Fig. 5. tfap2a and phox2a expression and NA neuron specification in the LC. (A) Lateral view and (B) dorsal view of a wild-type embryo at 33 hpf showing expression of tfap2a (red) and phox2a (blue) in the hindbrain. phox2a and tfap2a are expressed in a segmental pattern reflecting rhombomeres 1-7 (r1-r7). In the rhombomeres, small groups of neurons in the ventral medial expression domain of tfap2a co-express phox2a (black arrowheads; white arrowheads indicate the lateral expression of tfap2a). In the spinal cord, both genes are expressed widely. (C) Dorsal view of a wild-type embryo at 33 hpf. In the locus coeruleus, tfap2a (red) and phox2a (blue) are co-expressed. (D) Dorsal view of a wild-type embryo at 33 hpf. Double staining for th (blue) and tfap2a (red) confirmed the co-localization of th and phox2a in the LC. (E) Lateral view of a wild-type embryo at 38 hpf. In the ventral part of r6-r7, most of the cells co-express tfap2a (red) and phox2a (blue), while in the dorsal part, only tfap2a is expressed. (F) Dorsal view of the same embryo. Co-expression of tfap2a and phox2a continues in the medial line of tfap2a expression (black arrowheads; white arrowheads indicate the lateral expression of tfap2a). (G-J) Transversal sections of embryos at 33 hpf showing expression of tfap2a and phox2a at the level of the LC (G,I; left and right halves show left and right LC from different sections) and rhombomere 6-7 and the medulla (H,J). In I and J, the same sections as in G and H are shown, but the Fast Red dye used to detect tfap2a expression is visualized by fluorescence microscopy. The phox2a expression domains in LC and medulla are part of the tfap2a expression domain, albeit tfap2a is expressed at relatively low levels in the region of phox2a-expressing cells. (K) Lateral view of a wild-type embryo at 48 hpf. phox2a is expressed in the LC and anterior lateral line ganglia (allg) and sensory ganglia (sg). (L) Lateral view of a tfap2a mutant embryo at 48 hpf. The LC develops in the mutant embryo, as revealed by the expression of phox2a in this region. The white arrow highlights the absence of phox2a expression at the position of the sensory ganglia. (M) Dorsal view of the hindbrain of a wild-type embryo at 72 hpf showing th expression in the posterior hindbrain. (N) Dorsal view of a soulless/phox2a mutant embryo, with th expression in the posterior hindbrain that is not distinguishable from wild-type. (A-F,K-N), anterior towards the left; (G-J), transverse sections. LC, locus coeruleus; MO, medulla oblongata; sc, spinal cord; sg, sensory ganglia; tec, tectum; 1-7, rhombomeres 1-7.

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