Inhibition of the Pim1 Oncogene Results in Diminished Visual Function

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 results 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.Irish Research CouncilScience Foundation Irelan

Pim1 inhibition results in a reduced visual response at 5 dpf.

<p>(A) The OKR of Pim1 MO injected fish is significantly reduced. Larvae were treated with increasing concentrations of Pim1 inhibitor 2 or Pim1 inhibitor II ranging from 1 to 100 µM. Pim1 inhibitors were dissolved in 0.1% DMSO at all concentrations except the 100 µM concentration which was dissolved in 1% DMSO. (B) The ERG a-wave and b-wave are significantly decreased in 100 µM Pim1 inhibitor 2 treated fish. (C–J), significant reduction of OKR saccades in zebrafish treated from 3–5 dpf (“chronic”) or treated for 1 hour at 5 dpf (“acute”) using Pim1 inhibitors. The locomotor touch response of fish treated with Pim1 inhibitors is unaltered. p-values were calculated using Student's t test. *: p<0.05. **: p<0.01. ***: p<0.001.</p

Top 50 differentially expressed genes between 5 versus 3 dpf.

a<p>: these differentially expressed genes were verified using real-time PCR in our previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052177#pone.0052177-Yin1" target="_blank">[11]</a>.</p

Top 20 unknown differentially expressed genes between 5 versus 3 dpf and their human homologs identified using BLASTX.

<p>Top 20 unknown differentially expressed genes between 5 versus 3 dpf and their human homologs identified using BLASTX.</p

Localisation of Pim1 in the larval eye.

<p>(A) Immunohistochemical analysis of Pim1 protein (red) and DAPI (blue) in 3 and 5 dpf larval eyes reveals Pim1 expression throughout the neuoretina including the ganglion cell layer (arrow) and inner nuclear layer (arrowhead) at both timepoints. (B) Pim1 RNA is expressed in the INL, GCL and CMZ of 2 and 5 dpf larvae. (C) RT-PCR amplification of <i>pim1</i> from 5 dpf cDNA results in a 1059 bp band in control morpholino-injected larvae and a second ∼500 bp band in <i>pim1</i> splice site morpholino-injected larvae consistent with knockdown of <i>pim1</i>.(D) Pim1 expression is present in the ONL, INL and GCL of 5 dpf larvae injected with 0.025 mM standard control morpholino but highly reduced in 5 dpf larvae injected with 0.025 mM pim1 morpholino. White boxes indicate the areas magnified. MO, morpholino; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; CMZ, ciliary marginal zone; L, lens.</p

Summary of differentially expressed genes between 5 versus 3 dpf eyes using GO biological process annotation.

<p>A, B: GO summary for up-regulated genes between 5 versus 3 dpf. C, D: GO summary for down-regulated genes between 5 versus 3 dpf. A, C: pie chart presenting the number of differentially expressed genes in each GO category. B, D: bar plot presenting the significance of each GO term in Q-values. Q-value<0.05 was set as the significance threshold as depicted by the red dashed line.</p

Larvae treated with Pim1 inhibitors have a slightly reduced number of primary hyaloid vessels and eye size.

<p>(A) Representative images of whole zebrafish larvae treated using Pim1 inhibitors. (B) and (C) zebrafish (n = 17 to 20) treated with Pim1 inhibitor 2 from 3–5 dpf have normal hyaloid vasculature morphology, while treatment with Pim1 inhibitor II from 3–5 dpf slightly reduces the number of primary hyaloid vessels. Primary hyaloid vessels are pointed using asterisks. P-value was calculated using one way ANOVA with Dunnett's correction for multiple comparisons. *:ANOVA p<0.05. (D) Zebrafish larvae treated with Pim1 Inhibitor 2 from 3–5 dpf have a smaller eye. ***: Student's t test p<0.001. (E) Retinal lamination appears normal in the larvae with drug-treated from 3–5 dpf. Scale bars are 1 mm (A) and 50 mm (E).</p

Socs3a, Socs1 and Stat3 expression in 2–7 dpf retina.

<p>First five panels are immunohistochemical analysis showing increasing expression of Socs3a, Socs1 and Stat3 throughout the retina from 2–7 dpf. Minimal staining is observed in secondary antibody alone, pre-immune serum or morphant controls. The last three panels are in-situ hybridisations of <i>socs3a</i>, <i>socs1</i> and <i>stat3</i> genes on 2 and 7 dpf. No significant staining was observed in sense probe controls. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer.</p

Sequence and structure analysis of Pim protein family.

<p>(A) Neighbor-joining tree with 1000 bootstrap resamplings. Subgroups for Pim1, Pim2 and Pim3 proteins are highlighted in different colors. <i>C. elegans</i> Prk proteins were used to root the tree. (B) The 3-D structure of zebrafish Pim1 protein was predicted by homology modeling using Swiss-Model <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052177#pone.0052177-Arnold1" target="_blank">[29]</a> using the human PIM1 crystal structure 3BGP as the template. Estimated accuracy in Qmean value was colored from blue to red. A lower Qmean value indicates more reliable prediction. The Pim1 inhibitor 2 drug docking site was predicted using SwissDock <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052177#pone.0052177-Grosdidier1" target="_blank">[32]</a>. (C) Chemical structure of Pim1 inhibitor 2. (D) Chemical structure of Pim1 inhibitor II. (E) Sequence alignment of zebrafish Pim proteins with human PIM1 with assigned secondary structure. Residues within 4.5 Å of the Pim1 inhibitor 2 docking site are highlighted in grey in human and zebrafish Pim1.The Pim1 antibody-binding site is highlighted using a rectangle.</p

Up-regulation of gene and protein expression in the Jak-Stat signaling pathway from 3 to 5 dpf during eye development.

<p>(A) Red blocks are up-regulated genes between 5 versus 3 dpf. Green blocks are down-regulated genes between 5 versus 3 dpf. Grey blocks are genes not changed between 5 versus 3 dpf. White blocks are genes not targeted by the microarray or not in the zebrafish genome. The gene interaction network figure is from the KEGG database with modification adapted to zebrafish genes. (B) Signal intensities on the microarrays. *: q-value<0.05. **: q-value<0.01. (C) Real-time PCR results are depicted as relative abundance compared to lowest abundance sample. *: p-value<0.05. **: p-value<0.01. (D) Western blot and (E) densitometric analysis of protein samples extracted from zebrafish whole larvae shows that Socs1, Socs3a and Stat3 expression are up-regulated from 2 to 7 dpf.</p