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Fig. 2. F1 transgenic X. laevis carrying the 5′ flanking region of six6 only express GFP in the differentiated optic cup. (A-E) Expression pattern of endogenous six6 by whole-mount in situ hybridization (WISH). (A) At stage 12.5, six6 is undetectable, but by stage 15 (B) six6 transcript is detected in the eye field. (C) Strong six6 expression persists in stage 24 optic vesicles, but no expression is seen in the forebrain. (D) At stage 28, six6 is expressed in the pineal (arrowhead) and the presumptive pituitary/hypothalamic area (arrow), as well as the optic cups. (E) At stage 32, strong expression continues to be detected in the eyes. (F-J) Whole mount in situ hybridization of embryos carrying the Xt six6 R2R1→GFP transgene shows (F-G) no detectable expression of GFP RNA at eye field or (H) optic vesicle stages. (I-J) GFP transcripts were first detected at stage 28 in the optic cup (black arrow, eye), pineal (a) and ventral forebrain (b). (K-L) GFP fluorescence was first detected at stage 33/34 in eye and pineal; (L) brightfield image of embryo in panel K. (M) Stage 40 animals expressing GFP in eye (arrowhead) and brain (tectum and midbrain). (N) Bright field image of transgenic tadpole shown in (O) expressing GFP brightly in eyes, optic stalk (os) and synapsing on the tectum; weak fluorescence was also sometimes observed in the hindbrain. Yolk autofluorescence is also observed (K,M,O). |
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Fig S4. Deletion products were observed in the genomic DNA (gDNA) of embryos injected with guide RNAs but not in controls. (A, B) Deletions were observed in a subset of PCR products amplified from gDNA isolated from embryos injected with L5′/L3′ (100 pg/250 pg), S5′/S3′ (200 pg/250 pg) guides (sgRNA) and hCas9 (500 pg) RNA (red arrows). Deletion products were not observed when amplification was performed using wild-type (WT) gDNA as template, although some false priming products common to both WT uninjected and injected embryos were observed (gray arrows). (C-E) Six6 transcript is detected throughout the optic vesicles of wild-type (WT) embryos as well as embryos mosaic for R3 deletions (six6.LdeltaR3 or six6.Sdelta R3). Embryos shown were stained by in situ hybridization then cut in half at the level of the pineal to show six6 expression is detected throughout the optic vesicle. Dashed lines mark optic vesicle; bar, 200 µm. |
