Journal: Frontiers in Cell and Developmental Biology

Article Title: Characterization and neurogenic responses of primary and immortalized Müller glia

doi: 10.3389/fcell.2025.1513163

Figure Lengend Snippet: Immunostaining results of neuron-like cells derived from primary MG cells with various neuronal markers and retinal neuron markers. (A) Cells were stained with TuJ1 marker and neuronal markers: NeuroD1 and Map2. Scale Bars, 50 μm. (B) Cells were stained with TuJ1 marker and retinal neuron markers: Calbindin (primarily expressed in horizontal cell, as well as in some subtypes of amacrine and retinal ganglion cells); Brn3a and Rbpms (markers of retinal ganglion cells); Otx2 (marker of bipolar and immature photoreceptor). Scale Bars, 50 μm. (C) Cells were stained retinal neuron markers: HuC/D (expressed in amacrine and retinal ganglion cells, with transient expression in horizontal cells during development), Rhodopsin (marker of mature rod photoreceptor). (D) The percentage of cells derived from primary MG that were positive for Calbindin/TuJ1 or HuC/D among total cells on day 7. The data are presented as mean ± SD (n = 3). Scale Bars, 50 μm. 6C–Medium supplemented with 6 small molecules.

Article Snippet: The primary antibodies used in this study were rabbit sex determining region Y (SRY)-box9 (Sox9; Merck, Cat. #AB5535, 1:100), rabbit glutamine synthetase (GS; Abcam, Cat. #ab73593, 1:100), rabbit glutamate aspartate transporter (GLAST; Frontier Institute, Cat. #GLAST-Rb-Af660, 1:100), rabbit glial fibrillary acidic protein (GFAP; CST, Cat. #12389, 1:100), mouse nestin (Invitrogen, Cat. #14-5,843-82, 1:500), rabbit paired box 6 (Pax6; BioLegend, Cat. #BL-901301, 1:300), mouse class III beta-tubulin (TuJ1; Genetex, GT11710, 1:500), rabbit RNA-binding protein with multiple splicing (Rbpms; Abcam, Cat. # Ab194213 , 1:200), rabbit Brn3a (Abcam, Cat. # Ab245230 , 1:500), rabbit Calbindin (Proteintech, Cat. # 14479-1-AP, 1:200), mouse Rhodopsin (Abcam, Cat. # ab3267, 1:200), rabbit microtubule-associated protein 2 (Map2; EMD Millipore, Cat. # AB5622, 1:200), mouse HuC/D (Santa Cruz Biotechnology, Cat. # sc-515624, 1:200), rabbit NeuroD1 (CST, Cat. #D90G12-7,019, 1:200), and rabbit Otx2 (PGI, Cat. #13497-1-AP, 1: 500).

Techniques: Immunostaining, Derivative Assay, Staining, Marker, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Characterization and neurogenic responses of primary and immortalized Müller glia

doi: 10.3389/fcell.2025.1513163

Figure Lengend Snippet: Immunostaining results of neuron-like cells derived from QMMuC-1 cells with various neuronal markers and retinal neuron markers. (A) Cells were stained with TuJ1 marker and neuronal markers: NeuroD1 and Map2. Scale Bars, 50 μm. (B) Cells were stained with TuJ1 marker and retinal neuron markers: Calbindin (primarily expressed in horizontal cell, as well as in some subtypes of amacrine and retinal ganglion cells); Brn3a and Rbpms (markers of retinal ganglion cells); Otx2 (marker of bipolar and immature photoreceptor). Scale Bars, 50 μm. (C) Cells were stained retinal neuron markers: HuC/D (expressed in amacrine and retinal ganglion cells, with transient expression in horizontal cells during development), Rhodopsin (marker of mature rod photoreceptor). Scale Bars, 50 μm. 6C–Medium supplemented with 6 small molecules.

Article Snippet: The primary antibodies used in this study were rabbit sex determining region Y (SRY)-box9 (Sox9; Merck, Cat. #AB5535, 1:100), rabbit glutamine synthetase (GS; Abcam, Cat. #ab73593, 1:100), rabbit glutamate aspartate transporter (GLAST; Frontier Institute, Cat. #GLAST-Rb-Af660, 1:100), rabbit glial fibrillary acidic protein (GFAP; CST, Cat. #12389, 1:100), mouse nestin (Invitrogen, Cat. #14-5,843-82, 1:500), rabbit paired box 6 (Pax6; BioLegend, Cat. #BL-901301, 1:300), mouse class III beta-tubulin (TuJ1; Genetex, GT11710, 1:500), rabbit RNA-binding protein with multiple splicing (Rbpms; Abcam, Cat. # Ab194213 , 1:200), rabbit Brn3a (Abcam, Cat. # Ab245230 , 1:500), rabbit Calbindin (Proteintech, Cat. # 14479-1-AP, 1:200), mouse Rhodopsin (Abcam, Cat. # ab3267, 1:200), rabbit microtubule-associated protein 2 (Map2; EMD Millipore, Cat. # AB5622, 1:200), mouse HuC/D (Santa Cruz Biotechnology, Cat. # sc-515624, 1:200), rabbit NeuroD1 (CST, Cat. #D90G12-7,019, 1:200), and rabbit Otx2 (PGI, Cat. #13497-1-AP, 1: 500).

