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ARS2 is required for retinal progenitor cell S-phase progression and Müller glial cell fate specification.
摘要： During a developmental period that extends postnatally in the mouse, proliferating multipotent retinal progenitor cells produce one of seven major cell types (rod, cone, bipolar, horizontal, amacrine, ganglion, and Müller glial cells) as they exit the cell cycle in consecutive waves. Cell production in the retina is tightly regulated by intrinsic, extrinsic, spatial, and temporal cues and is coupled to the timing of cell cycle exit. ARS2 (also known as SRRT) is a component of the nuclear cap-binding complex involved in RNA Polymerase II transcription, and is required for cell cycle progression. We show that postnatal RPCs require ARS2 for proper progression through S phase, and ARS2 disruption leads to early exit from the cell cycle. Furthermore, we observe an increase in the proportion of cells expressing a rod photoreceptor marker, and a loss of Müller glia marker expression, indicating a role for ARS2 in regulating cell fate specification or differentiation. Knockdown of FLASH, which interacts with ARS2 and is required for cell cycle progression and 3’-end processing of replication-dependent histone transcripts, phenocopies ARS2 knockdown. These data implicate ARS2/FLASH-mediated histone mRNA processing in regulating RPC cell cycle kinetics and neuroglial cell fate specification during postnatal retinal development.
关键词： photoreceptor cells,Müller cells,retina,retinal progenitor cells,cell cycle,Ars2
更新于2025-09-19 17:15:36
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Photoreceptor Progenitors Depend Upon Coordination of <i>gdf6a</i> , <i>thrβ</i> , and <i>tbx2b</i> to Generate Precise Populations of Cone Photoreceptor Subtypes
摘要： PURPOSE. Replacing cone photoreceptors, the units of the retina necessary for daytime vision, depends upon the successful production of a full variety of new cones from, for example, stem cells. Using genetic experiments in a model organism with high cone diversity, zebrafish, we map the intersecting effects of cone development factors gdf6a, tbx2b, and thrb. METHODS. We investigated these genes of interest by using genetic combinations of mutants, gene knockdown, and dominant negative gene expression, and then quantified cone subtype outcomes (which normally develop in tightly regulated ratios). RESULTS. Gdf6a mutants have reduced blue cones and, discovered here, reduced red cones. In combined gdf6a/tbx2b disruption, the loss of gdf6a in heterozygous tbx2b mutants reduced UV cones. Intriguingly, when we disrupted thrb in gdf6a mutants by using a thrb morpholino, their combined early disruption revealed a lamination phenotype. Disrupting thrb activity via expression of a dominant negative thrb (dnthrb) at either early or late retinal development had differential outcomes on red cones (reduced abundance), versus UV and blue cones (increased abundance). By using dnthrb in gdf6a mutants, we revealed that disrupting thrb activity did not change gdf6a mutant cone phenotypes. CONCLUSIONS. Gdf6a loss directly affects blue and red cones and indirectly affects UV cones by increasing sensitivity to additional disruption, such as reduced tbx2b, resulting in fewer UV cones. The effects of thrb change through photoreceptor development, first promoting red cones and restricting UV cones, and later restricting UV and blue cones. The effects of gdf6a on UV, blue, and red cone development overlap with, but likely supersede, those of thrb.
关键词： BMP signaling,progenitor,color vision,retinal lamination,thyroid signaling,zebrafish,retinal development,determination,regeneration,cone photoreceptor
更新于2025-09-19 17:15:36
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Loss of Citron Kinase Affects a Subset of Progenitor Cells That Alters Late but Not Early Neurogenesis in the Developing Rat Retina
摘要： PURPOSE. To understand how loss of citron kinase (CitK) affects retinal progenitor cells (RPCs) in the developing rat retina. METHODS. We compared knockout (KO) and wild-type (WT) retinae by immunohistochemistry. The TdT-mediated dUTP terminal nick-end labeling (TUNEL) assay was performed to determine cell death. Pulse-chase experiments using 5-ethynyl-2’-deoxyuridine (EdU) were carried out to interrogate RPC behavior and in turn neurogenesis. RESULTS. Reverse transcription–polymerase chain reaction analysis showed that CitK was expressed at embryonic day (E)12 and was turned off at approximately postnatal day (P)4. Immunohistochemistry showed CitK being localized as puncta at the apical end of the outer neuroblastic layer (ONBL). Analyses during embryonic development showed that the KO retina was of comparable size to that of WT until E13. However, by E14, there was a reduction in the number of S-phase RPCs with a concomitant increase in TUNELt cells in the KO retina. Moreover, early neurogenesis, as re?ected by retinal ganglion cell production, was not affected. Postnatal analysis of the retina showed that ONBL in the KO retina was reduced to half the size of that in WT and showed further degeneration. Immunohistochemistry revealed absence of Islet1t bipolar cells at P2, which was further con?rmed by EdU pulse-chase experiments. The CitK KO retinae underwent complete degeneration by P14. CONCLUSIONS. Our study showed that CitK is not required for a subset of RPCs before E14, but is necessary for RPC survival post E14. This in turn results in normal early embryonic neurogenesis, but severely compromised later embryonic and postnatal neurogenesis.
