A systematic genome-wide analysis of zebrafish protein-coding gene function

Since the publication of the human reference genome, the identities of specific genes associated with human diseases are being discovered at a rapid rate. A central problem is that the biological activity of these genes is often unclear. Detailed investigations in model vertebrate organisms, typically mice, have been essential for understanding the activities of many orthologues of these disease-associated genes. Although gene-targeting approaches1, 2, 3 and phenotype analysis have led to a detailed understanding of nearly 6,000 protein-coding genes3, 4, this number falls considerably short of the more than 22,000 mouse protein-coding genes5. Similarly, in zebrafish genetics, one-by-one gene studies using positional cloning6, insertional mutagenesis7, 8, 9, antisense morpholino oligonucleotides10, targeted re-sequencing11, 12, 13, and zinc finger and TAL endonucleases14, 15, 16, 17 have made substantial contributions to our understanding of the biological activity of vertebrate genes, but again the number of genes studied falls well short of the more than 26,000 zebrafish protein-coding genes18. Importantly, for both mice and zebrafish, none of these strategies are particularly suited to the rapid generation of knockouts in thousands of genes and the assessment of their biological activity. Here we describe an active project that aims to identify and phenotype the disruptive mutations in every zebrafish protein-coding gene, using a well-annotated zebrafish reference genome sequence18, 19, high-throughput sequencing and efficient chemical mutagenesis. So far we have identified potentially disruptive mutations in more than 38% of all known zebrafish protein-coding genes. We have developed a multi-allelic phenotyping scheme to efficiently assess the effects of each allele during embryogenesis and have analysed the phenotypic consequences of over 1,000 alleles. All mutant alleles and data are available to the community and our phenotyping scheme is adaptable to phenotypic analysis beyond embryogenesis

LRRC50, a Conserved Ciliary Protein Implicated in Polycystic Kidney Disease

Cilia perform essential motile and sensory functions central to many developmental and physiological processes. Disruption of their structure or function can have profound phenotypic consequences, and has been linked to left-right patterning and polycystic kidney disease. In a forward genetic screen for mutations affecting ciliary motility, we isolated zebrafish mutant hu255H. The mutation was found to disrupt an ortholog of the uncharacterized highly conserved human SDS22-like leucine-rich repeat (LRR)-containing protein LRRC50 (16q24.1) and Chlamydomonas Oda7p. Zebrafish lrrc50 is specifically expressed in all ciliated tissues. lrrc50hu255H mutants develop pronephric cysts with an increased proliferative index, severely reduced brush border, and disorganized pronephric cilia manifesting impaired localized fluid flow consistent with ciliary dysfunction. Electron microscopy analysis revealed ultrastructural irregularities of the dynein arms and misalignments of the outer-doublet microtubules on the ciliary axonemes, suggesting instability of the ciliary architecture in lrrc50hu255H mutants. The SDS22-like leucine-rich repeats present in Lrrc50 are necessary for proper protein function, since injection of a deletion construct of the first LRR did not rescue the zebrafish mutant phenotype. Subcellular distribution of human LRRC50-EGFP in MDCK and HEK293T cells is diffusely cytoplasmic and concentrated at the mitotic spindle poles and cilium. LRRC50 RNAi knock-down in human proximal tubule HK-2 cells thoroughly recapitulated the zebrafish brush border and cilia phenotype, suggesting conservation of LRRC50 function between both species. In summary, we present the first genetic vertebrate model for lrrc50 function and propose LRRC50 to be a novel candidate gene for human cystic kidney disease, involved in regulation of microtubule-based cilia and actin-based brush border microvilli

Nitric oxide interacts with monoamine oxidase to modulate aggression and anxiety-like behaviour.

