A conserved enhancer upstream of the duplicated fugu hoxa2 co-orthologs displays subfunctionalization of regulatory activity between neural crest cells and hindbrain segments. (A) Schematic of an enhancer from the mouse Hoxa2 gene mapping the known regulatory elements. Boxes mark neural crest cell activity (NC) in green and r3/r5 activity (RE) in purple. (B-D) F₀ transgenic zebrafish embryos at 48 h post fertilization (hpf) expressing a GFP reporter gene under the control of enhancers from zebrafish hoxa2b (zf-Hoxa2b) (B), fugu hoxa2a (fr-Hoxa2a#1) (C) or fugu hoxa2b (fr-Hoxa2b#1) (D). (E-G) F₀ transgenic mouse embyos at 9.5 dpc expressing the LacZ reporter gene under the control of enhancers from mouse Hoxa2 (m-Hoxa2) (E), fugu hoxa2a (fr-Hoxa2a#1) (F) or fugu hoxa2b, fr-Hoxa2b#1 (G). Red=RFP expression driven by a Krox20 enhancer in r3 and r5; green=GFP expression; dotted line=otic vesicle (OV). In B-G neural crest (NC) cells are marked with arrows and r3/r5 with arrowheads.

A conserved enhancer upstream of the duplicated fugu hoxa2 co-orthologs displays subfunctionalization of regulatory activity between neural crest cells and hindbrain segments. (A) Schematic of an enhancer from the mouse Hoxa2 gene mapping the known regulatory elements. Boxes mark neural crest cell activity (NC) in green and r3/r5 activity (RE) in purple. (B-D) F₀ transgenic zebrafish embryos at 48 h post fertilization (hpf) expressing a GFP reporter gene under the control of enhancers from zebrafish hoxa2b (zf-Hoxa2b) (B), fugu hoxa2a (fr-Hoxa2a#1) (C) or fugu hoxa2b (fr-Hoxa2b#1) (D). (E-G) F₀ transgenic mouse embyos at 9.5 dpc expressing the LacZ reporter gene under the control of enhancers from mouse Hoxa2 (m-Hoxa2) (E), fugu hoxa2a (fr-Hoxa2a#1) (F) or fugu hoxa2b, fr-Hoxa2b#1 (G). Red=RFP expression driven by a Krox20 enhancer in r3 and r5; green=GFP expression; dotted line=otic vesicle (OV). In B-G neural crest (NC) cells are marked with arrows and r3/r5 with arrowheads.

Transgenic analysis of chimeric fugu hoxa2a/hoxa2b enhancers in zebrafish embryos define NC5 as a cis-element important in neural crest expression. (A-E) GFP reporter expression mediated by Hybrids 5-7 and isolated regions of hoxa2a (fr-Hoxa2a #2 and fr-Hoxa2a #3) in neural crest cells (NC) in pharyngeal arch tissue (arrows) or rhombomeres 3 and 5 (arrowheads) in 48hpf zebrafish embryos. In A-D: red=RFP expression in r3 and r5 driven by a control Krox20 enhancer; green=GFP expression. (F) Drawing of the hybrid constructs mapping the portion of the hoxa2a and hoxa2b enhancers in each hybrid or the small isolated regions compared to the annotated mouse Hoxa2 enhancer at top. The purple box= rhombomere elements; green box=neural crest elements; red=hoxa2a enhancer sequence; gray=hoxa2b enhancer sequence. The table at the right of each construct describes the F₀ transgenic expression data marking: presence (+) or absence () of GFP expression in neural crest or r3/r5; # of embryos with expression in neural crest and/or r3/5 (#relevant expression); # of embryos with any GFP expression; total # embryos injected. The number of stable transgenic lines created (F1s) are noted, far right.