Differentiation Of Retinal Progenitors Research Articles

The role of EZH2 in ocular diseases: a narrative review.

EZH2, acting as a catalytic subunit of PRC2 to catalyze lysine 27 in histone H3, induces the suppression of gene expression. EZH2 can regulate cell proliferation and differentiation of retinal progenitors, which are required for physiological retinal development. Meanwhile, an abnormal level of EZH2 has been observed in ocular tumors and other pathological tissues. This review summarizes the current knowledge on EZH2 in retinal development and ocular diseases, including inherited retinal diseases, ocular tumors, corneal injury, cataract, glaucoma, diabetic retinopathy and age-related retinal degeneration. We highlight the potential of targeting EZH2 as a precision therapeutic target in ocular diseases.

Interaction between SDF1 and CXCR4 Promotes Photoreceptor Differentiation via Upregulation of NFκB Pathway Signaling Activity in Pax6 Gene-Transfected Photoreceptor Precursors

Background: CCL2 (also known as monocyte chemoattractant protein 1) and CX3CR1 (also known as Fractalkine receptor)-deficient mice have damaged photoreceptors. Objectives: We examined the interaction of SDF1 and CXCR4 on the differentiation of retinal progenitors into rhodopsin-positive photoreceptors. Methods: Cloned retinal progenitors were obtained by Pax6 gene transfection of mouse iPS cells followed by serial dilution. Clones were selected by expression of nestin, Musashi1, Six3, and Chx10 mRNA. Cell surface protein expression was analyzed by flow cytometry. The levels of mRNA and intracellular protein were examined by real-time PCR and immunochemistry, respectively. Transient transfection experiments of retinal progenitors were conducted using a human rhodopsin promoter luciferase plasmid. Results: We selected 10 clones that expressed Six3, Chx10, Crx, Rx1, Nrl, CD73, and rhodopsin mRNA, which, except for rhodopsin, are photoreceptor precursor markers. Clones expressed both CD73 and CXCR4 on the cell surface and differentiated into rhodopsin-positive photoreceptors, which was reinforced by the addition of exogenous SDF1. A CXCR4 inhibitor AMD3100 blocked SDF1-mediated differentiation of progenitors into photoreceptors. SDF1 enhanced human rhodopsin promoter transcription activity, possibly via the NFκB pathway. Addition of SDF1 to the cell culture induced nuclear translocation of NFκB on retinal progenitor cell clones. Neonatal and newborn mouse retinas expressed SDF1 and CXCR4. Cells in the outer nuclear layer where photoreceptors are located expressed CXCR4 at P14 and P56. Cells in the inner nuclear layer expressed SDF1. Conclusions: These findings suggest that retinal progenitor cell differentiation was at least partly regulated by SDF1 and CXCR4 via upregulation of NFκB activity.

MiR-29a regulates the proliferation and differentiation of retinal progenitors by targeting Rbm8a.

During development, tight regulation of the expansion of retinal progenitor cells (RPCs) and their differentiation into neuronal and glial cells is important for retinal formation and function. Our study demonstrated that microRNA (miR)-29a modulated the proliferation and differentiation of RPCs by suppressing RBM8A (one of the factors in the exon junction complex). Particularly, overexpression of miR-29a reduced RPC proliferation but accelerated RPC differentiation. By contrast, reduction of endogenous miR-29a elicited the opposite effects. Overexpression of miR-29a repressed the translation of Rbm8a, thus negatively regulating RPC proliferation and promoting the neuronal and glial differentiation of RPCs, and knockdown of endogenous Rbm8a phenocopied the observed effects of miR-29a overexpression. Furthermore, a luciferase reporter assay showed that miR-29a directly interacted with the Rbm8a mRNA 3′UTR, which indicated that Rbm8a is the direct target of miR-29a. To further verify the result, co-overexpression of the Rbm8a 3′ UTR-wt (plasmids into which the Rbm8a 3′ UTR sequence had been introduced) and miR-29a in RPCs rescued the phenotype associated with miR-29a overexpression, reversing the promotion of differentiation and inhibition of proliferation. These results show a novel mechanism by which miR-29a regulates the proliferation and differentiation of RPCs through Rbm8a.

