Fig. 7.

Targeting Pax6 in the developing chick retina using RIA-CRISPR retrovirus. (A) The genomic locus for Pax6 in the chick with paired-box (green) and homeodomain (purple) indicated. Of gRNAs tested, the one targeting the splice acceptor site of exon 5 was most efficient. (B-E) The right side of gastrula-stage embryos was electroporated with constructs encoding Cas9, RFP and Pax6 gRNA. Transfected cells (C) lacked expression of Pax6 (D), as seen in the overlay image (B). Dashed line delineates the neural tube. Cross-sections through embryos electroporated with the single CRISPR plasmid targeting Pax6 were used to quantify fluorescence intensity of Pax6 in the neural tube; results reveal a significant difference in Pax6 expression between the knockout and control sides (E; *P<0.05, paired Student's t-test, n=4 sections from 2 representative embryos). (F,F′) Section through the eye at embryonic day 4 (F) injected with RIA-Cas9-GFP and RIA-H2B-RFP retroviruses. Labeled cells were evenly distributed (F′) through all layers of the developing retina. (G) The injection strategy for targeting Pax6 in retinal progenitor cell precursors residing in the optic vesicle. (H,H′) Citrine expression (H) in the developing eye (H′) was first observed 48 h post-infection. Dashed lines delineate the developing eye. (I-K′) Citrine-labeled cells (I,J,K) in both the retinal pigmented epithelium and the ganglion cell layers were negative for Pax6 expression (J′,K′, arrowheads). Intensity of Citrine expression together with endogenous Pax6 levels allowed identification of two clones in the retinal pigmented epithelium layer (J′): outlined in white – high Citrine, high Pax6; outlined in yellow – low Citrine, no Pax6. (L) Relative Citrine CTCF intensity per labeled cell was significantly higher in the ganglion cell layer (GCL) compared with the retinal pigmented epithelium (RPE) (*P<0.01, paired Student's t-test, n=9). (M-O′) 77.08±5.5% of Citrine-labeled cells (M,N,O) were biased towards an amacrine cell fate based on expression of Islet1 (N′,O′, arrowheads). Several migratory cells were observed in the inner nuclear layer. (P-Q′) 80.24±3.1% of Citrine-labeled cells (P,Q) in the ganglion cell layer failed to turn on Tubb3, a marker for differentiated mature neurons (Q′, arrowheads). ect, ectoderm; gcl, ganglion cell layer; inl, inner nuclear layer; nc, notochord; nt, neural tube; rpe, retinal pigmented epithelium. Error bars represent s.e.m.