FIGURE 5

Hoxb5b is sufficient to expand early enteric neural progenitors. (A) Schematized model of a zebrafish embryo highlighting the region of the gut tube, which is imaged in the following panels. (B–D) Whole mount IHC for Phox2b in 50 hpf control embryos [(B), n = 10] compared to embryos injected either with 15 pg [(C), n = 7] or 30 pg [(D), n = 9] of vp16-hoxb5b mRNA. Yellow arrow heads indicated POD localized Phox2b⁺ cells, white arrows designate terminal end of enteric NCC chain, which falls within the gut tract outlined with white dashes. (E,F) Quantified cell numbers from the same animals reveal a coordinate increase in Phox2b⁺ along the gut axis at 50 hpf trending with increasing vp16-hoxb5b mRNA amounts [(E), 15 pg: p = 2.834 × 10^–5; 30 pg: p = 0.00318]. Additionally, the number of Phox2b⁺ cells restricted to the POD also increased in response to elevated Hoxb5b activity [(F), 15 pg: p = 0.00328; 30 pg: p = 0.00068]. (G–I) Whole mount IHC on 3 dpf -8.3phox2bb:kaede embryos with antibodies against Elavl3/4 and Kaede, marking the enteric NCC lineage cells in vp16-hoxb5b overexpressing animals (15 pg n = 8; 30 pg n = 7) compared to uninjected sibling controls (n = 7). (J–L) Quantification of the number of enteric neural progenitors [(J), 15 pg: p = 0.00011; 30 pg: p = 4.09 × 10^–5) and differentiating enteric neurons [(K), 15 pg: p = 0.001352; 30 pg: p = 0.0001042] at 3 dpf show decreasing numbers of both cell populations. However, the total fraction of differentiating (Hu⁺) NCC-derived Kaede⁺ cells unchanged following elevated Hoxb5b activity [(L), 15 pg: p = 0.0.1282; 30 pg: p = 0.102]. Scale Bar (B,G): 100 μM.