Noggin 1 overexpression in retinal progenitors affects bipolar cell generation.

Waves of Bone Morphogenetic Proteins (BMPs) and their antagonists are present during initial eye development, but their possible roles in retinogenesis are still unknown. We have recently shown that noggin 1, a BMP antagonist, renders pluripotent cells able to differentiate into retinal precursors, and might be involved in the maintenance of retinal structures in the adult vertebrate eye. Here, we report that noggin 1, differently from noggin 2 and noggin 4, is expressed during all phases of Xenopus laevis retinal development. Gain-of-function experiments by electroporation in the optic vesicle show that overexpression of noggin 1 significantly decreases the number of bipolar cells in the inner nuclear layer of the retina, without significantly affecting the generation of the other retinal cell types. Our data suggest that BMP signaling could be involved in the differentiation of retinal progenitors into specific retinal subtypes during late phases of vertebrate retinal development.

Dicer Is Required for the Transition from Early to Late Progenitor State in the Developing Mouse Retina

MicroRNAs (miRNAs), small 19-25 nucleotide RNAs that influence gene expression through posttranscriptional regulation of mRNA translation and degradation, have recently emerged as important regulators of neural development. Using conditional knock-out of Dicer, an RNase III enzyme required for miRNA maturation, previous studies have demonstrated an essential role for miRNAs in mouse cortical, inner ear, and olfactory development. However, a previous study (Damiani et al., 2008) using a Chx10cre mouse to delete Dicer in retinal progenitors reported no defects in the retina before the second postnatal week, suggesting that miRNAs are not required for mouse retinal development. In an effort to further study the role of miRNAs during retinal development and resolve this apparent conflict, we conditionally knocked out Dicer using a different (alphaPax6cre) line of transgenic mice. In contrast to the previous study, we demonstrate an essential role for miRNAs during mouse retinal development. In the absence of Dicer in the embryonic retina, production of early generated cell types (ganglion and horizontal cells) is increased, and markers of late progenitors are not expressed. This phenotype persists into postnatal retina, in which we find the Dicer-deficient progenitors fail to generate late-born cell types such as rods and Müller glia but continue to generate ganglion cells. We further characterize the dynamic expression of miRNAs during retinal progenitor differentiation and provide a comprehensive profile of miRNAs expressed during retinal development. We conclude that Dicer is necessary for the developmental change in competence of the retinal progenitor cells.

Characterization of early retinal progenitor microenvironment: Presence of activities selective for the differentiation of retinal ganglion cells and maintenance of progenitors

The maintenance and differentiation of retinal progenitors take place in the context of the microenvironment in which they reside at a given time during retinal histogenesis. To understand the nature of the microenvironment in the developing retina, we have examined the influence of activities present during the early stage of retinal histogenesis on enriched retinal progenitors, using the neurosphere model. Early and late retinal progenitors, enriched as neurospheres from embryonic day 14 (E14) and E18 rat retina, respectively, were cultured in embryonic day 3 (E3) chick retinal conditioned medium, simulating the microenvironment present during early retinal histogenesis. Examination of the differentiation and proliferation of retinal progenitors revealed that the early microenvironment contains at least three regulatory activities, which are partitioned in different size fractions of the conditioned medium with different heat sensitivity. First, it is characterized by activities, present in heat stable <30 kDa fraction, that promote the differentiation of retinal ganglion cells (RGCs), the early born neurons. Second, it contains activities, present in heat-sensitive >30 kDa fraction, that regulate the number of early born neurons and maintain the pool of retinal progenitors. Third, it possesses activities, present in heat-sensitive <30 kDa fraction, that prevent the premature differentiation of early retinal progenitors into the late born neurons. Thus, our observations demonstrate the regulatory influence of microenvironment on the maintenance and differentiation of retinal progenitors and establish neurospheres as a viable model system for the examination of such influences.

