FIGURE 4

Temporally controlled overexpression of Hoxb5b during the first day in development is sufficient to expand the neural crest pool. (A) Schematized model of the hsp70l:gfp-hoxb5b;cryaa:dsRed genetic construct. (B) Illustration depicting specific periods of heat shock for embryo groups relative to classical hallmarks of zebrafish NCC development. (C,D) in situ hybridization using a probe for crestin in hsp70l:GFP-hoxb5b ⁺ embryos heat shocked at 14 hpf and fixed at 24 hpf, compared to GFP⁻ sibling controls treated in parallel. Dramatic expansion of the POD can be seen (yellow arrowheads) in the Hoxb5b overexpressing embryos, while more subtle expansion is noted in the cranial NCC (white arrowheads) and pre-otic crest (red arrowheads). (E–H) Similar to (C,D) in situ hybridization with a crestin probe in hsp70l:gfp-hoxb5b and GFP- sibling controls heat shocked at 22 hpf and fixed at 26–28 hpf, examining both crestin ⁺ domains both laterally (E,F) and dorsally (G,H). (I,J) Graphs depicting areas occupied by crestin staining in both GFP- control and Hoxb5b overexpressing embryos. Hoxb5b overexpression at 14 hpf was sufficient to expand NCC localization and qualitatively NCC number [(I), control n = 6; hsp70:GFP-hoxb5b ⁺ n = 8; p = 0.0234]. A later heat shock at 22 hpf—fixation at 26–28 hpf, also expanded vagal NCC localization [(J), control n = 10; hsp70:GFP-hoxb5b ⁺ n = 14; p = 0.0398], and increased crestin staining, indicative of an increase in NCC number. Scale bars (C,E,G): 100 μM