DIO3 coordinates photoreceptor development timing and fate stability in human retinal organoids
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PROVIDER: PRJNA1236401 | ENA |
REPOSITORIES: ENA
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Project description:How neuronal subtypes are generated at distinct times and proportions during human retinal development is poorly understood. Here, we investigated how rod and cone photoreceptor subtypes develop in human retinal organoids. We find that type 3 iodothyronine deiodinase (DIO3), an enzyme that degrades thyroid hormone (TH), is a master regulator of human photoreceptor developmental timing and cell fate stability. DIO3 is highly expressed in retinal progenitor cells (RPCs) and decreases as cells asynchronously differentiate into neurons, progressively lowering TH degradation during development. DIO3 mutant organoids display precocious S cone, L/M cone, and rod development, increased photoreceptor (PR) density, and conversion of S cones to L/M fate. Cell autonomous and non-autonomous mechanisms regulate DIO3 expression to maintain and locally coordinate levels among cells. In a computational model, a mechanism that couples TH levels and fate specification provides robustness to photoreceptor development compared to a probabilistic, cell-intrinsic mechanism. Our findings suggest a population-based mechanism for photoreceptor subtype developmental timing in which the relative proportion of progenitors to neurons decreases over time to relieve degradation of TH signaling, which advances photoreceptor development.
2025-08-18 | GSE292057 | GEO
Project description:Animals respond to environmental cues to time phenological events, but the intrinsic mechanism of circannual timing remains elusive. We used advanced transcriptomic sequencing and high frequency resolution during the initiation, maintenance and recovery of circannual timing for Siberian hamster energy balance to delineate the molecular architecture of a neuroendocrine seasonal clock. Three distinct phases of transcript changes were identified, and deiodinase type-3 (Dio3) expression was activated during the initiation phase. Targeted mutation of Dio3 resulted in a shortening of the circannual interval timer period. Hamsters that exhibit a form of naturally occurring disruption in Dio3 eliminated circannual interval timing. Our work demonstrates that the evolutionary conserved changes in hypothalamic tanycytes expression of Dio3 is critical for timing seasonal life history traits in chordates.
2025-09-12 | GSE274003 | GEO
Project description:Photoreceptor loss is a leading cause of blindness, but mechanisms underlying photoreceptor degeneration are not well understood. Treatment strategies would benefit from an improved understanding of gene-expression patterns directing photoreceptor development, as many genes are implicated in both development and degeneration. Neural retina leucine zipper (NRL) is critical for rod photoreceptor genesis and degeneration, with NRL mutations known to cause enhanced S-cone syndrome and retinitis pigmentosa. While murine Nrl loss has been characterized, studies of human NRL can identify important insights for human retinal disease. Here we utilized human organoid models of retinal development to molecularly define developmental alterations in a human model of NRL loss. Consistent with the function of NRL in rod fate specification, human retinal organoids lacking NRL develop S-opsin dominant photoreceptor populations. We report generation of two distinct S-opsin expressing populations in NRL null retinal organoids and identify MEF2C as a candidate regulator of cone development.
2020-01-15 | GSE143669 | GEO
Project description:Mutation of rod photoreceptor-enriched transcription factors is a major cause of inherited blindness. We identified the orphan nuclear hormone receptor ERRβ as selectively expressed in rod photoreceptors. Overexpression of ERRβ induces expression of rod-specific genes in retinas of both wildtype and in Nrl-/- mice, which lack rod photoreceptors. Mutation of ERRβ results in dysfunction and degeneration of rods, while inverse agonists of ERRβ trigger rapid rod degeneration, which is rescued by constitutively active mutants of ERRβ. ERRβ coordinates expression of multiple genes that are rate-limiting regulators of ATP generation and consumption in photoreceptors. Furthermore, enhancing ERRβ activity rescues photoreceptor defects that result from loss of the photoreceptor-specific transcription factor Crx. Our findings demonstrate that ERRβ is a critical regulator of rod photoreceptor function and survival, and suggest that ERRβ agonists may be useful in the treatment of certain retinal dystrophies. Affymetrix MOE430 microarrays were used to analyze the expression patterns of P21 mouse retinal tissues. The results were compared across the variable of Genotype, specifically ERRβ knockout versus wildtype.
2010-07-30 | E-GEOD-21944 | biostudies-arrayexpress
Project description:Purpose: Autologous cell replacement shows great promise for the treatment of inherited retinal degeneration. While mature differentiation protocols exist to produce retinal organoids from patient-derived stem cells, not all cell types present in these organoids are desirable for transplant. To increase the potency of future cell therapies, methods for isolating photoreceptors from dissociated retinal organoids are needed. In this work, we show how partial dissociation can be used to exploit the spatial organization of retinal organoids to produce highly pure photoreceptor populations without the use of specialized sorting devices or reagents such as xenobiotic antibodies. Methods: Retinal organoids were generated as we have described previously. For flow cytometry experiments, organoids were dissociated using papain for 30 minutes or for 90 minutes followed by trituration. For scRNAseq experiments, liberated cells were collected from papain-dissociating organoids at 20-minute intervals. At the conclusion of the experiment, remaining tissue was fully dissociated. A control 60-minute full dissociation was performed in parallel. Photoreceptor purity was assessed via flow cytometry and scRNAseq. Results: Photoreceptors desired for transplant are typically found in the outer layers of retinal organoids, suggesting that partial dissociation could selectively release these cells. Flow cytometry results indicating an increase in purity of CD133+ cells from 21.1% to 91.7% when cells were partially dissociated as compared to full dissociation. A time dependent release of photoreceptors, with a maximum purity of 96% CRX+ cells at 40 minutes as compared to 66% pure for traditional dissociation. Conclusions: By timing the dissociation of retinal organoids, highly pure photoreceptor cell populations suitable for cell replacement therapies can be obtained under cGMP without the use of sorting reagents or equipment.
