Functional testing of retinal DHSs near
, and the conservation between mouse and human. (A) The DNase I cleavage landscape is shown surrounding the Otx2 gene for P0, P7, and adult (Ad) retina to highlight developmental DHS dynamics. Selected Otx2 DHSs labeled with arrows. Tan shading highlights DHSs differentially active in the retina, cerebellum, and cerebral cortex. (B-G’) Panels show representative images of expression from Otx2 DHS reporter constructs (green) co-immunostained for transfection control plasmid (CHERRY, red) and endogenous OTX2 (white). Arrows (G’) highlight five example OTX2+ GFP+ co-expressing cells. Left two columns show expression from indicated constructs in electroporated P0 retinal explants cultured 24 h in vitro
(B-D’) (N = 3). Right two columns show expression from indicated constructs in retinas electroporated in vivo at P0 and harvested at P7 (E-G’) (N = 2 to 5). ONL, outer nuclear layer; NBL, neuroblastic layer; GCL, ganglion cell layer; INL, inner nuclear layer. Scale bar = 200 μm. (H) Quantification of electroporation data showing that nearly 100% of the cells expressing Otx2 DHS-2 or Otx2 DHS-15 also express OTX2 in P0 retina. (N = 3) *P < 0.01; error bars ± SD. (I) Alignment of human and mouse genomes showing the sequence (orange rectangles) conservation of DHSs surrounding the Nr2e3 gene, important in rod photoreceptor gene expression. (J) Comparison of functional and/or sequence conservation of human and mouse DHSs for the CNS (set of DHSs common to mature retina and brain), CNS-core (DHSs only active in the CNS), retina, cerebellum, adult whole brain (Brain), and cerebral cortex (Cortex). Red, DHS in mouse, orthologous region is a DHS in human; purple, DHS in mouse, orthologous region is not a DHS in human; green, DHS in mouse, no orthologous sequence in human. Right blow-up: genes associated with mouse cortex DHSs that have orthologous sequence but no activity in the human genome; numbers of DHSs near respective genes, expressed as the intensity of colored cells.