Vertical sections of the P7 retina were hybridized with riboprobes to cNotch1 (a and b; blue) and labeled for the glial marker 2M6 (c; red). Panel d is a merged image of panels b and c. Panels e–h are 7-fold enlargements of corresponding boxed areas in panel d. Arrows in panel b–h indicate cNotch1 and 2M6 double-positive cells. The scale bar (50μm) in panel a applies to panel a alone and that in panel b applies to panels b–d. mRNA was harvested from retinas, reverse-transcribed to generate cDNA, and amplified using (i) PCR or (j–l) real-time PCR. The PCR products were run on an agarose gel, and stained with ethidium bromide (i). Reactions minus the reverse transcriptase enzyme (RT−) were used as a negative control for each primer set. The percentage change in cDNA levels for cNotch1 (j), cHes1 (k) and cHes5 (l) were calculated from C(t) values that were normalized to GAPDH. Relative expression levels were measured for each mRNA at P0 in central retina (set as baseline) and at P7, P14 and P21 in central and peripheral retina and E3 whole retina. Significance of difference (* p<0.005 between two timepoints within the same region, # p<0.01 between central and peripheral retina at the same timepoint) was determined by using a two-factor ANOVA followed by a post hoc t-test. Abbreviations: RT – reverse transcriptase, INL – inner nuclear layer, IPL – inner plexiform layer.

Representative images of TUNEL-positive nuclei in vertical sections of retinas for eyes that were treated with vehicle before NMDA (a) or DAPT before NMDA (b). Histograms illustrate the mean (± SEM) number of dying cells in the INL in central, nasal or temporal regions of the retina (c and d). Numbers of TUNEL+ cells were counted in retinas treated with 150 μg NMDA (c) or 30 μg NMDA (d) with and without DAPT pre-treatment. Representative images of whole-mounted retinas that were treated with vehicle before colchicine (e) or DAPT before colchicine (f). Retinas were labeled with antibodies to Brn3a to identify ganglion cells. Histograms illustrate the mean (±SEM) number of ganglion cells per 0.55mm² in central, dorsal, nasal or temporal regions of the retina (g and h). Data sets were obtained from 6 animals per treatment, per timepoint. Significance of difference (*p<0.05, **p<0.01) was determined by using a two-tailed, unpaired Student’s t-test. Abbreviations: ONL – outer nuclear layer, INL – inner nuclear layer. The scale bar (50μm) in panel a applies to panels a and b and that in panel f applies to panels e and f.

mRNA was harvested from retinas obtained from eyes treated with saline (control) or 3 consecutive daily doses of insulin and FGF2 (a), saline (control) or 2 consecutive daily doses of insulin and FGF2 (b), saline (control) or FGF2 alone (b) and saline (control) or insulin alone (c). Individual doses of insulin and FGF2 were 1000 ng and 250 ng, respectively. mRNA was reverse-transcribed to generate cDNA and amplified using real-time PCR. Ct values were normalized to GAPDH and the fold difference between control and treated samples was determined using the ΔCt method and represented as a percentage change (a–d). Significance of difference (*p<0.03) was determined by using a two-tailed, unpaired Student’s t-test. Abbreviations: RT – reverse transcriptase.

DAPT suppresses the accumulation of p38 MAPK and pCREB in Müller glia that results from NMDA+FGF2 treatment. Images were obtained using identical camera exposures and microscope illumination settings. Retinas were processed for immunolabeling after treatment with 150 μg of NMDA and two consecutive daily doses of 250 ng FGF2 (control) or 250 ng FGF2 + 865 ng DAPT (treated). As described in the Methods, ImagePro 6.2 was used to obtain measurements of total area for pixel intensities >72 for pCREB and >65 for p38 MAPK (0 = black, 255 = saturated green), and the density sum. The small numbers and red areas in panels c, d, g and h indicate the pixels designed by ImagePro 6.2 that met the threshold criteria. Means and standard deviations are displayed in histograms for total area (e and i) and density sum (f and j) for areas with pixels above threshold. Significance of difference (**p<0.001) was determined by using a two-tailed, unpaired Student’s t-test. Abbreviations: ONL – outer nuclear layer, INL – inner nuclear layer, IPL – inner plexiform layer. The scale bar (50μm) in panel b applies to panels a –b and that in panel h applies to panels c–h.

