Zebrafish Hagoromo mutants upregulate fgf8 post-embryonically and develop neuroblastoma

We screened an existing collection of zebrafish insertional mutants for cancer susceptibility by histologic examination of heterozygotes at 2 years of age. As most mutants had no altered cancer predisposition, this provided the first comprehensive description of spontaneous tumor spectrum and frequency in adult zebrafish. Moreover, the screen identified four lines, each carrying a different dominant mutant allele of Hagoromo previously linked to adult pigmentation defects, which develop tumors with high penetrance and that histologically resemble neuroblastoma. These tumors are clearly neural in origin, although they do not express catecholaminergic neuronal markers characteristic of human neuroblastoma. The zebrafish tumors result from inappropriate maintenance of a cell population within the cranial ganglia that are likely neural precursors. These neoplasias typically remain small but they can become highly aggressive, initially traveling along cranial nerves, and ultimately filling the head. The developmental origin of these tumors is highly reminiscent of human neuroblastoma. The four mutant Hagoromo alleles all contain viral insertions in the fbxw4 gene, which encodes an F-box WD40 domain–containing protein. However, although one allele clearly reduced the levels of fbxw4 mRNA, the other three insertions had no detectable effect on fbw4 expression. Instead, we showed that all four mutations result in the postembryonic up-regulation of the neighboring gene, fibroblast growth factor 8 (fgf8). Moreover, fgf8 is highly expressed in the tumorigenic lesions. Although fgf8 overexpression is known to be associated with breast and prostate cancer in mammals, this study provides the first evidence that fgf8 misregulation can lead to neural tumors. (Mol Cancer Res 2009;7(6):841–50)National Cancer Institute (U.S.) (Grant CA106416

RNAi mediated myosuppressin deficiency affects muscle development and survival in the salmon louse (Lepeophtheirus salmonis)

Muscle activity is regulated by stimulatory and inhibitory neuropeptides allowing for contraction and relaxation. In Arthropods, one of the important myoinhibitors is Myosuppressin, belonging to FMRFamide-like peptides, that was shown to have inhibitory effects on visceral muscle contraction and to regulate vital physiological processes including reproduction or feeding. We have identified myosuppressin in salmon louse Lepeophtheirus salmonis (LsalMS) and systematically characterised its function and complex abnormalities emerging after LsalMS knockdown by RNAi in all developmental stages in this species. Immunohistochemistry analysis localized the LsalMS mainly to the central nervous system, but also to the vital organs within the alimentary tract and the reproductive system. The most striking feature of LsalMS deficiency during lice development was severe reduction of the muscle content, with abnormalities detected in both the visceral and skeletal muscles. Moreover, down-regulation of LsalMS affects moulting, spermatophore deposition and feeding by affecting development of the intestinal wall and increasing its contraction frequency.</p

Co-receptor RNAi and infection efficiency of various fish species.

<p>(<b>a</b>). Infection of Atlantic salmon (<i>Salmo salar</i>) given as percentage copepodids attached at three days and two weeks post infection. Significant reduced attachment (day three post infection) and settlement (two weeks post infection) was observed for the group treated with ds<i>Lsal</i>IR8b. Type of dsRNA treatment given under each bar. (<b>b</b>) Copepodids attached to the lumpfish 72h post infection (whit arrows). (<b>c</b>) Infection on the non-host Lumpfish (<i>Cyclopterus lumpus</i>). Number of copepodids attached to lumpfish 30 minutes and three days post infection. The group treated with ds<i>Lsal</i>IR25a showed significantly higher infection three days post infection. Type of dsRNA treatment displayed under each bar. (<b>d</b>) Number of copepodids attached to the Lumpfish during the experiments (from infection to 72h post infection). Average values for each time point are used. Each line indicates different types of dsRNA used (color code under the graph). (<b>e</b>) Number of copepodids attached to Ballan wrasse (<i>Labrus bergylta</i>) at 30 minutes post infection and three days post infection. Type of dsRNA used displayed under each bar. (<b>f</b>) Number of copepodids attached to the Ballan wrasse during the course of the experiments (from infection to 72h post infection). Average values for each time point are shown. Each line indicates different types of dsRNA used (color code under the graph). Each experiment was repeated between 3 and 6 times (n = 3–6) using copepodids treated with dsRNA (200 for Atlantic salmon, 150 for lumpfish and Ballan wrass). Asterisks show various levels of significance between the control group and each RNAi treated groups evaluated with two-way ANOVA followed by the post hoc Tukey-Kramer test: 2 asterisks (p < 0.01), 3 asterisks (p < 0.0001).</p

<i>Ls</i>IR25a expression in selected tissues from adult female and male.

