Genome-Wide Analysis of Müller Glial Differentiation Reveals a Requirement for Notch Signaling in Postmitotic Cells to Maintain the Glial Fate

Abstract

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.

Loss of Caveolin-1 Impairs Retinal Function Due to Disturbance of Subretinal Microenvironment

“…1). Cav-1 has been identified as a Müller glia-enriched transcript compared with retinal neurons (38), and its expression increases as Müller glia mature with the same temporal expression as aquaporin-4 and the inwardly rectifying potassium channel, Kir4.1 (37). In RPE, Cav-1 and morphologically identifiable caveolae localize to both the apical membranes facing photoreceptors and the basal RPE membranes facing the choroid (35), consistent with the localization shown in Fig.…”

“…In the outer retina (outer nuclear layer and outer plexiform layer), Cav-1 immunoreactivity is more intense. At the resolution of confocal microscopy, we are unsure if this immunoreactivity is derived solely from Müller glial cells or also from expression in photoreceptors because it is expressed in both cell types albeit enriched in the former (37,38). Based on localization in the outer retina, cofractionation with phototransduction proteins (32,33,44), and interaction, in vitro, with transducin ␣-subunits (30), we hypothesized that loss of Cav-1 might affect retinal function.…”

“…Caveolin-1 is expressed in several retinal cell types: photoreceptors (28 -33), retinal pigment epithelium (RPE) (34 -36), Müller glial cells (37,38), and retinal vascular endothelium (39 -43). In photoreceptors, several phototransduction proteins, including transducin, RGS-9, arrestin, guanylate cyclase, rhodopsin kinase, and the cyclic nucleotide-gated channel, cofractionate in Cav-1-enriched membranes (32,33,44), and Cav-1 and the ␣-subunit of transducin co-immunoprecipitate in a cholesterol-and guanine nucleotide-dependent manner (30).…”

Conserved microRNA pathway regulates developmental timing of retinal neurogenesis

“…Of these, 200 genes were increased by more than 1.5-fold in the Dicer-CKO retinal cells (Table S1) whereas 382 genes were down-regulated by a similar amount. Many of the genes that were down-regulated in the Dicer CKO cells (Table S2) have been previously shown to be expressed in the late progenitors, e.g., Ascl1, Dll3, Hes6 and Sox9 (3,14,19) further confirming that loss of Dicer blocked the developmental progression of the progenitor cells.…”

“…1D) from E12 to P0. To determine whether these miRNAs are expressed in retinal progenitors, we used FAC sorting to purify progenitors from a Hes5-GFP mouse line that expresses GFP in retinal progenitors (14), and then carried out RT-PCR. We found that let-7 and miR-9 are enriched in the Hes5-GFP(+) progenitors (Fig.…”