Tempo-spatial expression and subcellular localization of Cadm3. (A) The workflow of RNA-seq. (B) Heat-map of gene expression showing the top 20 highly expressed genes in WT donor compared with p15⁺ host RPCs. (C) The quantification of cadm3 expression in WT donor RPCs and p15⁺ host RPCs by real-time qPCR. Data represent mean ± SEM. (D) In situ hybridization showing the cadm3 expression in WT retinae at 24, 36, 48, and 72 hpf. At 72 hpf, the zoom-in image shows the ciliary marginal zone (CMZ). Areas within white dashed contour lines are retinae. (E) Images showing the localization of Cadm3 protein on cell membrane at 24 hpf in retinae by co-injecting cadm3-GFP and mCherry-UtrCH mRNAs. (F) The left panel shows a representative image of the asymmetric distribution of Cadm3 protein in WT donor RPCs in p15⁺ host retinae (n = 22) and the right panel shows a representative image of evenly distributed Cadm3 protein with ig1 domain deleted (Cadm3-ig1De) in WT donor RPCs in p15⁺ host retinae (n = 33). (G) Time-lapse of Cadm3-GFP in WT donor RPCs in p15⁺ host retinae. The dashed white lines in E–G represent apical and basal side of the retina. The yellow and white arrowheads in G respectively indicate the apical and basal terminals of Cadm3-GFP expression. (H) Summary schematic describing a proposed working model accounting for the distribution patterns of full-length (top) and ig1-deleted (bottom) Cadm3 proteins. Scale bars, 10 µm.