Figure 8

Spatiotemporal expression patterns of conserved annular ligament associated genes. (A) WISH expression analysis of annular ligament 1 (AL1) associated genes nusap1, frzb, cxcr4b, rrn2 and cdh5 at 24, 72 and 120 hpf displayed in dorsal and lateral views. (B) 10 µm cryosections of 120 hpf embryo eyes after WISH. (C) Two color FWISH depicting co-expression of nusap1(green)/scinla(red), frzb(green)/scinla(red), cxcr4b(green)/scinla, rrn2(green)/scinla(red) and cdh5(green)/scinla(red) at 120 hpf. DAPI (blue) was used to stain the nuclei. Volume projections in lateral view and single section dorsal views from 3D confocal stacks are displayed. White arrows indicate example regions of co-expression. Scale bar = 100 µm. Individual fluorescence channels are provided in Fig. S11A. (D) Average mean expression measurements from AL1 clusters for genes nusap1, frzb, cxcr4b rrn2, and cdh5 at each timepoint examined, 48-144 hpf. (E) WISH expression analysis of annular ligament 2 (AL2) associated genes mcm7, vim, lxn, fmoda and fabp11a at 24, 72 and 120 hpf displayed in dorsal and lateral views. (F) 10 µm cryosections of 120 hpf embryo eyes after WISH. (G) Two color FWISH depicting co-expression of mcm7(green)/scinla(red), vim(green)/scinla(red), lxn(green)/scinla, fmoda(green)/scinla(red) and fabp11a(green)/scinla(red) at 120 hpf. DAPI (blue) was used to stain the nuclei. Volume projections in lateral view and single section dorsal views from 3D confocal stacks are displayed. White arrows indicate example regions of co-expression. Scale bar = 100 µm. Individual fluorescence channels are provided in Fig. S11B. (H) Average mean expression measurements from AL2 clusters for genes mcm7, vim, lxn, fmoda and fabp11a over developmental time. ALP Annular ligament progenitor (yellow arrow), CEp corneal epithelium (blue arrow), CEn Corneal endothelium (green arrow), CSt Corneal stroma (green arrow).