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. 2018 Feb 28;30:38–51. doi: 10.1016/j.ebiom.2018.02.023

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© 2018 German Center for Neurodegenerative Diseases (DZNE)

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Fig. 1 — Müller glial cells (MGCs) undergo reprogramming and differentiate into CALR⁺ cells following NMDA-damage. (a) Experimental scheme. We used transgenic GFAP-Cre/R26Y mice. In these mice, ubiquitous expression of YFP is impeded by the presence of a floxed-STOP codon, which can be excised by Cre recombinase. Expression of Cre recombinase is driven by the glial-specific GFAP promoter. As a consequence, the YFP reporter allows to trace glial cells. We injected NMDA in the right eyes to induce retinal degeneration. Left eyes were injected with PBS, as controls. We characterized YFP⁺ cells at various time-points post-injection. (b) Representative immunostaining of retinal sections harvested from GFAP-Cre/R26Y mice sacrificed 24 hpi and 4 dpi. Higher magnification images (from the areas enclosed by the white boxes) are shown in the bottom panel. YFP⁺ cells (green) are also positive for GS (red), a well-known glial marker (onl, outer nuclear layer; inl, inner nuclear layer; gc, ganglion cells layer). Scale bar: 100 μm. (c) RT-PCR expression analysis of neural stem cell and retinal progenitor genes using total RNA harvested from FACS-sorted YFP⁺ cells of either PBS-treated (CTR) or NMDA-damaged (NMDA) retinae of GFAP-Cre/R26Y mice, 24 hpi and 4 dpi. Transcript levels are expressed as fold-changes relative to YFP⁺ cells sorted from PBS-injected retinae. Data are represented as mean ± S.E.M. (n = 4). Statistical analysis is based on unpaired Student's t-test (24 hpi: Pax6, p = 0.3533^ns;Nestin, p = 0.1704^ns; Chx10, p = 0.1009^ns; Six3, p = 0.3896^ns; Math3, p = 0.1157^ns; Math5, p = 0.5200^ns; Prox1, p = 0.8599^ns; Ascl1, p = 0.3695^ns; Sox2, p = 0.2774^ns; 4 dpi: Pax6, p = 0.2357^ns; Nestin, p = 0.3592^ns; Chx10, p = 0.0771^ns; Six3, p = 0.8952^ns; Math3, p = 0.4317^ns; Math5, p = 0.1218^ns; Prox1, p = 0.3917^ns; Ascl1, p = 0.2790^ns; Sox2, p = 0.0378^⁎). (d) Quantification of proliferating MGCs. Results are presented as number of counted PCNA⁺/YFP⁺ and phH3⁺/YFP⁺ cells per section, 24 hpi and 4 dpi, both for PBS-treated (CTR) and NMDA-damaged (NMDA) retinae. Data are represented as mean ± S.E.M. (n = 3). Statistical analysis is based on unpaired Student's t-test (phH3: 24 hpi, p = 0.0086^⁎⁎; 4 dpi, p = 0.0042^⁎⁎; PCNA: 24 hpi, p = 0.0103^⁎; 4 dpi, p = 0.0203^⁎). (e) Immunostaining of retinal flat mounts from GFAP-Cre/R26Y mice sacrificed 3 weeks post-injection. Representative fields from PBS-injected (CTR) and NMDA-damaged (NMDA) retinae. YFP⁺ cells (green) differentiating into CALR⁺ cells (red) are indicated by yellow arrows. Nuclei were counterstained with DAPI (blue). Scale bar: 20 μm. (f) Schematic representation of the method used for counting marker-positive cells in retinal flat mounts. GFAP-Cre/R26Y mice were used for these experiments. YFP⁺ and YFP⁺/CALR⁺ cells were counted in 10 random fields from at least three different retinal flat mounts for each treatment group. (g) Percentages of YFP⁺ cells expressing CALR, 3 wpi. Cells were counted in 6–10 random fields for each flat mount harvested from either PBS-injected (CTR) and damaged (NMDA) retinae. Data are represented as mean ± S.E.M. (n = 3). Statistical analysis is based on unpaired Student's t-test (p < 0.0001^⁎⁎⁎⁎).