Mouse Rab11‐FIP4 regulates proliferation and differentiation of retinal progenitors in a Rab11‐independent manner

We identified Rab11-family interacting protein 4 (Rab11-FIP4) as a gene strongly expressed in the developing mouse retina. The major transcript encoding a full-length protein, mRab11-FIP4A, was expressed predominantly in neural tissues; whereas an alternative transcript encoding an N-terminally truncated form of the protein, mRab11-FIP4B, was expressed ubiquitously as a minor form. Gain-of-function of mRab11-FIP4A in retina promoted cell cycle exit and increased subpopulations of retinal cells localized in the inner nuclear layer, such as bipolar cells and Müller glia. Reversal of the phenotype was observed in the loss-of-function experiment. Furthermore, Shh signaling was suggested to be involved in these functions. Analysis using truncation mutants revealed the essential role of the N-terminal region containing a conserved EF-hand motif for the retinal phenotypes induced by the expression of mRab11-FIP4A, whereas binding to Rab11 was dispensable, suggesting the involvement of a novel Rab11-independent mechanism for mRab11-FIP4A action in the regulation of retinal development.

Rab11-FIP4 is predominantly expressed in neural tissues and involved in proliferation as well as in differentiation during zebrafish retinal development

Rab11 family interacting protein 4 (Rab11-FIP4) was initially identified in humans as an Rab11-binding protein, but its biological function has remained unknown. We cloned the zebrafish orthologue of Rab11-FIP4 (zRab11-FIP4) and analyzed its function in vivo by using antisense morpholino. zRab11-FIP4 was expressed as 2 alternative transcripts, i.e., the longer A-form predominantly expressed in neural tissues and the shorter B-form expressed ubiquitously; and in situ hybridization revealed that the A-form was the dominant form. In the developing retina, zRab11-FIP4 was expressed in progenitors throughout the retina at early stages; and then, along with the differentiation, the expression became gradually restricted to the ganglion cell layer and ciliary marginal zone. zRab11-FIP4A knockdown embryos exhibited eye phenotypes similar to those of the shh mutant, such as a small eye with impaired cell proliferation and the delay in cell-cycle exit and differentiation of retinal progenitors. The lack of induction of p57kip2 and enhanced expression of cyclin D1 were observed in the morphant retina. Importantly, the delay in cell-cycle exit was rescued by ectopic expression of either p57Kip2 or dominant-negative PKA, suggesting that Rab11-FIP4A plays pivotal roles in retinal development by regulating Shh signaling and a mechanism acting in parallel with Shh signaling in the control of cell-cycle exit.

Fabrication of degradable polymer scaffolds to direct the integration and differentiation of retinal progenitors

Retinal progenitor cells (RPCs) are self-renewing cells capable of differentiating into the different retinal cell types including photoreceptors, and they have shown promise as a source of replacement cells in experimental models of retinal degeneration. We hypothesized that a biodegradable polymer scaffold could deliver these cells to the subretinal space in a more organized manner than bolus injections, while also providing the graft with laminar organization and structural guidance channels. We fabricated highly porous scaffolds from blends of poly( l-lactic acid) and poly(lactic- co-glycolic acid) using a variety of techniques to produce pores oriented normal to the plane of the scaffold. RPCs were seeded on the polymer scaffolds and cultured for 14 days. Seeded scaffolds were then either fixed for characterization or used in an explant or in vivo rat model. The scaffolds were fully covered by RPCs in 3 days. Attachment of RPCs to the polymer scaffold was associated with down-regulation of immature markers and up-regulation of markers of differentiation. This suggests that the scaffold may promote differentiation of RPCs. The seeded cells elaborated cellular processes and aligned in the scaffold in conjunction with degenerating retinal explants. The cells also exhibited morphologies consistent with photoreceptors including a high degree of polarization of the cells. This data suggests that the scaffold may be a means to assist in the promotion of photoreceptor phenotypes. Implantation of the seeded scaffold into the rat eye is associated with increased RPC survival. Taken together, these data suggest that these polymer scaffolds provide a useful means for delivering RPCs to the subretinal space and may assist in the formation of retinal cell phenotypes, although it is clear that more cues are needed to direct the differentiation of RPCs into functional photoreceptors.