This figure draws from several studies to construct putative modes of action for insulin/IGF, FGF2 and neuronal damage on Müller glia in the chick retina (Fischer et al., 2002b; Fischer et al., 2004b; Fischer et al., 2009b; Fischer et al., 2009a)
(a) Effects of insulin/IGF1 alone: insulin/IGF1 signaling via the NIRG cells and/or microglia secondarily enhances effectors of MAPK-signaling, i.e p38 MAPK (IGF1-mediated) and cFos, Egr1 and pERK (insulin-mediated) in Müller glia in addition to increasing neuronal cell death (magenta arrows). It also modestly decreases Notch and related genes (magenta dashed arrows). Low levels of Notch-signaling in Müller glia inhibits the neuron-supporting functions of Müller glia, exacerbating neuronal death (black arrows) during damage (orange arrows). In addition, low levels of Notch-signaling enhance de-differentiation and progenitor-like properties (black arrows), promoting Müller glial proliferation during damage.
(b) Effects of FGF2 alone: FGF2 has Notch-dependent and Notch-independent effects.
(1) Notch-independent effects – FGF/MAPK-signaling induces the accumulation of pERK1/2, p38 MAPK, pCREB, cfos and Egr1 in Müller glia, which may stimulate the Müller glia to become neuroprotective and provide support to neurons in damaged retinas (blue arrows). pERK1/2 and Egr1 promote Müller glial de-differentiation and proliferation in a Notch-independent manner (green arrows).
(2) Notch-dependent effects – FGF/MAPK-signaling induces upregulates expression of Notch and associated genes (pink dashed arrows). Low levels of Notch-signaling in an undamaged or moderately damaged retina “prime” the glia to proliferate (black arrows). FGF2-mediated Müller glial proliferation requires some retinal damage and active Notch-signaling, which promotes accumulation of p38 MAPK and pCREB (red arrows), which may make the glia more progenitor-like, inducing Müller glial proliferation during damage (orange arrow).
(c) Combined effects of insulin/IGF1 and FGF2: insulin/IGF1 signaling in the NIRG cells/microglia and FGF/MAPK-signaling in the Müller glia together upregulate expression of Notch and its downstream effectors (pink arrows). Insulin/IGF1-signaling, FGF/MAPK-signaling and upregulated Notch-signaling together induce the de-differentiation and proliferation of Müller glia in the absence of damage.

mRNA was harvested from central and peripheral retinas obtained from eyes injected with vehicle (Control) or DAPT (Treated). mRNA was reverse-transcribed to generate cDNA and amplified using (a) PCR or (b) real-time PCR. The PCR products were run on an agarose gel, and stained with ethidium bromide (a). For real-time PCR, C(t) values were normalized to GAPDH and the fold difference between control and treated samples was determined using the ΔCt method and represented as a percentage change from baseline (b). Representative images from retinas harvested 24 hrs after the last dose of vehicle or DAPT (c–j). Vertical sections of the P7 retina were labeled for Sox9 (c and d), GFAP (e and f), transitin (g and h) or vimentin (i and j). Abbreviations: RT – reverse transcriptase, ONL – outer nuclear layer, INL – inner nuclear layer, IPL – inner plexiform layer. The scale bar (50μm) in panel j applies to panels c– j.

Eyes were injected with three consecutive daily doses of insulin and FGF2 or saline (control) staring at P4, BrdU at P7 and harvested at P8. Vertical sections of retina were hybridized with riboprobes to cNotch1 (a; blue) and subsequently labeled for PCNA (b; red) and BrdU (c; green). Panel d is a merged image of panels a – c. Panels e–g are 3.5-fold enlargements of corresponding boxed areas in panel d. Control retinas did not show an upregulation of cNotch1 (h), PCNA (i) or BrdU (j). White arrows in panel b indicate PCNA positive cells. Yellow arrows in panel c and d indicate BrdU-positive cells. The asterisks (*) in panels e–g indicate cells labeled for cNotch1, PCNA and BrdU-positive cells. Abbreviations: ONL – outer nuclear layer, INL – inner nuclear layer, IPL – inner plexiform layer. The scale bar (50μm) in panel a applies to panels a –c and h–j and the scale bar in panel d (50μm) applies to d alone.

Eyes were treated with 150 μg of NMDA at P4 followed by two consecutive daily doses of saline (control) or 250 ng FGF2 (treated) at P5 and P6, BrdU at P6 and harvested 24 hrs later at P7. Vertical sections of the P7 retina were labeled for Sox9 (a, e; magenta), BrdU (b, f; green) and PCNA (c, g; red). Panel d is a merged image of panels a – c. Panel h is a merged image of panels e–g. Panels d and h are 3.5-fold enlargements of panels a–c and e–g, respectively. Arrows in panels e–h indicate Sox9-, BrdU- and PCNA- triple-positive cells. The histograms in panel i represent the mean (±SEM) number of BrdU- and PCNA-positive cells per 15000μm² of the retinas. Vertical sections from control and treated retinas were labeled for 2M6 (red) and p38 MAPK (green; j–o) or pCREB (green; p–u). Significance of difference (**p<0.001) was determined by using a two-tailed, unpaired Student’s t-test. Arrows indicate 2M6-positive Müller glia labeled for p38 MAPK (m – o) or pCREB (s– u). Abbreviations: INL – inner nuclear layer. The scale bar (50μm) in panel g applies to panels a – c, e–g and that in panel u applies to panels j– u.

Eyes were injected with 150 μg NMDA before or after two consecutive daily doses of 250 ng FGF2 (control) or 250 ng FGF2 and 865 ng DAPT (treated), BrdU at P6 (or P7) and harvested at P7. Vertical sections of the P7 retina were labeled for Sox9 (a, e; magenta), BrdU (b, f; green) and PCNA (c, g; red). Panel d is a merged image of panels a –c. Panel h is a merged image of panels e–g. Panels d and h are 4-fold enlargements of panels a–c and e–g, respectively. Arrows in panels a–h indicate Sox9-, BrdU-, and PCNA- positive triple-labeled cells and arrowheads indicate Sox9- and PCNA- double-positive cells. The histograms in panel i represent the number of Sox9-, BrdU- and PCNA- triple positive cells per 15000μm² of retina. Significance of difference (**p<0.001) was determined by using a two-tailed, unpaired Student’s t-test. Abbreviations: INL – inner nuclear layer. The scale bar (50μm) in panel g applies to panels a–c and e–g.