Techniques: Immunostaining, Derivative Assay, Staining, Marker, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Characterization and neurogenic responses of primary and immortalized Müller glia

doi: 10.3389/fcell.2025.1513163

Figure Lengend Snippet: Immunostaining results of neuron-like cells derived from ImM10 cells with various neuronal markers and retinal neuron markers. (A) Cells were stained with TuJ1 marker and neuronal markers: NeuroD1 and Map2. Scale Bars, 50 μm. (B) Cells were stained with TuJ1 marker and retinal neuron markers: Calbindin (primarily expressed in horizontal cell, as well as in some subtypes of amacrine and retinal ganglion cells); Brn3a and Rbpms (markers of retinal ganglion cells); Otx2 (marker of bipolar and immature photoreceptor). Scale Bars, 50 μm. (C) Cells were stained retinal neuron markers: HuC/D (expressed in amacrine and retinal ganglion cells, with transient expression in horizontal cells during development), Rhodopsin (marker of mature rod photoreceptor). Scale Bars, 50 μm. 6C–Medium supplemented with 6 small molecules.

Article Snippet: The primary antibodies used in this study were rabbit sex determining region Y (SRY)-box9 (Sox9; Merck, Cat. #AB5535, 1:100), rabbit glutamine synthetase (GS; Abcam, Cat. #ab73593, 1:100), rabbit glutamate aspartate transporter (GLAST; Frontier Institute, Cat. #GLAST-Rb-Af660, 1:100), rabbit glial fibrillary acidic protein (GFAP; CST, Cat. #12389, 1:100), mouse nestin (Invitrogen, Cat. #14-5,843-82, 1:500), rabbit paired box 6 (Pax6; BioLegend, Cat. #BL-901301, 1:300), mouse class III beta-tubulin (TuJ1; Genetex, GT11710, 1:500), rabbit RNA-binding protein with multiple splicing (Rbpms; Abcam, Cat. # Ab194213 , 1:200), rabbit Brn3a (Abcam, Cat. # Ab245230 , 1:500), rabbit Calbindin (Proteintech, Cat. # 14479-1-AP, 1:200), mouse Rhodopsin (Abcam, Cat. # ab3267, 1:200), rabbit microtubule-associated protein 2 (Map2; EMD Millipore, Cat. # AB5622, 1:200), mouse HuC/D (Santa Cruz Biotechnology, Cat. # sc-515624, 1:200), rabbit NeuroD1 (CST, Cat. #D90G12-7,019, 1:200), and rabbit Otx2 (PGI, Cat. #13497-1-AP, 1: 500).

Techniques: Immunostaining, Derivative Assay, Staining, Marker, Expressing

Journal: bioRxiv

Article Title: Heterochronic transcription factor expression drives cone-dominant retina development in 13-lined ground squirrels

doi: 10.1101/2025.04.25.650540

Figure Lengend Snippet: Conserved TFs bind to species-specific enhancers to promote Cone specification in 13LGS. ( A ) Schematic illustrating annotation of cis-regulatory elements in RPCs and Photoreceptor Precursors by integration of scATAC-Seq and CUT&RUN 13LGS and mouse datasets. ( B ) Heatmaps show annotated accessible regulatory elements in both 13LGS and mouse. Promoters, activated enhancers (AEs), and poised enhancers (PEs) which are associated with histone markers associated with genes in clusters C2 and C3, which are selectively active in 13LGS RPCs and/or photoreceptor precursors. Shading indicates CUT&TAG signal for the corresponding histone modification within 2kb of the scATAC-Seq peak center. Bar plots displaying the number of each category of regulatory element in each species that are conserved or species-specific. ( C ) Dot plots showing the enrichment of binding sites for Otx2 and Neurod1, TFs which are broadly expressed in both neurogenic RPC and rod and cone precursors, which are enriched in both evolutionarily-conserved cis-regulatory elements in both species. ( D ) Bar plots showing the number of evolutionarily-conversed and species-specific enhancers per TSS in four cone-promoting genes between 13LGS and mouse. (E) The gene regulatory networks regulating Thrb expression in 13LGS and mouse late N. RPCs. ( F ) An example of a Thrb-related regulon and its corresponding scATACseq and CUT&RUN tracks. The arrow indicates the consistent regulatory relationships between GRNs prediction and experimental validations. ( G ) The epigenetic model of cone specification in 13LGS and mouse.

Article Snippet: Then 0.5 µg of the following antibodies were added to each respective reaction: IgG control (EpiCypher, 13-0042), H3K4me1 (EpiCypher, 13-0057), H3K4me3 (EpiCypher, 13-0041), H3K27ac (EpiCypher, 13-0059), H3K27me3 (EpiCypher, 13-0055), NeuroD1 (Cell Signaling, 62953), Otx2 (R&D Systems, BAF1979), or Otx2 (Atlas Antibodies, HPA000633).

Techniques: Modification, Binding Assay, Expressing