Fig. 9.

Hh signaling is required before 18 hpf for the induction of branchiomotor neurons. Panel A shows a lateral view, and panels F–I show dorsal views of the hindbrain, with anterior to the left. (A) In a 15-hpf wild-type embryo, gli2 is expressed at all axial levels throughout the dorsoventral extent of the hindbrain. The asterisk marks low level of fgf3 expression at the mid-hindbrain boundary, which was used to orient embryos for sectioning (see Materials and methods for details). (B and C) Cross-sections (dorsal is up) showing that gli2 is expressed in the ventral aspects of the neural tube in rhombomeres 5 and 6, but at lower levels than in the dorsal neural tube. nc, notochord. (D and E) Quantification of embryos with normal numbers of GFP-expressing nV motor neurons in r2 (D) and nVII neurons in r4–r7 (E) following cyclopamine (CyA) treatment beginning at the times indicated (hpf). There is no effect on nV neuron number in r2 when CyA treatment is initiated at 12 hpf or later (2 experiments; 20 embryos per experiment). There is no effect on nVII neuron number when CyA treatment is initiated at 18 hpf or later (2 experiments). (F) In a 48-hpf wild-type embryo treated with ethanol (EtOH) from 6 hpf, islet antibody labeling reveals that the number and organization of branchiomotor neurons, including nV (arrow), nVII (white arrowhead) and nX (black arrowhead), are unaffected. (G) In an embryo treated with CyA from 6 hpf, islet-labeled branchiomotor neurons are absent. (H) In an embryo treated with CyA from 12 hpf, nV neurons in r2 are mostly present (arrow; light staining), nVII neurons are greatly reduced in number (arrowhead), and nX neurons are absent. (I) In an embryo treated with CyA from 18 hpf, the nV (arrow), nVII (white arrowhead), and nX neurons (black arrowhead; out of focus) are mostly unaffected. oto, otocyst.