Nitric oxide (NO) is a gaseous neurotransmitter that has important behavioural functions in the vertebrate brain. In this study we compare the impact of decreased nitric NO signalling upon behaviour and neurobiology using both zebrafish and mouse. nitric oxide synthase mutant (nos1(-/-)) zebrafish show significantly reduced aggression and an increase in anxiety-like behaviour without altered production of the stress hormone cortisol. Nos1(-/-) mice also exhibit decreased aggression and are hyperactive in an open field test. Upon reduction of NO signalling, monoamine neurotransmitter metabolism is reduced as a consequence of decreased Monoamine oxidase activity. Treatment of nos1(-/-) zebrafish with the 5-HT receptor 1A agonist 8-OH-DPAT rescues aggression and some aspects of anxiety-like behaviour. Taken together, the interplay between NO and 5-HT appears to be critical to control behaviour. Our cross-species approach challenges the previous notion that reduced neuronal NOS leads to increased aggression. Rather, Nos1 knock-out can also lead to decreased aggression in some situations, a finding that may have implications for future translational research

Human TGCT samples.

<p>Genotypic analysis was performed on human TGCT samples (n = 51); a total of 30 seminomas (SE) and five spermatocytic seminomas (SS) were initially analyzed. These samples were expanded with 15 fTGCT samples, including eight seminomas, which were isolated from patients known to have a familial background of seminomas. Non-SE = non-seminoma, EC = embryonic carcinoma, (im)Te = (immature) teratoma, YS = yolk sac tumor, Ch = choriocarcinoma. Patients carrying alleles identified in this study are indicated.</p

Heterozygous <i>lrrc50^Hu255h</i> zebrafish are predisposed to testicular tumor formation.

<p>(A) Incidences of tumors extracted from randomly selected male controls derived from ENU-mutagenesis (n = 104) and heterozygote male <i>lrrc50^Hu255h</i> (n = 30) zebrafish between 24–44 months of age are summarized in pie charts. Tumor formation presented as both TGCT and non-TGCT (sporadic tumors in somatic tissues) in the <i>lrrc50^Hu255h</i> cohort (90%) is significantly elevated from TGCT (16.3%) formation in the controls (<i>P</i><0.0001). No alternative tumor types were noticeable in the control group. (B) An age-matched wild-type zebrafish compared to a TGCT bearing <i>lrrc50+/−</i> zebrafish (skin around tumor removed; merge of three images). (C) Wild-type testes are composed of two tubular arms forming the paired gonad. The tissue architecture is severely disrupted in the tumor.</p

Characterization of <i>lrrc50^Hu255h</i> zebrafish TGCT suggests analogy to human seminoma.

<p>(A–D) Histological characterization of wild-type testis (left panels) and <i>lrrc50^H255h</i> tumors (right panels). Two magnifications shown A, B (largest in insert) all scale bars; 50 µm. The characterization indicates the presence of predominantly early germ cells and loss of differentiated germ cells in the tumors. (A) Morphological tissue analysis of toluidine blue stained sections indicates the presence of all stages of spermatogenesis in normal tissue (extensively described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003384#pgen.1003384.s003" target="_blank">Figure S3A</a>), and shows a dramatic loss of differentiated germ cells in the tumor. (B) IHC characterization with proliferation marker α-<i>phospho</i>-HistoneH3 (pH3) shows synchronously dividing cell-clusters in normal tissue, indicative of differentiated germ cells. Early SPG is the only germ cell that can divide as a single cell, and the tumors show mostly single proliferating cells. Increased pH3 staining suggests the tumor tissue is highly proliferative (quantified in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003384#pgen.1003384.s004" target="_blank">Figure S4A</a>). (C) IHC using α-Ziwi (strong cytoplasmic expression). In normal tissue, Ziwi expression is restricted to early SPG and gradually and diffusely lost differentiated germ cells. With the exception of somatic tissue, the tumors are almost completely composed of early SPG. (D) IHC with meiosis marker α-γ-H2Ax shows normal tissue that is composed of various stages of differentiation, whereas these are predominantly absent in tumor tissue. (E) Chromatograms of WT zebrafish, heterozygote <i>lrrc50^Hu255h</i> and <i>lrrc50^Hu255h</i> tumors. We observe a loss of the remaining wild-type allele c.263T>A/p.Lys88* in 44.4% of the tumors (LOH).</p

Localization and expression of LRRC50.