<p>(<b>a</b>) In situ hybridization of paraffin sections of antennae dissected from adult salmon louse male. Antisense probes for <i>LsalIR25a</i> label neuronal cell bodies scattered in the male antennae. No specific labeling was detectable with the sense control. Specifically labeled cell bodies are indicated with an arrow. The cuticle in each section is non-specifically labeled in blue. (<b>b</b>) Q-PCR data shows relative expression in organs/tissues relative to the entire adult animal. <i>Lsal</i>IR25a is highest expressed in antennae. In the remaining body, the highest expression is detected in the mouth and the brain and low level of transcripts is detected in reproductive tissues (testes and ovaries), intestine and subcuticular tissue. Quantification of gene expression was performed on samples consisting of 10–20 dissected organs (n = 3). Error bars show standard deviation.</p

Copepodid, infectious stage of <i>L</i>.<i>salmonis</i> and antenna-specific expression of IRs: Co-receptors and antennal IRs.

<p>(<b>a</b>). Free living, infectious stage of <i>L</i>. <i>salmonis</i> with marked sensory organs—first antennae. (<b>b</b>). Close up of copepodid’s first antenna in SEM. Long sensilla are located mainly on the most distal segment. <b>(c)</b>. Expression of co-receptors and antennal IRs in antennae and remaining body tissue. Expression levels are given as a fold difference compared to intact animal. Bars indicate standard deviation. Analysis was performed on three parallels, where each antennal sample consisted of antennae pairs dissected from 500 animals, and the remaining body samples consisted of 100 dissected animals. On the right top, drawing of copepodid showing first antennae dissection for expression analysis.</p

Systematic identification and characterization of stress-inducible heat shock proteins (HSPs) in the salmon louse (Lepeophtheirus salmonis)

Salmon lice (Lepeophtheirus salmonis) are parasitic copepods, living mainly on Atlantic salmon and leading to large economical losses in aquaculture every year. Due to the emergence of resistances to several drugs, alternative treatments are developed, including treatment with hydrogen peroxide, freshwater or thermal treatment. The present study gives a first overview of the thermotolerance and stress response of salmon lice. Sea lice nauplii acclimated to 10 °C can survive heat shocks up to 30 °C and are capable of hardening by a sublethal heat shock. We searched in the genome for heat shock protein (HSP) encoding genes and tested their inducibility after heat shock, changes in salinity and treatment with hydrogen peroxide, employing microfluidic qPCRs. We assessed 38 candidate genes, belonging to the small HSP, HSP40, HSP70 and HSP90 families. Nine of these genes showed strong induction after a non-lethal heat shock. In contrast, only three and two of these genes were induced after changes in salinity and incubation in hydrogen peroxide, respectively. This work provides the basis for further work on the stress response on the economically important parasite L. salmonis

Treatment with dsRNA against co-receptors.

<p>(<b>a</b>) Exon/intron organization of IR co-receptor with underlined regions targeted by dsRNA. Fragments are complementary to exon sequences (black bars) only. Size of each fragments are given in brackets. For <i>Lsal</i>IR25a and <i>Lsal</i>IR8b two different fragments were used. (<b>b</b>) Silencing efficacy for each tested fragment (F) is presented as a relative expression of targeted gene. Type of treatment is indicated under each bar, and the gene tested above each graph. Error bars shows standard deviation. Each batch contained 100 copepodids, n = 6 and the experiment was repeated 3 times for each gene. Asterisks show significant differences (p < 0,05). Statistical evaluation of differences in mRNA level between the control group and the dsRNA treated group, was performed by Independent-Samples T-Test independently for each gene. (<b>c</b>) Mutual interactions between co-receptors. Expression levels (given as relative expression) for all co-receptors were tested for each dsRNA treatment. The type of dsRNA treatment is given under each bar and tested genes are given above each graph. Error bars indicate standard deviation. Each batch contained 100 copepodids, n = 5. Each experiment was repeated 3 times. Asterisks indicate significant difference (p < 0.05). Statistical evaluation of differences in mRNA level of each gene between the control group and the dsRNA treated group, was performed by Independent-Samples T-Test, for each gene independently. (<b>d</b>) The influence of co-receptors down-regulation on antennal IRs. Six genes show the lowest expression levels in samples treated with ds<i>Lsal</i>IR8b. Tested genes indicated above graph, type of treatment stated under each bar. Graphs show relative expression of each gene in comparison to the control, treated with dsCYP185. Expression of each gene in the control sample was set as 1 and omitted in this graph for clarity. Error bars show standard deviation. Each batch contained 100 copepodids, n = 4. Experiment was repeated 4 times.</p

Ionotropic receptors signal host recognition in the salmon louse (<i>Lepeophtheirus salmonis</i>, Copepoda)

