Journal: Development (Cambridge, England)

Article Title: Evolutionary conservation of VSX2 super-enhancer modules in retinal development

doi: 10.1242/dev.202435

Figure Lengend Snippet: hR0-36 Δ/Δ retinal organoids have defects in proliferation and neuronal differentiation . (A) Micrographs of stage 2 retinal organoids for each genotype. The heterogeneity of hR0-36 Δ/Δ retinal organoids are apparent at this stage. (B) Scatter dot plot of organoid area in mm 2 . Each dot represents a single retinal organoid measurement. VSX2 –/– and hR0-36 Δ/Δ retinal organoids are significantly smaller than wild-type retinal organoids (unpaired two-tailed t -test, **** P <0.0001). (C) Representative micrographs of wild-type retinal organoid at stage 2 showing EdU (red) and nuclear stain (blue). Arrows indicate representative EdU-positive cells. (D) EdU scoring at stage 2 for each genotype (** P <0.01). Data are mean±s.d. (E) Representative micrograph of stage 2 retinal organoids stained for activated caspase 3 (red) as a marker of cell death (arrows). Nuclei are stained with DAPI (blue). (F) Percentage of activated caspase 3-positive nuclei. There was no significant difference across genotypes. Data are mean±s.d. (G) Principal component analysis plot of bulk RNA-seq from stage 2 retinal organoids. Each dot represents a distinct biological replicate. (H) Scatterplot of Log2-FPKM from bulk RNA-seq for VSX2 –/– and hR0-36 Δ/Δ retinal organoids. Each dot is an individual gene; the red line is the linear regression with a correlation coefficient of 0.976. (I) Volcano plot of hR0-36 Δ/Δ retinal organoids versus wild type with some highlighted upregulated ( DCT , MITF and TYR ) and downregulated ( VSX2 , ARR3 , CRX , RD3 and RCVRN ) genes. (J) FPKM values for representative genes in hR0-36 Δ/Δ and wild-type retinal organoids (unpaired two-tailed t -test; * P <0.05, ** P <0.01; n.s., not significant). Data are mean±s.d. FPKM, fragments per kilobase per million reads. Scale bars: 0.5 mm in A; 25 µm in C; 5 µm in E.

Article Snippet: EdU labeling was performed per manufacturer's instructions (Click-iT EdU imaging kit, Invitrogen, C10340), and DNA was stained with 0.2 μg/ml DAPI (Sigma-Aldrich).

Techniques: Two Tailed Test, Staining, Marker, RNA Sequencing Assay