Figure 1

Col2a1a is expressed in the ocular neural crest during early development. Wholemount in situ hybridization in WT Casper (roy−/−; nacre−/−) zebrafish embryos during development at 24, 36, 48, 60, 72 and 96 hpf. Col2a1a gene expression was detected using a colorimetric assay (Vector Blue Substrate Kit, Vector Laboratories) that is both chromogenic (blue) and fluorescent (Far Red/Cy5). The sections were mounted in a media containing DAPI (gray). Lateral (A–F) brightfield wholemount images show that col2a1a expression initiates within the hindbrain (black arrow, (A) notochord at 24 hpf with dorsoposterior (white arrows, (B–F)) and ventral (blue arrows, (B–F)) progression into the ocular and craniofacial expression during eye and jaw development. The yellow arrow (C) highlights col2a1a expression in the ocular fissure at 48 hpf. Ventral (B’–F’) brightfield wholemount images show col2a1a expression in the developing jaw. The ventral-anterior progression of col2a1a expression is indicated (blue dashed arrows). The red arrows (D’) highlight col2a1a expression in the optic nerve at 60 hpf. By 96 hpf (F’), profound col2a1a expression was detected in the developing jaw and pharyngeal arches (PA). A ventral image of embryonic col2a1a expression at 24 hpf (A’) was not obtained because the embryo is tightly bound to the yolk mass at this stage of development, making it difficult to image the ventral side. Mc, Meckel’s cartilage; pq, palatoquadrate; ch, ceratohyal; bh, basihyal; hs, hyosympletic. Transverse cephalic sections and fluorescence (FL) microscopy analyses (A”–F”) provide additional information for significant ocular col2a1a expression in the anterior segment, with apparent expression in the anterior segment [iris (Ir), iris outflow tract (IOT), hyaloid vasculature (black arrows), sclera (Sc), periocular mesenchyme (PM), and optic nerve (ON)] at 48 hpf (C”). The dashed line in the lateral wholemount brightfield image of a 48 hpf embryo indicates the orientation of the plane of section, which passes perpendicular to the spinal column and extends in the rostral-caudal direction. (G,H) Wholemount colorimetric in situ hybridization, followed by GFP immunostaining in Tg(sox10::EGFP) and Tg(foxd3::EGFP) zebrafish embryos during early development at 48 hpf revealed the colocalization of col2a1a expression with neural crest cell markers in the developing jaw (G) [Meckel’s cartilage (Mc), trabeculae (T), and quadrate (Q)] and anterior segment (H) (hyaloid vasculature (white arrows), iris outflow tract (IOT), sclera (Sc), and periocular mesenchyme (PM)).