Inhibition of the Pim1 Oncogene Results in Diminished Visual Function Jun Yin1, Lisa Shine2, Francis Raycroft3, Sudhakar Deeti2, Alison Reynolds2, Kristin M. Ackerman3, Antonino Glaviano2, Sean O’Farrell2, Olivia O’Leary2, Claire Kilty2, Ciaran Kennedy2, Sarah McLoughlin2, Megan Rice2, Eileen Russell2, Desmond G. Higgins1, David R. Hyde3, Breandan N. Kennedy2* 1 UCD School of Medicine and Medical Science, UCD Conway Institute, University College Dublin, Dublin, Ireland, 2 UCD School of Biomolecular and Biomedical Science, UCD Conway Institute, University College Dublin, Dublin, Ireland, 3 Department of Biological Sciences and the Center for Zebrafish Research, University of Notre Dame, Notre Dame, Indiana, United States of America Abstract Our objective was to profile genetic pathways whose differential expression correlates with maturation of visual function in zebrafish. Bioinformatic analysis of transcriptomic data revealed Jak-Stat signalling as the pathway most enriched in the eye, as visual function develops. Real-time PCR, western blotting, immunohistochemistry and in situ hybridization data confirm that multiple Jak-Stat pathway genes are up-regulated in the zebrafish eye between 3–5 days post-fertilisation, times associated with significant maturation of vision. One of the most up-regulated Jak-Stat genes is the proto-oncogene Pim1 kinase, previously associated with haematological malignancies and cancer. Loss of function experiments using Pim1 morpholinos or Pim1 inhibitors result in significant diminishment of visual behaviour and function. In summary, we have identified that enhanced expression of Jak-Stat pathway genes correlates with maturation of visual function and that the Pim1 oncogene is required for normal visual function. Citation: Yin J, Shine L, Raycroft F, Deeti S, Reynolds A, et al. (2012) Inhibition of the Pim1 Oncogene Results in Diminished Visual Function. PLoS ONE 7(12): e52177. doi:10.1371/journal.pone.0052177 Editor: Ryan Thummel, Wayne State University School of Medicine, United States of America Received May 14, 2012; Accepted November 14, 2012; Published December 26, 2012 Copyright: ß 2012 Yin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors thank the Irish Research Council for Science Engineering and Technology (IRCSET) Graduate Education Programme (GREP), and Science Foundation Ireland (SFI) SFI 04/IN3/B559 (BK), and SFI 06/RFP/BIM052 (BK) for funding support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Introduction The first electrical responses from the retina have been detected as early as 72 hpf [7]. These responses are also small in amplitude, Our objective was to investigate the molecular genetics requiring high intensity stimuli. Zebrafish electroretinograms regulating maturation of visual function in vertebrates. Develop- (ERG) are typically recorded from 5 dpf larvae in which responses ment of the zebrafish visual system is rapid with morphogenesis of are more robust [8]. the optic vesicles beginning at ,10 hours post-fertilisation (hpf) Here, we avail of Affymetrix GeneChip technology to globally [1]. Rapid proliferation and progressive lamination follows. By profile genes with significant differential expression in the zebrafish ,72 hpf, most retinal cell types are distinguishable, and lamina- eye between 3–5 dpf, as visual function matures. Interestingly, tion of the retina does not significantly change from 3–5 days post- significantly enhanced expression of Jak-Stat signalling genes, a fertilisation (dpf). However, progression from a morphologically pathway typically associated with interferon and cytokine signal- developed eye, to an eye with robust visual function occurs ling, correlates with maturation of visual function [9]. Pim1–2 between 3–5 dpf [2,3]. kinases, proto-oncogenes and downstream components of Jak-Stat A light-evoked locomotor response is detected in zebrafish at signalling, unexpectedly displayed differential expression in the ,68 hpf [3]. This startle response likely recapitulates an escape developing eye [10]. Pharmacological and genetic inhibition of response invoked by the shadow of an approaching predator [4]. Pim1 kinase results in a specific disruption of visual behaviour and Initially known as the shadow-induced startle response, it was first retinal function. These results highlight a novel role for the Pim1 assessed by placing larvae in a petri dish, extinguishing a light kinase in visual function. source for 1 second and observing whether larvae moved in response. The related visual motor response (VMR) is assessed Materials