Now showing 1 - 4 of 4
  • Publication
    A method for isolation of cone photoreceptors from adult zebrafish retinae
    Background: Cone photoreceptors are specialised sensory retinal neurons responsible for photopic vision, colour perception and visual acuity. Retinal degenerative diseases are a heterogeneous group of eye diseases in which the most severe vision loss typically arises from cone photoreceptor dysfunction or degeneration. Establishing a method to purify cone photoreceptors from retinal tissue can accelerate the identification of key molecular determinants that underlie cone photoreceptor development, survival and function. The work herein describes a new method to purify enhanced green fluorescent protein (EGFP)-labelled cone photoreceptors from adult retina of Tg(3.2gnat2:EGFP) zebrafish. Results: Methods for dissecting adult zebrafish retinae, cell dissociation, cell sorting, RNA isolation and RNA quality control were optimised. The dissociation protocol, carried out with ~30 retinae from adult zebrafish, yielded approximately 6 × 106 cells. Flow cytometry cell sorting subsequently distinguished 1 × 106 EGFP+ cells and 4 × 106 EGFP− cells. Electropherograms confirmed downstream isolation of high-quality RNA with RNA integrity number (RIN) >7.6 and RNA concentration >5.7 ng/µl obtained from both populations. Reverse Transcriptase-PCR confirmed that the EGFP-positive cell populations express known genetic markers of cone photoreceptors that were not expressed in the EGFP-negative cell population whereas a rod opsin amplicon was only detected in the EGFP-negative retinal cell population. Conclusions: This work describes a valuable adult zebrafish cone photoreceptor isolation methodology enabling future identification of cone photoreceptor-enriched genes, proteins and signalling networks responsible for their development, survival and function. In addition, this advancement facilitates the identification of novel candidate genes for inherited human blindness.
      611Scopus© Citations 6
  • Publication
    Histone Deacetylase: Therapeutic Targets in Retinal Degeneration
    Previous studies report that retinitis pigmentosa (RP) patients treated with the histone deacetylase inhibitor (HDACi) valproic acid (VPA) present with improved visual fields and delayed vision loss. However, other studies report poor efficacy and safety of HDACi in other cohorts of retinal degeneration patients. Furthermore, the molecular mechanisms by which HDACi can improve visual function is unknown, albeit HDACi can attenuate pro-apoptotic stimuli and induce expression of neuroprotective factors. Thus, further analysis of HDACi is warranted in pre-clinical models of retinal degeneration including zebrafish. Analysis of HDAC expression in developing zebrafish reveals diverse temporal expression patterns during development and maturation of visual function.
    Scopus© Citations 12  175
  • Publication
    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.
      351Scopus© Citations 20
  • Publication
    Genes and signaling networks regulated during zebrafish optic vesicle morphogenesis
    Background: The genetic cascades underpinning vertebrate early eye morphogenesis are poorly understood. One gene family essential for eye morphogenesis encodes the retinal homeobox (Rx) transcription factors. Mutations in the human retinal homeobox gene (RAX) can lead to gross morphological phenotypes ranging from microphthalmia to anophthalmia. Zebrafish rx3 null mutants produce a similar striking eyeless phenotype with an associated expanded forebrain. Thus, we used zebrafish rx3-/- mutants as a model to uncover an Rx3-regulated gene network during early eye morphogenesis. Results: Rx3-regulated genes were identified using whole transcriptomic sequencing (RNA-seq) of rx3-/- mutants and morphologically wild-type siblings during optic vesicle morphogenesis. A gene co-expression network was then constructed for the Rx3-regulated genes, identifying gene cross-talk during early eye development. Genes highly connected in the network are hub genes, which tend to exhibit higher expression changes between rx3-/- mutants and normal phenotype siblings. Hub genes down-regulated in rx3-/- mutants encompass homeodomain transcription factors and mediators of retinoid-signaling, both associated with eye development and known human eye disorders. In contrast, genes up-regulated in rx3-/- mutants are centered on Wnt signaling pathways, associated with brain development and disorders. The temporal expression pattern of Rx3-regulated genes was further profiled during early development from maternal stage until visual function is fully mature. Rx3-regulated genes exhibited synchronized expression patterns, and a transition of gene expression during the early segmentation stage when Rx3 was highly expressed. Furthermore, most of these deregulated genes are enriched with multiple RAX-binding motif sequences on the gene promoter. Conclusions: Here, we assembled a comprehensive model of Rx3-regulated genes during early eye morphogenesis. Rx3 promotes optic vesicle morphogenesis and represses brain development through a highly correlated and modulated network, exhibiting repression of genes mediating Wnt signaling and concomitant enhanced expression of homeodomain transcription factors and retinoid-signaling genes.
      485Scopus© Citations 20