<p>LRRC50 antibody ab75163 (reproducible with alternative LRRC50 antibodies) shows a dynamic, cell cycle dependent, distribution in RPE-hTERT cells (representative for other tested mammalian cell lines). Panels represent LRRC50 (green) at various stages, counterstained with DAPI (blue) and acetylated-α-tubulin (Ac-tub, red). Optical sections in A–C are 3 µm. (A) In ciliated serum-starved cells, LRRC50 localizes to the peripheral centrosome/basal body region dorsal of the axoneme (red). Scale bar 10 µm. (B) In mitotic cells, LRRC50 remains associated with the duplicated centrosomes, as indicated with γ-tubulin (red). Inserts b1,2 demonstrate a peri-centrosomal localisation. Scale bar 2 µm. (C) Temporal localization to the midbody in cytokinesis. Scale bar 10 µm. (D) During mitosis LRRC50 dynamically associates with condensed chromosomes (extensively described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003384#pgen.1003384.s005" target="_blank">Figure S5B</a>). Counterstain CREST (red) marks kinetochores. Image is a maximum intensity projection of deconvoluted stacks. Scale bar 2 µm. (E) Dynamic <i>LRRC50</i> mRNA expression (correlating with dynamic localization) with error bars as standard deviation. T47D cells synchronized at the G1/S transition with thymidine show a strong <i>LRRC50</i> transcript up-regulation upon release, specifically in the cell population entering early S-phase (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003384#pgen.1003384.s006" target="_blank">Figure S6A</a>). Intriguingly; although the protein remains stable during mitosis (shown in D) the transcript is rapidly down-regulated, and restored to basal levels. (F) Expression profiling of <i>LRRC50</i> mRNA expression with error bars of standard deviation in a library of mouse cDNA tissues normalized to full mouse reference pool. Testis expression shown in red as the expression level (>26.000%) strongly exceeds the normalized value.</p

LRRC50 missense changes are functional nulls in a zebrafish model of development.

<p>(A) Live embryo images of <i>lrrc50</i> morphants. Morpholino (MO) mediated suppression of <i>lrrc50</i> gives rise to gastrulation defects in mid-somitic zebrafish embryos that can be categorized according to previously established objective scoring criteria <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003384#pgen.1003384-OToole1" target="_blank">[19]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003384#pgen.1003384-Davis1" target="_blank">[57]</a>. Representative lateral and dorsal views are shown (top and bottom panels respectively). (B) Scoring of <i>in vivo</i> complementation; WT embryos were injected with MO and/or human mRNA and scored at the 8–9 somite stage according to phenotypes shown in panel A. Gln307Glu and Thr590Met are not significantly different (NS; c²) from MO alone suggesting that both are functional nulls, n = 44–79 embryos/injection repeated twice with masked scoring. (C) Representative images of flat mounted <i>in situ</i> hybridized (ISH) zebrafish embryos labeled with a cocktail of <i>krox20</i>, <i>myoD</i>, and <i>pax2a</i> riboprobes. Arrows indicate measurement parameters for morphometric analyses shown in panel D; length (<i>l</i>) was measured as the anterior-posterior distance from the first to the last appreciable somite; width (<i>w</i>) was measured as the distance spanning the lateral tips of the fifth somites counted from anterior to posterior. (D) Morphometric quantification of Gln307Glu and Thr590Met gastrulation defects. Images of flat-mounted embryos age-matched at 9 somites were measured in two dimensions (as shown in panel C); the ratios of medial-lateral (width; <i>w</i>) versus anterior-posterior (length; <i>l</i>) measurements are shown for randomly chosen embryos from live scoring experiments (panels A and B) for ISH. <i>LRRC50</i> Gln307Glu and Thr590Met do not produce a significant rescue in comparison to MO alone (NS; t-test), corroborating the <i>in vivo</i> scoring results in panel B (n = 9–13 embryos/injection). Error bars indicate standard deviation of the mean.</p