both luteinizing hormone and GnRH receptor.</p

URP1 and URP2 are equipotent to induce intracellular calcium mobilization in a <i>h</i>UT-transfected CHO cell line.

<p>Representative dose-response curves of <i>h</i>UII (●), <i>m</i>URP (■), URP1 (▲) and URP2 (▼) on the intracellular calcium mobilization <b>(A)</b>. The values are expressed as percentages of the baseline and each point is the mean (± S.E.M.) of 3 replicates. Experimental data were fitted using a four-parameter logistic equation. The potencies of 7–13 independent experiments for each peptides were plotted as—Log(EC₅₀) with box and whiskers <b>(B)</b>. Values were considered as statistically different as assessed by analysis of variance followed by Tukey’s post-test, n.s., not significant, *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001, ****<i>p</i> < 0.0001.</p

<i>urp1</i>⁺ cells express <i>pkd2l1</i>, a specific marker of spinal cerebrospinal fluid- contacting neurons in zebrafish.

<p>Simultaneous expression of <i>urp1</i> and <i>pkd2l1</i> revealed by double fluorescent ISH (TAMRA, red for <i>urp1</i> and FITC, green for <i>pkd2l1</i>) on 24 hpf-embryo <b>(A)</b> and adult spinal cord sections <b>(B)</b>. <i>pkd2l1</i> mRNA is distributed in two rows of cells along the rostro-caudal axis of the spinal cord both in embryo and adult <b>(A2, B2)</b>. All the <i>urp1</i>⁺ cells are <i>pkd2l1</i>⁺<b>(A1,3</b>, <b>B1,3)</b> but only the ventral <i>pkd2l1</i>⁺ cells are <i>urp1</i>⁺. The white dash line indicates the central canal. <b>A</b>, lateral views; <b>B</b>, sagittal sections with dorsal up. Scale bars: 20μm.</p

<i>urp1</i>⁺ cells in the hindbrain are cholinergic neurons expressing ChAT in adult zebrafish.

<p><i>urp1</i> expression revealed by fluorescent ISH (TAMRA, red) on coronal sections of adult brain, together with a fluorescent immunostaining for ChAT (Alexa 488, green). <i>urp1</i>⁺ cells express ChAT. Scale bars: 100 μm.</p

<i>urp1</i> mRNA is restricted to the ventral spinal cord and hindbrain at early stages of development in zebrafish.

<p>Expression of <i>urp1</i> revealed by ISH (BM purple, violet) on nacre embryos at 22 <b>(A)</b>, 28 <b>(B)</b> and 48 hpf <b>(C)</b>. At 22 hpf and 28 hpf, <i>urp1</i>⁺ cells occur only in the spinal cord at the level of the lateral floor plate <b>(A, B)</b>, while from 48 hpf, they are mainly visible in the hindbrain <b>(C)</b>. <b>A1</b>, <b>B1</b>, <b>B5</b> and <b>C1</b>, dorsal views; <b>A2</b>, <b>B2, B3</b> and <b>C2</b>, lateral views with dorsal up; <b>B4</b>, coronal section with dorsal up; all embryos oriented anterior left; <b>B3</b> and <b>B5</b> are details at higher magnifications of B2 and B1, respectively. Ch, chord; Cc, central canal; Nt, neural tube; Scale bar: 100 μm.</p

Both <i>urp1</i>⁺ and <i>urp2</i>⁺ cells are GABAergic neurons in the zebrafish embryo.

<p><i>urp1</i><b>(A)</b> and <i>urp2</i><b>(B)</b> expression revealed by fluorescent ISH (FITC, green) on 24 hpf-embryo, together with a fluorescent immunostaining for GAD_65/67 (Alexa Fluor 546, red). Both <i>urp1</i>⁺ and <i>urp2</i>⁺ cells are GAD⁺ (arrows). Note that only ventral KA (KA”) cells are doubly stained. In contrast, dorsal KA (KA’) cells are GAD⁺ but do not express <i>urp1</i> (arrowhead). The white dash line indicates the central canal. <b>A</b> and <b>B</b>, coronal sections with dorsal up. Scale bars: 15 μm.</p

<i>urp1</i> and <i>urp2</i> mRNAs are exclusively detected in the brain and spinal cord in adult zebrafish.

