Journal: eLife

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

doi: 10.7554/eLife.108485

Figure Lengend Snippet: ( A ) Transcriptional start site enrichment profiles of scATAC-Seq datasets. Lines are colored by time points. ( B ) Fragment size distribution of scATAC-Seq datasets. Individual time points are indicated by colored lines. ( C ) Heatmap showing the Pearson correlation between gene expression and gene accessibility for each retina cell type. ( D ) Heatmap of cell type-specific genes. ( E ) Heatmap of cell type-specific peaks. ( F ) Heatmap of cell type-specific motifs. ( G ) Examples of transcription factor (TF) footprint profiles for Pou4f2, Crx, Nfix, and Onecut1 in indicated scATAC-Seq cell types. ( H ) Examples of chromVAR score are shown for Otx2, Pou2f2, Nfix, and Neurod1 using scATAC-Seq datasets. ( I ) The relative abundance of retinal cell types in scRNA- and scATAC-Seq is different between developing 13LGS and mouse retina.

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: Gene Expression

Journal: eLife

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

doi: 10.7554/eLife.108485

Figure Lengend Snippet: Fluorescent in situ hybridization showing co-expression of ( A ) Pou2f1 and Otx2 or ( B ) Zic3 , Rxrg , and Otx2 in P1, P5, P10, and P24 retinas. Insets show higher power images of highlighted areas. ( C ) Zic3, Rxrg, and Otx2 fluorescent in situ hybridization from P24 with matched ( C’ ) negative controls. ( D ) Pou2f1 and Otx2 fluorescent in situ hybridization from P24 with matched ( D’ ) negative controls. ( E ) Quantification of the fraction of Otx2 -positive cells in the outer neuroblastic layer (P1, P5) and ONL (P10, P24) that also express Zic3 . ( F ) Immunohistochemical analysis of Mef2c and Otx2 expression in P1, P5, P10, and P24 retinas. ( G ) Mef2c and Otx2 immunohistochemistry from P24 with matched ( G’ ) negative controls. Negative controls for fluorescent in situ hybridization omit the probe and for immunohistochemistry omit primary antibodies. Scale bars, 10 µm (S2A–F), 50 µm (S2G), and 5 µm (inset). Cell counts in E were analyzed using one-way ANOVA analysis with Sidak multiple comparisons test and 95% confidence interval. **p < 0.01, ****p < 0.0001, and ns = non-significant. N = 3 independent experiments. DAPI, 4′,6-diamidino-2-phenylindole; NbONL, neuroblastic outer nuclear layer; ONL, outer nuclear layer; INL, inner nuclear layer. Arrowheads indicate cells co-expressing indicated markers.

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: In Situ Hybridization, Expressing, Immunohistochemical staining, Immunohistochemistry

Journal: eLife

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

doi: 10.7554/eLife.108485

Figure Lengend Snippet: Immunohistochemistry showing GFP and ( A ) Nrl, ( B ) Otx2, ( C ) Sox9, or ( D ) Tfap2a expression in P8 mouse retinal explants electroporated with a plasmid expressing GFP alone (Empty) or GFP in a bicistronic transcript with MEF2C ( MEF2C ). Scale bars, 50 µm. DAPI, 4′,6-diamidino-2-phenylindole; GFP, green fluorescent protein; ONL, outer nuclear layer; INL, inner nuclear layer.

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: Immunohistochemistry, Expressing, Plasmid Preparation

Journal: eLife

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

doi: 10.7554/eLife.108485

Figure Lengend Snippet: ( 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 2 kb 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 photoreceptor precursors, which are enriched in both conserved cis -regulatory elements in both species. ( D ) Bar plots showing the number of conserved and species-specific enhancers per transcription start site (TSS) in four cone-promoting genes between 13LGS and mouse. ( E ) The gene regulatory networks (GRNs) regulating Thrb expression in 13LGS and mouse late N. RPCs. ( F ) An example of a Thrb-related regulon and its corresponding scATAC-Seq and CUT&RUN tracks. The arrow indicates the consistent regulatory relationships between GRN 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