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Fig. Ext. 8 a,b, Dr_dlx5a/6a-p1:GFP and Hs_DLX5/6-p1:GFP are not active in the known ventral arch CNCC expression domain of dlx5a in 1.5 dpf zebrafish. White arrow indicates activity of Hs_DLX5/6-p1:GFP in the first endodermal pouch. At 14 dpf, both enhancers are active at the bases of gills but not in elastic cartilage. sox10:dsRed labels CNCCs at 1.5 dpf and cartilage at 14 dpf. c, RNA in situ hybridization for Dlx5 in frontal sections of an E15.5 mouse head shows expression in developing outer-ear canal (arrowhead) and pinna (arrow). Magnified box region highlights absence of expression in the epiglottis (green arrow) and hyaline hyoid cartilages (yellow arrowheads). d, Endogenous fluorescence of X-gal reaction product shows Dr_dlx5a/6a-p1:lacZ labels outer ear mesenchyme (arrow) adjacent to outer-ear canal (white solid outline) but not the pinna (white dashed outline) in E15.5 mouse embryo. e, Frontal section of whole-mount X-gal stained Dr_dlx5a/6a-p1:lacZ mouse embryo. Magnified boxed regions show expression in mesenchyme (black arrows) associated with elastic cartilage (dashed outlines) lining the external auditory meatus (asterisk), and absence of expression in the epiglottis (green arrow). f, RNA in situ hybridization for Gata3 in horizontal sections of E13.5 mouse head. Magnified boxed region highlights expression in the mesenchyme (white arrow) and epithelium (yellow arrow) of the pinna, and ear canal epithelia (yellow arrowhead). g, RNA in situ hybridization for gata3 in horizontal sections of 14 dpf zebrafish. Magnified boxed region highlights expression in gill-filament mesenchyme and epithelia. h, Compared to activity of unaltered Hs_NRP2-p1:GFP transgene in gill filament cartilages at 17 dpf, loss of DLX sites in Hs_NRP2-p1_DLXdel:GFP abolishes gill activity. sox10:dsRed labels cartilage. i, Schematic of DLX motif deletions. Scale bars, 50?µm (a,b,g,h), 100?µm (c-f).