2024-09-02 | GSE266526 | GEO
Project description:Due to the high energy demands and characteristic morphology, retinal photoreceptor cells require a specialized lipid metabolism for survival and function. This study focused on the roles of saturated fatty acids and their metabolism. A frameshift mutation of lysophosphatidylcholine acyltransferase 1 (Lpcat1), introducing saturated fatty acids into lysophosphatidylcholine to produce disaturated phosphatidylcholine (PC), has been reported to cause spontaneous retinal degeneration in mice (rd11 mice). In this study, we performed a detailed characterization of Lpcat1 in the retina and found that Lpcat1 deficiency induces light-independent and photoreceptor-specific apoptosis in mice. Lipidomic analyses of the retina and isolated photoreceptor outer segment (OS) suggested that loss of Lpcat1 affects disaturated PC production and the proper cellular fatty acid flux, presumably by altering saturated fatty acyl-CoA availabilities. Furthermore, we demonstrated that Lpcat1 deletion increased mitochondrial reactive oxygen species (ROS) levels in photoreceptor cells, but not in other retinal cells without affecting the OS structure and trafficking of OS-localized proteins. These results suggest that the LPCAT1-dependent production of disaturated PC is critical for metabolic adaptation during photoreceptor maturation. Our findings highlight the therapeutic potential of saturated fatty acid metabolism in photoreceptor cell degeneration-related retinal diseases.
2022-01-01 | GSE184817 | GEO
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. Keywords: genetic modification
2006-08-18 | GSE5338 | GEO
Project description:Mutation of rod photoreceptor-enriched transcription factors is a major cause of inherited blindness. We identified the orphan nuclear hormone receptor ERRβ as selectively expressed in rod photoreceptors. Overexpression of ERRβ induces expression of rod-specific genes in retinas of both wildtype and in Nrl-/- mice, which lack rod photoreceptors. Mutation of ERRβ results in dysfunction and degeneration of rods, while inverse agonists of ERRβ trigger rapid rod degeneration, which is rescued by constitutively active mutants of ERRβ. ERRβ coordinates expression of multiple genes that are rate-limiting regulators of ATP generation and consumption in photoreceptors. Furthermore, enhancing ERRβ activity rescues photoreceptor defects that result from loss of the photoreceptor-specific transcription factor Crx. Our findings demonstrate that ERRβ is a critical regulator of rod photoreceptor function and survival, and suggest that ERRβ agonists may be useful in the treatment of certain retinal dystrophies.
2010-07-02 | GSE21944 | GEO
Project description:Background: To identify and functionally annotate cell type-specific gene expression in the human retinal pigment epithelium (RPE), a key tissue involved in age-related macular degeneration and retinitis pigmentosa. Methodology: RPE, photoreceptor and choroidal cells were isolated from selected freshly frozen healthy human donor eyes using laser microdissection. RNA isolation, amplification and hybridization to 44k microarrays was carried out according to Agilent specifications. Bioinformatics was carried out using Rosetta Resolver, David and Ingenuity software. Principal Findings: Our previous 22k analysis of the RPE transcriptome showed that the RPE has high levels of protein synthesis, strong energy demands, is exposed to high levels of oxidative stress and a variable degree of inflammation. We currently use a complementary new strategy aimed at the identification and functional annotation of RPE-specific expressed transcripts. This strategy takes advantage of the multilayered cellular structure of the retina and overcomes a number of limitations of previous studies. In triplicate, we compared the transcriptomes of RPE, photoreceptor and choroidal cells and we deduced RPE specific expression. We identified at least 114 entries with RPE-specific gene expression. Thirty-nine of these 114 genes also show high expression in the RPE, comparison with the literature showed that 85% of these 39 were previously identified to be expressed in the RPE. In the group of 114 RPE specific genes there was an overrepresentation of genes involved in (membrane) transport, vision and ophthalmic disease. More fundamentally, we found RPE-specific involvement in the RAR-activation, retinol metabolism and GABA receptor signaling pathways. Conclusions: In this study we provide a further specification and understanding of the RPE transcriptome by identifying and analyzing genes that are specifically expressed in the RPE. Six RPE samples, three phot samples, three choroid samples
2010-06-08 | E-GEOD-20191 | biostudies-arrayexpress
Project description:While dysfunction and/or death of light-detecting photoreceptor cells underlies most inherited retinal dystrophies, knowledge of the species-specific details of human rod and cone photoreceptor cell development remains limited. Here, we generate retinal organoids using induced pluripotent stem cells (iPSC) derived from a patient with genetic photoreceptor disease, an isogenic control, and an unrelated control. Organoids were sampled using single-cell RNA sequencing across the developmental window encompassing photoreceptor specification, emergence, and maturation; up to 260 days of in vitro differentiation. Using single-cell transcriptomics data, we reconstruct the rod photoreceptor developmental lineage and identify a branchpoint in development unique to the disease state that gives rise to a divergent rod photoreceptor cell population. We show that the rod-specific transcription factor NR2E3 is required for the proper expression of genes involved in phototransduction, including expression of the light-sensitive protein rhodopsin, which is absent in divergent rods. NR2E3-null rods additionally misexpress several cone-specific phototransduction genes at both the transcript and protein level. Using joint multimodal single-cell sequencing on late-stage retinal organoids, we further identify the specific putative regulatory sites where rod-specific factors act to steer rod and cone photoreceptor cell development. Importantly, these findings are strikingly different than that observed in rodent models of disease. Together, these data provide a roadmap of human photoreceptor development and leverage patient iPSCs to define the specific roles of rod transcription factors in photoreceptor cell emergence and maturation.
2024-04-01 | GSE236197 | GEO