FIGURE 3

Elevated Hoxb5b activity expands the expression domains of vagal markers foxd3 and phox2bb during the first day in development. (A,B) in situ hybridization against foxd3, a marker for multipotent NCCs at this stage, in 32 hpf embryos injected with 30 pg vp16-hoxb5b, were compared to WT controls. (C,D) in situ hybridization against phox2bb, an autonomic NCC marker, in 32 hpf embryos injected with 30 pg vp16-hoxb5b, compared to WT controls. The POD is denoted with a white arrow head. (E–G) Quantified POD foxd3 expression area (E, control n = 7; vp16-hox5b n = 10; p = 0.00571) or phox2bb expression area (F, control n = 7; vp16-hox5b n = 8; p = 0.138), as noted in representative images by black bars. The discrete POD expression domain is quantified from these same embryos as shown in (G) (p = 0.00563). Areas are normalized to control mean. (H–K) IHC detection of Phox2b⁺ cells viewed along the lateral axis of 32 hpf embryos reveal that WT controls (n = 7) already have a nascent population [(H), white arrowheads]. vp16-hoxb5b overexpression, at either 15 pg [(I), n = 9] or 30 pg [(J), n = 5] of mRNA injected, expands the Phox2b⁺ vagal NCCs (white arrowheads). (K) Counted Phox2b⁺ cells from 3 dimensional micrographs reveal increasing cells with the amount of vp16-hoxb5b mRNA (15 pg: p = 3.03 × 10^–5; 30 pg: p = 2.61 × 10^–5). Scale bar (A,H): 100 μM.