Zebrafish Prion Protein PrP2 Controls Collective Migration Process during Lateral Line Sensory System Development

<div><p>Prion protein is involved in severe neurodegenerative disorders but its physiological role is still in debate due to an absence of major developmental defects in knockout mice. Previous reports in zebrafish indicate that the two prion genes, <i>PrP1</i> and <i>PrP2</i>, are both involved in several steps of embryonic development thus providing a unique route to discover prion protein function. Here we investigate the role of PrP2 during development of a mechano-sensory system, the posterior lateral line, using morpholino knockdown and PrP2 targeted inactivation. We confirm the efficiency of the translation blocking morpholino at the protein level. Development of the posterior lateral line is altered in <i>PrP2</i> morphants, including nerve axonal outgrowth and primordium migration defects. Reduced neuromast deposition was observed in <i>PrP2</i> morphants as well as in <i>PrP2^−/−</i> mutants. Rosette formation defects were observed in <i>PrP2</i> morphants, strongly suggesting an abnormal primordium organization and reflecting loss of cell cohesion during migration of the primordium. In addition, the adherens junction proteins, E-cadherin and ß-catenin, were mis-localized after reduction of PrP2 expression and thus contribute to the primordium disorganization. Consequently, hair cell differentiation and number were affected and this resulted in reduced functional neuromasts. At later developmental stages, myelination of the posterior lateral line nerve was altered. Altogether, our study reports an essential role of PrP2 in collective migration process of the primordium and in neuromast formation, further implicating a role for prion protein in cell adhesion.</p></div

Primodium disorganisation and absence of rosette formation.

<p><b>A′.</b> Schematic representation of a normal <i>claudinB-GFP</i> embryo at 30 hpf and detailed organization of the primordium with rosette structure. Red spot indicates a normal concentration point of actin. <b>A, B.</b> Phalloidin staining (Phalloidin-TRITC) in control embryo <i>claudinB-GFP</i>, at 30 hpf, is observed in muscle cells and within the primodium at the center of the rosette (arrows), on the apical side. <b>C, D.</b> Phalloidin-TRITC staining in <i>PrP2</i>-MO, no rosette structure is observed and no actin concentration is found. <b>E, F.</b> Higher magnification shows the co-localization of central actin concentration with <i>claudinB-GFP</i> at the rosette center in control. In morphants, cell disorganization is observed and no actin concentration is observed associated with the absence of a rosette. <b>G–I.</b> Phalloidin staining and DAPI nuclei labeling highlight the primordium and rosette center (arrows) in control embryos. <b>J–L.</b> In <i>PrP2^−/−</i> mutants, actin apical localization in rosette was severely reduced or barely detectable (arrow) and primordium organization at the periphery was impaired: loose cells were visible on the border (arrowheads). <b>I′, L′.</b> In <i>PrP2^−/−</i> mutant, the primordium position was often delayed and the first neuromast deposited close to the ear. <b>M</b>. Quantification of rosette number was established in control (n = 20), <i>PrP2</i>-MO (n = 84) and <i>PrP2^−/−</i> mutant (n = 28) using actin staining at the center, **: p<0.01, ***: p<0.001, Student t test. See also associated <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113331#pone.0113331.s002" target="_blank">Movies S1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113331#pone.0113331.s006" target="_blank">S5</a>.</p

Delocalisation of E-cadherin and beta-catenin in primordium cells in absence of PrP2.

<p><b>A</b>. Schematic representation of <i>claudinB-GFP</i> embryo at 30 hpf, primordium structure, and transversal view of a rosette showing the level of confocal focus plan. <b>B–B″</b>. In control embryos at 30 hpf, E-cadherin is expressed at the membrane level at the basal side of the primordium. <b>C–C″.</b> In morphant embryos, E-cadherin is observed in the cytoplasm and cellular membrane at basal and apical levels. The staining pattern is more punctuated in morphant compared to control. <b>D, E.</b> In control embryos, beta catenin expression is observed at the membrane level and at the center of rosette structures. <b>F, G</b>. In morphant, small rounded primordium shows a membrane homogeneous pattern with no rosette.</p

Loss of PLL nerve fasciculation and associated myelination in absence of PrP2.

