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1.  p53 is required for the developmental restriction in Müller glial proliferation in mouse retina 
Glia  2012;60(10):1579-1589.
Müller glia are normally mitotically quiescent cells, but in certain pathological states they can reenter the mitotic cell cycle. While several cell cycle regulators have been shown to be important in this process, a role for the tumor suppressor, p53, has not been demonstrated. Here, we investigated a role for p53 in limiting the ability of Müller glia to proliferate in the mature mouse retina. Our data demonstrate that müller glia undergo a developmental restriction in their potential to proliferate. Retinal explants or dissociated cultures treated with EGF become mitotically quiescent by the end of the second postnatal week. In contrast, Müller glia from adult trp53−/+ or trp53−/− mice displayed a greater ability to proliferate in response to EGF stimulation in vitro. The enhanced proliferative ability of trp53 deficient mice correlates with a decreased expression of the mitotic inhibitor Cdkn1a/p21cip and an increase in c-myc, a transcription factor that promotes cell cycle progression. These data show that p53 plays an essential role in limiting the potential of Müller glia to re-enter the mitotic cycle as the retina matures during postnatal development.
doi:10.1002/glia.22377
PMCID: PMC3422417  PMID: 22777914
cell cycle; proliferation; retina
2.  Three-Dimensional Neuroepithelial Culture from Human Embryonic Stem Cells and Its Use for Quantitative Conversion to Retinal Pigment Epithelium 
PLoS ONE  2013;8(1):e54552.
A goal in human embryonic stem cell (hESC) research is the faithful differentiation to given cell types such as neural lineages. During embryonic development, a basement membrane surrounds the neural plate that forms a tight, apico-basolaterally polarized epithelium before closing to form a neural tube with a single lumen. Here we show that the three-dimensional epithelial cyst culture of hESCs in Matrigel combined with neural induction results in a quantitative conversion into neuroepithelial cysts containing a single lumen. Cells attain a defined neuroepithelial identity by 5 days. The neuroepithelial cysts naturally generate retinal epithelium, in part due to IGF-1/insulin signaling. We demonstrate the utility of this epithelial culture approach by achieving a quantitative production of retinal pigment epithelial (RPE) cells from hESCs within 30 days. Direct transplantation of this RPE into a rat model of retinal degeneration without any selection or expansion of the cells results in the formation of a donor-derived RPE monolayer that rescues photoreceptor cells. The cyst method for neuroepithelial differentiation of pluripotent stem cells is not only of importance for RPE generation but will also be relevant to the production of other neuronal cell types and for reconstituting complex patterning events from three-dimensional neuroepithelia.
doi:10.1371/journal.pone.0054552
PMCID: PMC3554725  PMID: 23358448
3.  Genome-Wide Analysis of Müller Glial Differentiation Reveals a Requirement for Notch Signaling in Postmitotic Cells to Maintain the Glial Fate 
PLoS ONE  2011;6(8):e22817.
Previous studies have shown that Müller glia are closely related to retinal progenitors; these two cell types express many of the same genes and after damage to the retina, Müller glia can serve as a source for new neurons, particularly in non-mammalian vertebrates. We investigated the period of postnatal retinal development when progenitors are differentiating into Müller glia to better understand this transition. FACS purified retinal progenitors and Müller glia from various ages of Hes5-GFP mice were analyzed by Affymetrix cDNA microarrays. We found that genes known to be enriched/expressed by Müller glia steadily increase over the first three postnatal weeks, while genes associated with the mitotic cell cycle are rapidly downregulated from P0 to P7. Interestingly, progenitor genes not directly associated with the mitotic cell cycle, like the proneural genes Ascl1 and Neurog2, decline more slowly over the first 10–14 days of postnatal development, and there is a peak in Notch signaling several days after the presumptive Müller glia have been generated. To confirm that Notch signaling continues in the postmitotic Müller glia, we performed in situ hybridization, immunolocalization for the active form of Notch, and immunofluorescence for BrdU. Using genetic and pharmacological approaches, we found that sustained Notch signaling in the postmitotic Müller glia is necessary for their maturation and the stabilization of the glial identity for almost a week after the cells have exited the mitotic cell cycle.
doi:10.1371/journal.pone.0022817
PMCID: PMC3149061  PMID: 21829655
4.  BAF60c is a component of the neural progenitor specific BAF complex in developing retina 
Remodeling of the chromatin network plays an important role regulating embryonic development as well as differentiation. The SWI/SNF complex is an ATP dependent chromatin-remodeling complex. It consists of several proteins, including an ATPase subunit, either Brg1 or Brm. Two subunits of this complex Baf53a and Baf45 have been previously identified as being neural progenitor specific. In this study, we show that Baf60c, another important part of this large complex, acts in a similar neural-progenitor specific manner. We show that during development Baf60c is expressed in neural progenitors in human retinas as well as mouse retina, cortex and spinal cord. Baf60c expression is lost during neural differentiation and its over-expression keeps the progenitors in a proliferative state through its interaction with the Notch pathway. Finally, we show that Baf60c is re-expressed in the Müller glial cells that reenter the cell cycle after neurotoxic damage.
doi:10.1002/dvdy.21697
PMCID: PMC2707830  PMID: 18816825
Chromatin remodeling; Baf60c; retina development; retinal regeneration; neural development; Swi/Snf
5.  Cell-Specific Differential Modulation of Human Trabecular Meshwork Cells by Selective Adenosine Receptor Agonists 
Experimental eye research  2006;84(1):126-134.
