Dataset Information

E3 SUMO ligase PIAS3 fine-tunes dorsoventral patterning and visual response of cones in the mouse retina but is not essential for photoreceptor cell fate specification

ABSTRACT: Differentiation of distinct neurons in the developing retina is controlled by combinatorial action of a small subset of transcription factors and signalling molecules. Protein inhibitor of activated STAT3 (PIAS3) has been implicated in guiding the specification of both rod and cone photoreceptors through posttranslational modification of key retinal transcription factors. To investigate its role during retinal development, we deleted exon 2-5 of the mouse Pias3 gene, which resulted in complete loss of the PIAS3 protein. Pias3-/- mice did not exhibit any overt phenotype, and retinal lamination appeared normal by histology even at 18 months. We detected reduced photopic b-wave amplitude by electroretinography (ERG) analysis following green light stimulation of Pias3-/- retina at postnatal day (P) 21, suggesting a compromised visual response of medium wavelength (M) cones. No change was evident in response of short wavelength (S) cones or rod photoreceptors until 7 months. Immunohistochemistry demonstrated altered distribution of cone photoreceptors as revealed by increased S-opsin expression in the M-cone dominant dorsal retina. Transcriptome profiling of P21 and 18-month old Pias3-/- retina revealed aberrant expression of genes associated with photoreceptor function. Our studies suggest redundancy in SUMOylation-associated transcriptional control mechanisms and identify a specific though limited role of PIAS3 in modulating spatial patterning and optimal function of cone photoreceptor subtypes in the mouse retina.

ORGANISM(S): Mus musculus

PROVIDER: GSE94011 | GEO | 2017/05/29

SECONDARY ACCESSION(S): PRJNA363051

REPOSITORIES: GEO

ACCESS DATA

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Project description:Rod and cone photoreceptors in mammalian retina are generated from common pool(s) of neuroepithelial progenitors. NRL, CRX and NR2E3 are key transcriptional regulators that control photoreceptor differentiation. Mutations in NR2E3, a rod-specific orphan nuclear receptor, lead to loss of rods, increased density of S-cones, and supernormal S-cone-mediated vision in humans. To better understand its in vivo function, NR2E3 was expressed ectopically in the Nrl-/- retina, where post-mitotic precursors fated to be rods develop into functional S-cones similar to the human NR2E3 disease. Expression of NR2E3 in the Nrl-/- retina completely suppressed cone differentiation and resulted in morphologically rod-like photoreceptors, which were not functional. Gene profiling of FACS-purified photoreceptors confirmed the role of NR2E3 as a strong suppressor of cone genes and an activator of a subset of rod genes (including rhodopsin) in vivo. Ectopic expression of NR2E3 in cone precursors and differentiating S-cones of wild type retina also generates rod-like cells. The dual regulatory function of NR2E3 is not dependent upon the presence of NRL and/or CRX, but on the timing and level of its expression. Our studies reveal a critical role of NR2E3 in establishing functional specificity of post-mitotic photoreceptor precursors during retinal neurogenesis. Experiment Overall Design: We mated the Crx::Nr2e3/Nrlko mice with the Nrl::GFP transgenic mice, in which the expression of GFP is driven by an Nrl promoter. Mouse retinas were dissected at 4 wk. GFP+ photoreceptors were enriched by FACS (FACSAria, BD Biosciences, Franklin Lakes, NJ). RNA was extracted from 1~5x105 flow-sorted cells using Trizol (Invitrogen). Total RNA (40-60 ng) was used for linear amplification with Ovation Biotin labeling system (Nugen), and 2.75 ug of biotin-labeled fragmented cDNA was hybridized to mouse GeneChips MOE430.2.0 (Affymetrix) having 45,101 probesets (corresponding to over 39,000 transcripts, and 34,000 annotated mouse genes). Experiment Overall Design: Four independent samples were used at 4 weeks. We normalized Experiment Overall Design: Crx::Nr2e3/Nrl-ko-Gfp data along with Nrl-ko-Gfp 4 weeks samples ( 4 replicates, refer to Series submission GSE4051). The normalized data was then subjected to two stage analysis based on False Discovery Rate Confidence Interval (FDR-CI) for screening differentially expressed genes (24, 27) with a minimum fold change of 4.

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

E3 SUMO ligase PIAS3 fine-tunes dorsoventral patterning and visual response of cones in the mouse retina but is not essential for photoreceptor cell fate specification

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets