168mg l tricaine s solution

All animals were euthanized by immersion in a 168mg/L tricaine-S solution (MP Biomedicals). Adult skeletons (Fig. 1): specimens were fixed for 3–7 days in 10% Normal Buffered Formalin (NBF). Skeletal staining was conducted according to^{39 (link)}. Larvae skeletons: 15dpf larvae, 1 month-old/1.4cm (TrL) and 2 month-old/1.7cm (TrL) juveniles were fixed for 3 days in 4% paraformaldehyde. Specimens stained with alizarin red and alcian blue as described with 60 mM MgCl₂^{40 (link)}. Live bone staining by calcein green and alizarin red was conducted as described in Kimmel et al., 2010 with modifications^{41 (link)}. Fish were stained for 24 hours in calcein green, returned to tanks for 14 days, and then chased with alizarin red for 24 hours. Fish were then returned to tanks for 24 hours before fixation overnight in 10% NBF at 4C and stored in 100% ethanol in the dark at 4°C until trypsin digestion as previously described^{41 (link)}. Preparations were phenotyped on either a Leica M165FC dissecting microscope or a Zeiss Axioskop 2 FS Plus compound microscope. The dissecting microscope was equipped with a Planapo 1.6X M-series objective, a Prior L200 fluorescent light source and a Leica DC7000T camera controlled by LAS X software. The compound microscope was equipped with a Qimaging Micropublisher 6 camera controlled by Ocular software.

All animals were euthanized by immersion in a 168 mg/L tricaine-S solution (MP Biomedicals) prepared in tank water prior to fixation. For staining on fixed tissues, larvae (8 dpf, 6 mm SL and 10 mm SL) were fixed in 4% PFA and stained with alizarin red and alcian blue or alcian alone as described^{57 (link)} with 60 mM MgCl₂. Fixed adults were stained as described^{58 (link)} with modifications^{57 (link)} for acid free stain. Live bone staining by calcein green and alizarin red was conducted as described previously^{59 (link)} with modifications.^{60 (link)} Fish were stained for 24 hours in calcein green, returned to tanks for 14 days, and then chased with alizarin red for 24 hours. Fish were then returned to tanks for 24 hours before fixation overnight in 10% normal buffered formalin at 4° C and stored in 100% ethanol in the dark at 4°C until trypsin digestion as previously described.^{60 (link)} Preparations were phenotyped on either a Leica M165FC dissecting microscope or a Zeiss Axioskop 2 FS Plus compound microscope. The dissecting microscope was equipped with a Planapo 1.6X M-series objective, a Prior L200 fluorescent light source and a Leica DC7000T camera controlled by LAS X software. The compound microscope was equipped with a Qimaging Micropublisher 6 camera controlled by Ocular software.