Fig. 5

Ectopic gene expression in 1-day old shh injected embryos. Embryos injected with capped shh RNA at 25 or 50 ng/μl (inj; C,E,F,G,I) or controls (wt; A,B,D,H) were analyzed by in situ hybridization. Embryos were fixed at 24-26 hpf (A-G) or at 30 hpf (H,I). (A) Double hybridization with zp-50 (purple) and shh (red) shows close proximity of expression in the ventroanterior brain. (B,C) shh injection causes disorganized and elongated zp- 50 expression domains in the ventroanterior diencephalon (small arrowheads). Prominent ectopic expression is visible in the dorsal thalamus and the midbrain. No aberrant zp-50 expression is observed along the midbrain/hindbrain boundary of injected embryos (asterisk). zp-50 expression is reduced in the telencephalon of injected embryos (large arrowheads). (D) Uninjected embryo stained for axial. (E,F) Two different examples of shh-injected embryos double labeled for zp-50 (purple) and axial (red). Besides ectopic zp- 50 expression, axial expression shows in these embryos a dorsal expansion of the finger-like domain in the dorsal thalamus (arrow). The relatively modest axial overexpression in the midbrain seen in E is more frequently seen than the more widespread expression in the midbrain tegmentum (arrowhead) visible in F. (G) shh-injected embryo double labeled for zp-50 (purple) and pax-2 (red). pax-2 expression in the otic vesicle and in the zp- 50-free gap at the midbrain/hindbrain boundary (asterisk) is not obviously affected by shh misexpression. However, pax-2 expression is dramatically widened in the optic stalk (cf. Fig. 3D). (H,I) Injection of shh RNA eliminates several hlx-1 expression domains in the midbrain. Residual hlx-1 midbrain expression is shifted dorsally like the expression domains in the hindbrain. d, diencephalon; dt, dorsal thalamus; h, hindbrain; m, mesencephalon; os, optic stalk; ov, otic vesicle; t, telencephalon; vt, ventral thalamus.