<div><p>A remarkable feature of many parasites is a high degree of host specificity but the mechanisms behind are poorly understood. A major challenge for parasites is to identify and infect a suitable host. Many species show a high degree of host specificity, being able to survive only on one or a few related host species. To facilitate transmission, parasite’s behavior and reproduction has been fine tuned to maximize the likelihood of infection of a suitable host. For some species chemical cues that trigger or attract the parasite in question have been identified but how metazoan parasites themselves receive these signals remains unknown. In the present study we show that ionotropic receptors (IRs) in the salmon louse are likely responsible for identification of a specific host. By using RNAi to knock down the expression level of different co-receptors, a significant change of infectivity and settlement of lice larvae was achieved on Atlantic salmon. More remarkably, knock down of the IRs changed the host specificity of the salmon louse and lice larvae settled at a significant rate on host that the wild type lice rejected within minutes. To our knowledge, this has never before been demonstrated for any metazoan parasite. Our results show that the parasites are able to identify the host quickly upon settlement, settle and initiate the parasitic life style if they are on the right host. This novel discovery opens up for utilizing the host recognition system for future parasite control.</p></div

IRs expression levels through <i>L</i>. <i>salmonis</i> life cycle and in selected tissues in adult lice.

<p>(<b>a</b>). Relative expression level of three co-receptors and selected antennal IR genes in all developmental stages. The highest expression is in planktonic copepodids for all tested genes. Quantification was performed on batches of 100 nauplia I, nauplia II, copepodids, 20 chalimus I, 10 chalimus II, 4 preadult I females, 2 preadult II females, 1 adult female, 10 preadult I males, 4 preadult II males and 1 adult male (n = 5). Error bars show standard deviation. Abbreviations: nI—nauplia I, nII—nauplia II, cop—copepodid, chI—chalimus I, chII—chalimus II, paI—preadult I (both sexes), paII—preadult II (both sexes), a–adult (both sexes). (<b>b</b>). Comparison of transcripts level of antennal IRs in adults of both sexes tested by qRT-PCR. All co-receptors and 8 out of 13 antennal genes reveal higher expression in males than in females. Expression-PCR was performed on 1 adult female or male (n = 5). Error bars show standard deviation. Each louse was analysed separately and standard deviations represent individual differences. Asterisks indicate statistically significant differences in expression level between male and female (p < 0.05). Statistical analysis was performed using Independent-Samples T-Test. Comparison was performed for each gene independently. (<b>c</b>). Transcript level of IR genes in the copepodid stage. mRNA level for each tested gene is presented as a ΔC_T value. Expression-PCR was performed on batches of 100 copepodids (n = 5). Error bars show standard deviation. (<b>d</b>). Comparison of C_T values of <i>Ls</i>IR25a and reference gene <i>Ls</i>EF1A in all developmental stages. <i>Ls</i>EF1A is expressed at the same level in all developmental stages whereas the C_T values for <i>Ls</i>IR25a vary considerably and are the lowest in the copepodid stage. Q-PCR were done on same samples as in Fig 2a (n = 6).</p

Diversity in cell motility reveals the dynamic nature of the formation of zebrafish taste sensory organs

Taste buds are sensory organs in jawed vertebrates, composed of distinct cell types that detect and transduce specific taste qualities. Taste bud cells differentiate from oropharyngeal epithelial progenitors, which are localized mainly in proximity to the forming organs. Despite recent progress in elucidating the molecular interactions required for taste bud cell development and function, the cell behavior underlying the organ assembly is poorly defined. Here, we used time-lapse imaging to observe the formation of taste buds in live zebrafish larvae. We found that tg(fgf8a.dr17)-expressing cells form taste buds and get rearranged within the forming organs. In addition, differentiating cells move from the epithelium to the forming organs and can be displaced between developing organs. During organ formation, tg(fgf8a.dr17) and type II taste bud cells are displaced in random, directed or confined mode relative to the taste bud they join or by which they are maintained. Finally, ascl1a activity in the 5-HT/type III cell is required to direct and maintain tg(fgf8a.dr17)-expressing cells into the taste bud.We propose that diversity in displacement modes of differentiating cells acts as a key mechanism for the highly dynamic process of taste bud assembly.Fil: Soulika, Marina. Ecole Normale Supérieure; FranciaFil: Kaushik, Anna-Lila. Ecole Normale Supérieure; FranciaFil: Mathieu, Benjamin. Ecole Normale Supérieure; FranciaFil: Lourenço, Raquel. Ecole Normale Supérieure; FranciaFil: Komisarczuk, Anna Z.. University of Sydney; AustraliaFil: Romano, Sebastián Alejo. Ecole Normale Supérieure; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Jouary, Adriene. Ecole Normale Supérieure; FranciaFil: Lardennois, Alicia. Ecole Normale Supérieure; FranciaFil: Tissot, Nicolas. Université Paris Diderot - Paris 7; FranciaFil: Okada, Shinji. The University of Tokyo; JapónFil: Abe, Keiko. The University of Tokyo; JapónFil: Becker, Thomas S.. University of Sydney; AustraliaFil: Kapsimali, Marika. Ecole Normale Supérieure; Franci