<p><b>A–C</b>. In control larvae at 5 dpf, <i>sox10-GFP</i> cells are tightly organized along the PLL nerve (<i>nbt-dsred</i>). <b>D–F</b>. Double morphant <i>PrP2/p53</i> displayed an enlarged PLL nerve (with defasciculated axons, arrows) and rounded Schwann cells (arrowheads). <b>G–G</b>′. In 5 dpf larvae MBP labeling is observed in close apposition to <i>sox10-GFP</i> cells and formed a homogeneous line. <b>H–H′</b>. MBP labeling is altered and partially missing while Schwann cells are disorganized. <b>I–I′</b>. At 7 dpf, control larvae display tightly and regularly organized <i>sox10-GFP</i> positive cells. <b>J–K′</b>. In <i>PrP2/p53</i> morphants (J–J″), loosened Schwann cell processes are observed (arrows) as well as rounded cells (arrowheads) in <i>PrP2</i> morphants (K–K′). Results obtained from five independent experiments (n = 160 embryos).</p

PrP2 decrease expression results in abnormal PLL development.

<p><b>A</b>. In control embryos at 48 hours post fertilization (hpf) from <i>nbt-dsred</i> line that labels neurons and axons, the PLL nerve develops from the PLL ganglia until the tip of the tail (arrow). <b>B–C</b>. In <i>PrP2</i>-MO embryos, a range of defects for the PLL nerve is observed with premature arrest (B, C). <b>D</b>. Control PLL nerve fibers are tightly while <i>PrP2</i>-MO fibers exhibit abnormal branches were observed in severe cases (<b>E</b>). <b>F</b>. In control <i>claudinB-GFP</i> embryos at 48 hpf all derivatives issuing from the primordium express GFP and appear normal. Five regularly spaced neuromasts are present, with 3 terminal neuromasts at the tip of the tail. Due to embryo transparency, the neuromasts on the other side of the embryo are also visible. In <i>PrP2</i>-MO (<b>G</b>) and <i>PrP2</i>-MO/<i>p53</i>-MO (<b>H</b>) injected embryos (in order to avoid off-target defects), neuromast numbers are reduced, and often irregularly spaced. <b>I–J</b>. Western blot analysis of PrP2 expression using SAF84 antibody shows a decrease of the PrP2 protein after morpholino injections (n = 3 independent experiments).</p

Decreased hair cell number of PLL neuromast in <i>PrP2</i>-MO.

<p><b>A–C.</b> In control embryos at 48 hpf, <i>Brn3-GFP</i> fluorescence labels hair cells of neuromasts and ear. High magnification of the first 3 neuromasts and zoom of the first neuromast show 8 hair cells. <b>D–F</b>. Morphants displays reduced number of neuromasts. High magnification shows smaller hair cells number. <b>G</b>. Quantification indicates significant reduction of hair cells/neuromast, independently of the neuromast position (mean hair cell number: 5.5±0.4, n = 36, compare to control 7.9±0.2, n = 20, p<0.001).</p

PrP2 is involved in PLL development.

<p><b>A, C.</b> Alkaline phosphatase staining of the trunk neuromasts (arrows) in wild type embryos at 72 hpf. <b>C</b>. High magnification of boxed area in A. <b>B, D.</b> In <i>Prp2^−/−</i> mutant, less neuromasts are visible with the first neuromast displaced anteriorly (<b>B</b>, white arrow) <b>D</b>. High magnification of boxed area in B. <b>E–G.</b> In Control <b>(E)</b>, <i>PrP2</i>-MO/<i>p53</i>-MO (<b>F</b>) and <i>PrP2</i>-MO (<b>G</b>), the total neuromast number is decreased in morphants. <b>H.</b> Quantification of total neuromast number shows a significant decrease in morphants and mutants compared to control. <b>I.</b> Neuromast position along the somite axis, scheme adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113331#pone.0113331-Matsuda1" target="_blank">[28]</a>. In <i>Prp2^−/−</i> mutant, L1 is retrieved anteriorly to the normal L1 position while in morphants, the present neuromasts are displaced posteriorly. *: p<0.05, ***: p<0.001.</p

Loss of cell-cell contact and disorganization of collective migration of the primordium.

<p>Select time points from time-lapse recording of primordium migration in <i>claudinB-GFP</i> embryos. In control embryos, primordium migration is continuous, with a neuromast deposition (time point 2h30) and out of view at the time point 4h30 (not shown). In contrast in one <i>PrP2</i> morphant example, representative of the observed phenotypes, primordium migration shows a progressive rounded shape and arrest. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113331#pone.0113331.s007" target="_blank">Movies S6</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113331#pone.0113331.s008" target="_blank">S7</a>.</p