Journal: PNAS Nexus

Article Title: Transient YAP activation uncovers the neurogenic potential of proliferative mammalian Müller glia

doi: 10.1093/pnasnexus/pgag188

Figure Lengend Snippet: Intersectional genetic lineage tracing of proliferative YAP5SA+ MG cells. A and B) Schematic illustrating the overall experimental design. A) Adult Glast-CreERT2 +/tg ; R26R-Ai65 ki/ki mice are injected with tamoxifen to induce Cre recombination of the loxP-flanked STOP cassette in the R26R-Ai65 locus. Intravitreal delivery of AAV, encoding FlpO, recombines the FRT-flanked stop cassette, leading to tdTomato expression specifically in MG that received the AAV. B) If the AAV also carries Yap5SA , the tdTomato+ MGs will proliferate and incorporate EdU in their nuclei. Upon subsequent divisions, the cells will lose AAV particles, exit the cell cycle, but retain both the tdTomato and EdU labels as lineage markers for postmitotic daughter cells. C) Map of the mKate2 negative control AAV and immunofluorescent (IF) image showing that, after 14 days postintravitreal injection, tdTomato is expressed in SOX9+ MGs. D) Map of the Yap5SA AAV and IF image showing that tdTomato and FLAG (fused to the N-terminus of YAP5SA) are expressed in SOX9+ MGs. E) Schematic summarizing how the previously published Yap5SA transgenic mouse drives MG spontaneous proliferation. F) EdU labeling showing that Yap5SA AAV induces spontaneous proliferation of tdTomato+ MGs, whereas mKate2 AAV does not. G) Representative cytometry scatter plots and quantification showing a significant increase of tdTomato+/EdU+ cells in retinas infected with Yap5SA AAV. H) IF and I) qRT-PCR showing Yap5SA AAV-dependent upregulation of Cyclin D1 . All quantified data are shown as mean ± SD and n ≥ 3 biologically independent samples per group. Significant differences between groups were determined using an unpaired t test with Welch correction. A P -value of <0.05 was considered significant. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Scale bars = 20 μm. Created in BioRender. Poché (2026) https://BioRender.com//9qf4bbm .

Article Snippet: Rosa26-Ai65 +/ki (JAX stock #021875) ( ) and Glast-CreERT2 +/tg ( ) mice were maintained on a C57BL/6J background and genotyped by PCR using established protocols.

Techniques: Injection, Expressing, Negative Control, Transgenic Assay, Labeling, Cytometry, Infection, Quantitative RT-PCR

Journal: PNAS Nexus

Article Title: Transient YAP activation uncovers the neurogenic potential of proliferative mammalian Müller glia

doi: 10.1093/pnasnexus/pgag188

Figure Lengend Snippet: Immunofluorescent and single-cell transcriptomic profiling of MG daughter cell fate. A) IF of the bipolar cell marker SCGN showing the colocalization with a subset of EdU+/tdTomato+ cells in three independent retinas, 56 days post- Yap5SA AAV. B) Representative cytometry scatter plots and quantification of the % EdU+/tdTomato+/SCGN+ cells of the total EdU+/tdTomato+ population driven by Yap5SA AAV. C) Representative cytometry scatter plots and quantification from C57BL/6J mice 28 days after infection with either control or Yap5SA AAV, showing an increase in the % of SCGN+ cells that are also EdU+, specifically in the Yap5SA AAV-infected retinas. D) UMAP of integrated scRNA-seq data from FACS-sorted tdTomato+ cells from control or Yap5SA AAV-infected retinas. E) scPoli reference mapping of the high-quality, filtered MGs. Query cells are projected onto the MRCA latent space, overlaid onto the reference MG (MG MRCA) and background reference cells (Other MRCA). F) Distribution of the scPoli Certainty Score for the filtered query cells, demonstrating high and comparable mapping confidence to the reference MG class. White boxes indicate the interquartile range and median. G) Dot plot of average expression between conditions for canonical MG markers. Scale bars = 20 μm.

Article Snippet: Rosa26-Ai65 +/ki (JAX stock #021875) ( ) and Glast-CreERT2 +/tg ( ) mice were maintained on a C57BL/6J background and genotyped by PCR using established protocols.

Techniques: Single Cell, Marker, Cytometry, Infection, Control, Expressing