<p>Tissue distribution of <i>urp1</i> and <i>urp2</i> mRNAs assessed by RT-PCR. Parallel amplification of zebrafish β-actin mRNA served as internal control. NTC, non-template control.</p

Spinal GFP⁺ axon motor projections of transgenic <i>uts2b</i>-GFP <i>X. laevis</i> tadpoles co-localize with α-bungarotoxin, a marker of postsynaptic neuromuscular junctions.

<p>Combined fluorescence of GFP (<b>A</b>) and immuno-labeling of α-bungarotoxin (<b>B</b>) at the level of spinal axon motor projections in a whole-mount stage 57 transgenic tadpole. <b>C</b>. Merged image obtained when GFP fluorescence and α-bungarotoxin staining were superimposed. Postsynaptic nicotinic acetylcholine receptors labeled by α-bungarotoxin and GFP⁺ motor neuron axons overlap (arrowheads). Lateral views, rostral to the left.</p

<i>urp1</i> mRNA is found in the caudal part of the hindbrain in adult zebrafish.

<p>Expression of <i>urp1</i> revealed by fluorescent ISH (FITC, green) on coronal sections of adult brain <b>(A)</b>. <i>urp1</i> mRNA is visible in neurons located in the intermediate reticular formation <b>(A1</b>) and the region of the glossopharyngeal-vagal motor nerve nuclei (<b>A2–A3</b>). More caudally, at the level of the junction between hindbrain and spinal cord, <i>urp1</i> mRNA occurs at the ventral edge of the central canal <b>(A4)</b>. Schematic sagittal view of an adult zebrafish brain depicting the distribution of <i>urp1</i> mRNA (red dots). Levels of sections shown in A are indicated. The anatomical structures are designated according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119290#pone.0119290.ref038" target="_blank">38</a>] <b>(B)</b>. CC, cerebellar crest; C, central canal; CCe, corpus cerebelli; DON, dorsal octavolateralis nucleus; EW, Edinger-Westphal nucleus; FLo, facial lobe; Ha, habenula; H, hypothalamus; IMRF, intermediate reticular formation; MO, medulla oblongata; NC, commissural nucleus of Cajal; nIX-X, glossopharyngeal-vagal motor nerve nuclei; OB, olfactive bulbs; OC, optic chiasma; P, pallium; PN, preopic nucleus; RV, rhombencephalic ventricle; SCsm, spinal cord somatomotor neurons; SP, subpallium; T, thalamus; TO, tectum opticum; TL, torus longitudinalis; TP, posterior tuberculum; TS, torus semicircularis; VLo, vagal lobe. Scale bars: 100 μm.</p

Both <i>urp1</i>⁺ and <i>urp2</i>⁺ cells in the spinal cord are GABAergic neurons in adult zebrafish.

<p>Simultaneous expression of <i>urp1</i> and <i>urp2</i> revealed by double fluorescent ISH (TAMRA, red for <i>urp1</i> and Cy5, white for <i>urp2</i>) in adult spinal cord, coupled to a fluorescent immunostaining for GAD_65/67 (Alexa Fluor 488, green) <b>(A</b>, <b>B)</b>. Both <i>urp1</i>⁺ and <i>urp2</i>⁺ cells are GAD⁺<b>(A, B)</b>. Arrows designate triple-stained cells <b>(A, B)</b>. Note the occurrence of some doubly-positive cells (<i>urp2</i>/GAD) that do not contain any <i>urp1</i> (arrowhead) <b>(A)</b>. Asterisks designate GABAergic interneurons located at the dorsal part of the spinal cord. The white dash line indicates the central canal. <b>A</b>, sagittal section with dorsal up; <b>B</b>, coronal section with dorsal up. Scale bars: 15 μm.</p