and Methods using an automated system which uses an infrared camera to quantify the movement of larvae in response to lights turned on or Microarray experiment off [4]. Another visual response, the optokinetic response (OKR) Zebrafish were maintained according to standard procedures on represents the ability of zebrafish to detect contrasting patterns a 14 h light/10 h dark cycle at 28uC. Embryos were obtained by and is detected from 73 hpf [3,5]. The initial OKR is slow and natural spawning and developmental stages established by time sporadic, but improves so that by 96 hpf, larvae track the drum and morphological criteria. Microarray experiments were per- analogous to adult fish and by 5 dpf, the response is adult-like [6]. formed as previously described [11]. Eyes were dissected from 3, 4 PLOS ONE | www.plosone.org 1 December 2012 | Volume 7 | Issue 12 | e52177 Inhibition of Pim1 Diminishes Visual Function and 5 days post fertilization (dpf) zebrafish larvae. Total RNA was Histological Analysis extracted and labeled using a two-cycle target labelling protocol Whole larvae were fixed overnight in a solution of 4% (Affymetrix, Santa Clara, USA) and hybridised with Affymetrix paraformaldehyde and 2.5% gluteraldehyde diluted in 0.1 M Zebrafish Genome Arrays. Three biological replicates per time Sorenson phosphate buffer (pH 7.3) at room temperature. point were used with equal amounts of RNA. The 3, 4 and 5 dpf Samples were then post-fixed in 1% osmium tetroxide in 0.1 M eyes microarray data set was deposited in GEO with accession ID Sorenson phosphate buffer for 1 hour at room temperature, GSE19320. All experimental protocols were approved by the dehydrated in ascending concentrations of ethanol to 100% and UCD Animal Research Ethics Committee, and the University of embedded in epon resin according to standard methods. Semi-thin Notre Dame Animal Care and Use Committee. (1 mm) sections were cut using a glass knife and a Reichert-Jung Ultracut E microtome and visualised by light microscopy using a Zebrafish genome reannotation and probe remapping Nikon E80i transmission microscope Gene annotation was based on the zebrafish genome version 9 (Zv9) and integrating gene transcript collections from multiple Immunoblot Analysis genome annotation databases [11]. Transcript data from the Immunoblots were performed similar to previously described RefSeq, GenBank and Ensembl databases were downloaded from (Kassen et al., 2007). Protein was harvested from ,30 larvae, the UCSC genome browser [12]. Transcripts were clustered into homogenized in 15 ml of extraction buffer (16 PBS/10% genes from overlapping coding exons. A customized probe Glycerol/1% Triton X-100/5 mM KPO4/0.05 mM EDTA/16 remapping was performed as previously described [11]. In order Complete Protease Inhibitor Cocktail Tablet (Roche; Indianapo- to take advantage of the human genome annotation, human- lis, IN) and a tyrosine and serine/threonine phosphatase inhibitor zebrafish homology data were downloaded from Ensembl [13], cocktail mix (Phosphatase Inhibitor Cocktails 2 and 3, Sigma; St. BioMart [14], ZFIN [15], and NCBI HomoloGene [16]. These Louis, MO) and stored at 220uC. After SDS-PAGE, proteins homology databases were combined with the zebrafish genome were electrotransferred to a PVDF H-Bond membrane (Amer- annotation databases. Where no functional annotation for a sham; Pis-cataway, NJ) and blocked in 16PBS/0.1% Tween-20/ transcript could be found, cDNA sequences were searched against 5% non-fat dry milk overnight at 4uC. The membrane was the NCBI refseq_protein database using blastx [17]. The highest incubated with immunopurified anti-Stat3 polyclonal antisera scoring human homologs were identified with at least 30% identity (1:5,000) [24], anti-Socs1 polyclonal antisera (1:5,000), anti-Socs3a to the query sequence over at least 30% sequence length. Human polyclonal antisera (1:2,000) or an anti-actin monoclonal antibody KEGG pathway [18] and Gene Ontology [19] annotations were (1:10,000, Calbiochem; San Diego, CA) overnight at 4uCin combined with zebrafish annotations for gene set analysis. Human blocking buffer. The membranes were washed in 16 PBS/0.1% retinal disease information was downloaded from RETNET [20]. Tween-20 (3610 min), and incubated for 1 hr at room temper- ature with either an anti-rabbit or anti-mouse HRP-conjugated Microarray data analysis secondary antibody (1:10,000, Amersham). The membranes were The Bioconductor package, gcrma, was used to normalize and washed in 16 PBS/0.1% Tween-20 (3610 min) and the summarize microarrays signal