A method for isolation of cone photoreceptors from adult zebrafish retinae

Abstract

BackgroundCone 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.ResultsMethods 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.ConclusionsThis 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.

HDAC6 inhibition by tubastatin A is protective against oxidative stress in a photoreceptor cell line and restores visual function in a zebrafish model of inherited blindness

“… 32 , 33 Previous data confirm that in zebrafish, hdac6 is expressed in the larval eye during maturation of visual function, 19 and specifically in cone photoreceptors as determined by microarray analysis of cone-enriched transcripts from adult zebrafish. 34 The data are deposited in the NCBI Gene Expression Omnibus (GEO) database, under accession number GSE86155. Furthermore, in a recent study Pinho et al 35 have demonstrated that zebrafish HDAC6 shows high sequence identity with the mammalian homolog at its deacetylase active site.…”

Transcripts within rod photoreceptors of the Zebrafish retina

“…The dataset was validated by qPCR of 45 transcripts, and many transcripts present in rods were not previously recognized as rod-enriched. Analysis of FACS-sorted fluorescent rods from transgenic zebrafish appears to be an excellent approach for expanding our knowledge of rod biology, and in the future may be applied to other photoreceptor subpopulations [ 63 ], since there are numerous transgenic tools available that selectively fluorescently label specific cone subtypes [ 64 , 65 ].…”

Diverse Signaling by TGFβ Isoforms in Response to Focal Injury is Associated with Either Retinal Regeneration or Reactive Gliosis

“…At different time points after injury induction (1, 3, and 7 dpli), both retinas of three gfap:gfap-GFP zebrafish per time point were dissected and kept in cold diethyl pyrocarbonate phosphate-buffered saline (DEPC-PBS) (Glaviano et al 2016 ). Enzymatic dissociation was initiated by 0.05% trypsin (ThermoFisher Scientific) diluted in DEPC-PBS at 37 °C for 10 min.…”