关键词： CitK,retina,bipolar cells,progenitor cells
更新于2025-09-10 09:29:36
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<i>In vitro</i> and <i>in vivo</i> Proteomic Comparison of Human Neural Progenitor Cell-Induced Photoreceptor Survival
摘要： Retinal degenerative diseases are some of the leading causes of blindness with few treatments. Various cell-based therapies have aimed to slow the progression of vision loss by preserving light-sensing photoreceptor cells. A subretinal injection of human neural progenitor cells (hNPCs) into the Royal College of Surgeons (RCS) rat model of retinal degeneration has aided in photoreceptor survival, though the mechanisms are mainly unknown. Identifying the retinal proteomic changes that occur following hNPC treatment will lead to better understanding of neuroprotection. To mimic the retinal environment following hNPC injection, a co-culture system of retinas and hNPCs was developed. Less cell death occurred in RCS retinal tissue co-cultured with hNPCs than in retinas cultured alone, suggesting that hNPCs provide retinal protection in vitro. Comparison of ex vivo and in vivo retinas identified NRF2-mediated oxidative response signaling as an hNPC-induced pathway. This is the first study to compare proteomic changes following treatment with hNPCs in both an ex vivo and in vivo environment, further allowing the use of ex vivo modeling for mechanisms of retinal preservation. Elucidation of the protein changes in the retina following hNPC treatment may lead to the discovery of mechanisms of photoreceptor survival and its therapeutic for clinical applications.
关键词： retinal degeneration,stem cells,transplantation,Human neural progenitor cells,neuroprotection
更新于2025-09-04 15:30:14
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The Effect of Transient Local Anti-inflammatory Treatment on the Survival of Pig Retinal Progenitor Cell Allotransplants
摘要： The development of photoreceptor degenerative disorders requires the identification of the optimal cell source and immunosuppressive regimen in a large animal model. Allotransplants are not acutely rejected in swine subretinal space, although it is not known if survival can be improved with immunosuppression. Here we investigated the survival and integration of expanded pig retinal progenitor cells (pRPCs) in normal recipients with and without transient anti-inflammatory suppression. pRPCs were derived from the neural retina of E60 GFP transgenic pigs, expanded for six passages, characterized, and transplanted into the subretinal space of 12 pigs. Six recipients received a single intravitreal injection of rapamycin and dexamethasone. pRPCs expressed the photoreceptor development genes Sox2, Pax6, Lhx2, Crx, Nrl, and Recoverin in vitro. Transplanted cells were identified in 9 out of 12 recipients 4 weeks after the injection. pRPCs integrated primarily into the photoreceptor inner segment layer and outer nuclear layer with single cells present in the inner nuclear layer. Donor cells remained recoverin-positive and acquired rhodopsin. We did not observe any signs of graft proliferation. The immunosuppression did not affect the survival or distribution of grafts. No macrophage infiltration or loss of retinal structure was observed in either group. Local immunosuppression with rapamycin and dexamethasone does not improve the outcome of pRPC allotransplantation into the subretinal space. Survival and integration of pRPC together with the lack of graft proliferation suggests that allogeneic RPC transplantation without transient immunosuppression is a favorable approach for photoreceptor cell replacement.
关键词： rapamycin,retina,retinal progenitor cells,cell therapy,photoreceptors
更新于2025-09-04 15:30:14
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Transplantation of retinal progenitor cells from optic cup-like structures differentiated from human embryonic stem cells in vitro and in vivo generation of retinal ganglion-like cells
摘要： Human embryonic stem cells (hESCs) have the potential to differentiate along the retinal lineage. We have efficiently differentiated human pluripotent stem cells (hPSCs) into optic cup-like structures by using a novel retinal differentiation medium (RDM).The purpose of this study was to determine whether the retinal progenitor cells (RPCs) derived from hESCs can integrate into the host retina and differentiate into retinal ganglion cells (RGCs) in vivo. In the present study, hESCs (H9-GFP) were induced to differentiate into optic cup-like structures by using our novel differentiation system. The RPCs extracted from the optic cup-like structures were transplanted into the vitreous cavity of N-Methyl-D-aspartic acid (NMDA)-treated mice. Sham-treated eyes received the same amount of retinal differentiation medium (RDM). The host retinas were analyzed by triple immunofluorescence on the 4th and 5th weeks after transplantation. The optic cup-like structures were efficiently differentiated from hESCs by using our novel differentiation system in vitro for 6-8 weeks. The RPCs extracted from the optic cup-like structures migrated and integrated into the GCL of the host retina. Furthermore, the remaining transplanted cells were spread over the GCL and had a complementary distribution with host residual RGCs in the GCL of the mouse retina. Surprisingly, some of the transplanted cells expressed the RGC-specific marker Brn3a. These findings demonstrated that the RPCs derived from hESCs could integrate into the host GCL and differentiate into retinal ganglion-like cells in vivo, suggesting that RPCs can be used as an ideal source in supplying countless RGC and ESC-based replacement therapies may be a promising treatment to restore vision in patients with degenerative retinal diseases.
关键词： retinal ganglion cells,human embryonic stem cells,transplantation,optic cup-like structures,retinal progenitor cells
更新于2025-09-04 15:30:14