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Fig. 7
Diencephalic precursors are required for separation of the single eye field. (A-F) Dorsal views, anterior to the top; (A-D) tailbud stage. (A,B) Diagram showing gene expression domains: otx2, red; opl, blue; mar, green; cyclops, grey. (A) The Type V ablation is marked by the red rectangle. Type V ablations, like unoperated and sham ablations never produced cyclopia. (B) The Type I ablation at neural plate stage is marked by the black rectangle. This type of ablation removed the entire mar protrusion and flanking regions. (C) Embryo with Type I ablation, fixed immediately after operation and hybridized in situ with probes for hgg-1 (red, polster; Thisse et al., 1994) and mar (black) mRNAs. (D) Embryo with Type I ablation, fixed immediately after operation and hybridized in situ with a probe for cyclops mRNA. (E,F) 12-somite stage. (E) Unoperated embryo. Retinal precursors had separated in unoperated embryos by this stage (n=15), and only the optic stalk (arrowhead) connected the eye primordia. Sham operated embryos (n=9) are indistinguishable from unoperated siblings (not shown). (F) Cells in the protrusion of the mar expression domain were ablated at neural plate stage (Type I as in A-C). A single retinal field persisted in operated embryos (12-somite stage, n=15), which closely resembled the morphology of cyclops mutant embryos at the same developmental stage (not shown). (G,H) 30h, side views, anterior to the left, dorsal to the top. (G) shh expression marks ventral diencephalon in sham (not shown) and unoperated embryos (arrowhead). (H) Operated embryos (Type I ablation; 10 out of 13 embryos) lack a ventral diencephalon, as indicated by the absence of sonic hedgehog (black) expressing cells (arrowhead) at 30h, and are completely cyclopic. Prospective brain regions: t, telencephalon; e, primordial eye (field); vd, ventral diencephalon; d, dorsal/posterior diencephalon; m, midbrain; mhb, mid-hindbrain boundary. Scale bars, 100 μm.