Activation of A1 and A2A subtype adenosine receptors (AR) likely exert opposing effects on outflow of aqueous humor, and thereby, on intraocular pressure. Selective agonists of adenosine receptor (AR) subtypes have previously been applied to trabecular meshwork (TM) and Schlemm’s canal (SC) cells to identify the site(s) of differential purinergic modulation. However, the apparent changes in volume monitored by previously measuring projected cell area might have partially reflected cell contraction and relaxation. In addition, whole-cell current responses of the TM cells previously described were highly variable following application of selective A1, A2A and A3 agonists. The complexity of the electrophysiologic responses may have reflected cell heterogeneity of the populations harvested from collagenase digestion of TM explants. We now report measurements of TM-cell volume using calcein fluorescence quenching, an approach independent of contractile state. Furthermore, we have applied selective AR agonists to a uniform population of human TM cells, the hTM5 cell line. A1, but not A2A or A3, AR agonists triggered TM-cell shrinkage. Both A1 and A2A AR agonists produced reproducible increases in TM-cell whole-cell currents of similar magnitude.
The results suggest that previous measurements of explant-derived TM cells may have reflected a range of responses from phenotypically different cell populations, and that the opposing effects of A1 and A2A agonists on outflow resistance are not likely to be mediated by actions on a single population of TM cells. These opposing effects might reflect AR responses by two or more subpopulations of TM cells, by TM and SC cells or by inner-wall SC cells, alone.
doi:10.1016/j.exer.2006.09.007
PMCID: PMC1764820  PMID: 17070802
6.  The Light Peak of the Electroretinogram Is Dependent on Voltage-gated Calcium Channels and Antagonized by Bestrophin (Best-1) 
The Journal of General Physiology  2006;127(5):577-589.
Mutations in VMD2, encoding bestrophin (best-1), cause Best vitelliform macular dystrophy (BMD), adult-onset vitelliform macular dystrophy (AVMD), and autosomal dominant vitreoretinochoroidopathy (ADVIRC). BMD is distinguished from AVMD by a diminished electrooculogram light peak (LP) in the absence of changes in the flash electroretinogram. Although the LP is thought to be generated by best-1, we find enhanced LP luminance responsiveness with normal amplitude in Vmd2−/− mice and no differences in cellular Cl− currents in comparison to Vmd2+/+ littermates. The putative Ca2+ sensitivity of best-1, and our recent observation that best-1 alters the kinetics of voltage-dependent Ca2+ channels (VDCC), led us to examine the role of VDCCs in the LP. Nimodipine diminished the LP, leading us to survey VDCC β-subunit mutant mice. Lethargic mice, which harbor a loss of function mutation in the β4 subunit of VDCCs, exhibited a significant shift in LP luminance response, establishing a role for Ca2+ in LP generation. When stimulated with ATP, which increases [Ca++]I, retinal pigment epithelial cells derived from Vmd2−/− mice exhibited a fivefold greater response than Vmd2+/+ littermates, indicating that best-1 can suppress the rise in [Ca2+]I associated with the LP. We conclude that VDCCs regulated by a β4 subunit are required to generate the LP and that best-1 antagonizes the LP luminance response potentially via its ability to modulate VDCC function. Furthermore, we suggest that the loss of vision associated with BMD is not caused by the same pathologic process as the diminished LP, but rather is caused by as yet unidentified effects of best-1 on other cellular processes.
doi:10.1085/jgp.200509473
PMCID: PMC2151522  PMID: 16636205
7.  The Ternary Rab27a–Myrip–Myosin VIIa Complex Regulates Melanosome Motility in the Retinal Pigment Epithelium 
Traffic (Copenhagen, Denmark)  2007;8(5):486-499.
The retinal pigment epithelium (RPE) contains melanosomes similar to those found in the skin melanocytes, which undergo dramatic light-dependent movements in fish and amphibians. In mammals, those movements are more subtle and appear to be regulated by the Rab27a GTPase and the unconventional myosin, Myosin VIIa (MyoVIIa). Here we address the hypothesis that a recently identified Rab27a- and MyoVIIa-interacting protein, Myrip, promotes the formation of a functional tripartite complex. In heterologous cultured cells, all three proteins co-immunoprecipitated following overexpression. Rab27a and Myrip localize to the peripheral membrane of RPE melanosomes as observed by immunofluorescence and immunoelectron microscopy. Melanosome dynamics were studied using live-cell imaging of mouse RPE primary cultures. Wild-type RPE melanosomes exhibited either stationary or slow movement interrupted by bursts of fast movement, with a peripheral directionality trend. Nocodazole treatment led to melanosome paralysis, suggesting that movement requires microtubule motors. Significant and similar alterations in melanosome dynamics were observed when any one of the three components of the complex was missing, as studied in ashen- (Rab27a defective) and shaker-1 (MyoVIIa mutant)-derived RPE cells, and in wild-type RPE cells transduced with adenovirus carrying specific sequences to knockdown Myrip expression. We observed a significant increase in the number of motile melanosomes, exhibiting more frequent and prolonged bursts of fast movement, and inversion of directionality. Similar alterations were observed upon cytochalasin D treatment, suggesting that the Rab27a–Myrip–MyoVIIa complex regulates tethering of melanosomes onto actin filaments, a process that ensures melanosome movement towards the cell periphery.
doi:10.1111/j.1600-0854.2007.00548.x
PMCID: PMC1920545  PMID: 17451552
melanosome motility; Myrip; Rab27a; RPE

Results 1-7 (7)