Dataset Information

Rab28 is localised to photoreceptor outer segments, regulates outer segment shedding but is not linked to retinal degeneration in zebrafish

ABSTRACT: The photoreceptor outer segment is the canonical example of a modified and highly specialised cilium, with an expanded membrane surface area in the form of discs or lamellae for efficient light detection. Many ciliary proteins are essential for normal photoreceptor function and cilium dysfunction often results in retinal degeneration leading to impaired vision. Herein, we investigate the function and localisation of the ciliary G-protein RAB28 in zebrafish cone photoreceptors. CRISPR-Cas9 generated rab28 mutant zebrafish display a reduction in shed outer segment material in the RPE at 1 month post fertilisation (mpf), but otherwise normal retinal structure and visual function up to 12 mpf. Cone photoreceptorspecific transgenic reporter lines show Rab28 localises almost exclusively to outer segments, independently of nucleotide binding. Co-immunoprecipitation analysis demonstrates tagged Rab28 interacts with components of the phototransduction cascade, including opsins, Phosphodiesterase 6C and Guanylate Cyclase 2D. Our data shed light on RAB28 function in cones and provide a model for RAB28-associated cone-rod dystrophy

INSTRUMENT(S): Q Exactive

ORGANISM(S): Danio Rerio (zebrafish) (brachydanio Rerio)

TISSUE(S): Eye, Photoreceptor Layer Of Retina

DISEASE(S): Retinal Degeneration

SUBMITTER: David Matallanas

LAB HEAD: Brendan Kennedy

PROVIDER: PXD017523 | Pride | 2020-04-09

REPOSITORIES: Pride

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Publications

Genetic Deletion of Zebrafish Rab28 Causes Defective Outer Segment Shedding, but Not Retinal Degeneration.

Carter Stephen P SP Moran Ailís L AL Matallanas David D McManus Gavin J GJ Blacque Oliver E OE Kennedy Breandán N BN

Frontiers in cell and developmental biology 20200317

The photoreceptor outer segment is the canonical example of a modified and highly specialized cilium, with an expanded membrane surface area in the form of disks or lamellae for efficient light detection. Many ciliary proteins are essential for normal photoreceptor function and cilium dysfunction often results in retinal degeneration leading to impaired vision. Herein, we investigate the function and localization of the ciliary G-protein RAB28 in zebrafish cone photoreceptors. CRISPR-Cas9 genera ...[more]

PMID: 32258030

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Project description:The loss of cone photoreceptor cells, which are critical for optimal daylight vision, have the great impact on vision during retinal degenerations. Retinal differentiation of human induced pluripotent stem cell (hiPSC) sources could provide a renewable source of cone photoreceptors towards developing a cone cell replacement therapy to treat blindness. Demonstration of comparable gene expression profiles between human foetal and stem cell-derived cones at equivalent stages is required to progress the cell transplantation approach into the patient, as it is hypothesised stem cell-derived cones are required to show high levels of developmental recapitulation of the in vivo generated cones. In this study, the AAV2/9.pR2.1.GFP reporter was used to specifically label L/M-opsin cone photoreceptors in human foetal retinal samples, obtained from the MRC-Wellcome Trust Human Developmental Biology Resource, at a range of developmental stages. The L/M-opsin cone population represent the majority cone cell types in the adult human retina and are the only photoreceptors present within the fovea. Using fluorescence activated cell sorting, L/M-opsin GFP+ cones and GFP- retinal populations, alongside total foetal retinal samples containing all retinal cell tytpes, were isolated and processed for bulk RNA sequencing and downstream comparative analysis. Using DESeq2 differential gene expression analyses, statistically significant genes enriched within the GFP+ human foetal LM-opsin cone populations were determined which led to the identification of a cone enriched gene signature of human L/M-opsin cone photoreceptors. The AAV2/9.pR2.1.GFP reporter was applied to hiPSC-derived retinal cultures to isolate and process cone-like cell populations for RNA sequencing using the same strategy developed within the human foetal retina. Applying the cone enriched gene signature to the transcriptome of hiPSC-derived GFP+ samples at equivalent developmental stages revealed some expression similarities in genes found to be enriched within the late foetal L/M-opsin cone photoreceptors. This analysis overall revealed an intermediate stage of cone differentiation was achieved within the hiPSC-derived samples and the comparison to human foetal L/M-opsin gene express profiles suggesting further differentiation of hiPSC-derived sample is required.

Dataset Information

Rab28 is localised to photoreceptor outer segments, regulates outer segment shedding but is not linked to retinal degeneration in zebrafish

Publications

Genetic Deletion of Zebrafish Rab28 Causes Defective Outer Segment Shedding, but Not Retinal Degeneration.

Similar Datasets

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