Evolution of Thalamic Sensory Centers in Amniotes: Phylogeny and Functional Adaptation

This chapter is a continuation of our previous study of the forebrain evolution in vertebrates using some new tests allowing evolutionary transformations to be revealed. As such tests, we chose the expression of calcium-binding proteins as neuronal functional markers and the metabolic activity of cytochrome oxidase, characterizing the level of neuronal activity. Here, we report the results of our study of the thalamic visual and auditory centers in reptiles (turtles, Emys orbicularis and Testudo horsfieldii) and birds (pigeon, Columba livia) with a special focus on differences in their parallel visual thalamofugal and tectofugal channels and auditory lemniscal and extralemniscal channels. A comparison with data obtained in other Sauropsida amniotes was drawn to elucidate the role of phylogenetic and functionally adaptive factors determining variable distribution of calcium-binding proteins and metabolic activity, as well as to identify evolutionary conservative and plastic traits in the organization of these thalamic sensory centers

Comparative Distribution and In Vitro Activities of the Urotensin II-Related Peptides URP1 and URP2 in Zebrafish: Evidence for Their Colocalization in Spinal Cerebrospinal Fluid-Contacting Neurons

International audienceUrotensin II (UII) is an evolutionarily conserved neuropeptide initially isolated from teleost fish on the basis of its smooth muscle-contracting activity. Subsequent studies have demonstrated the occurrence of several UII-related peptides (URPs), such that the UII family is now known to include four paralogue genes called UII, URP, URP1 and URP2. These genes probably arose through the two rounds of whole genome duplication that occurred during early vertebrate evolution. URP has been identified both in tetrapods and teleosts. In contrast, URP1 and URP2 have only been observed in ray-finned and cartilaginous fishes, suggesting that both genes were lost in the tetrapod lineage. In the present study, the distribution of urp1 mRNA compared to urp2 mRNA is reported in the central nervous system of zebrafish. In the spinal cord, urp1 and urp2 mRNAs were mainly colocalized in the same cells. These cells were also shown to be GABAergic and express the gene encoding the polycystic kidney disease 2-like 1 (pkd2l1) channel, indicating that they likely correspond to cerebrospinal fluid-contacting neurons. In the hindbrain, urp1-expressing cells were found in the intermediate reticular formation and the glossopharyngeal-vagal motor nerve nuclei. We also showed that synthetic URP1 and URP2 were able to induce intracellular calcium mobilization in human UII receptor (hUT)-transfected CHO cells with similar potencies (pEC50=7.99 and 7.52, respectively) albeit at slightly lower potencies than human UII and mammalian URP (pEC50=9.44 and 8.61, respectively). The functional redundancy of URP1 and URP2 as well as the colocalization of their mRNAs in the spinal cord suggest the robustness of this peptidic system and its physiological importance in zebrafish

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

<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

<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

<i>urp1</i> mRNA occurs in cells located along the ventral edge of the central canal of spinal cord in adult zebrafish.

<p>Expression of <i>urp1</i> revealed by ISH (BM purple, violet) on free-floating sections of adult spinal cord. <i>urp1</i>⁺ cells form a quasi-continuous line at the ventral edge of the central canal <b>(A)</b>. <i>urp1</i>⁺ cells are in close contact to the lumen of the central canal (arrowhead) <b>(B)</b>. <b>A1</b> and <b>A2</b>, lateral sections with dorsal up; <b>B</b>, coronal section with dorsal up. <i>urp1</i>⁺ cells boxed in <b>A1</b> are shown in <b>A2</b> at higher magnification. M, melanocytes. Scale bars: 50 μm.</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>⁺ 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

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