(A-J”) Confocal fluorescent images of Tg(sox10:mem-tdEosFP)-NC cells (green). Panels A-H represent time series of embryos viewed dorsally (A-D) and laterally (E-H) at 15- (A, E), 18- (B, F), 21- (C, G) and 24 hpf (D, H). (A, E) 1°NC cells (dotted line) spread over the proximal surface of the eye (e). Orientations: proximal (P), distal (Di), rostral (R), caudal (C), dorsal (D), ventral (V). (B, C, F and G) Clusters of 2°NC cells (within the magenta circle) are observed next to the diencephalon (d) and mesencephalon (m) at 18 hpf. t: telencephalon; Ln: lens. (D, H) 2°NC clusters dissolve and individual cells migrate into the distal side of the eye. (I) 2°NC cells that migrate into the distal side of the eye exhibit mesenchymal morphology with filopodia. (J-J”) At 40 hpf, the nascent AS of the eye harbours mem-tdEosFP-positive NC cells. Transverse (J’) and horizontal (J”) optical sections along the dashed lines through the lens show the localization of NC cells beneath the surface epithelium (Ep, stippled line). Scale bars: A-H, 100 μm; I-K4, 50 μm. (K1-4) Photoconversion of Tg(sox10:mem-tdEosFP) embryos at 17 hpf visualizes 1°NCs and 2°NCs separately in magenta and green, respectively. Lateral views of time lapse fluorescent images were merged over the bright field images. The eye primordium and lens are indicated by dotted lines. Note that 2°NC cells (orange line, K1) continue to express sox10 reporter, resulting in a white colour in the presence of photoconverted mem-tdEosFP (magenta). 2°NC cells located at the dorsal edge of the eye primordium (asterisk, K2) migrate into the distal eye compartment (arrowheads, K3-4). (L) Summary of the period of active sox10 expression in 1° and 2°NC cells elucidated by serial photoconversion experiments (S2 Fig). (M) Schematics showing the position of 1°NC (magenta) and 2°NC cells (green) at 18, 21 and 24 hpf.

Dorsal views of a Tg(sox10:h2a-tdEosFP;wnt-rep) embryo highlighting (A-E) h2a-tdEosFP-positive NC cells and (A’-E’) Wnt reporter expression (white) in h2a-tdEosFP-positive NC cells (blue) showing colocalized Wnt reporter with nuclei from sox10-positive NC cells. At 13–14 hpf, 2°NC cells in the medial region (magenta circles, A-B) express Wnt-rep (A’-B’) at higher levels than 1°NC cells. This Wnt-rep expression is transient and followed by a reduction at 15 hpf (C’). At 18–21 hpf, 2°NC cells develop into bilateral clusters at the level of the diencephalon and mesencephalon (magenta circles, D-E). These cells express high levels of Wnt-rep (D-E’). Orientation: anterior left. (F) Mean total intensity (expressed in arbitrary unit, AU) profile of Wnt reporter expression in sox10-NC cells during 10–33 hpf shows 1^st and 2^nd wave of Wnt activity. (G) 2°NC cells (green) express Wnt reporter (magenta) prior to and during migration into the distal side of the eye (arrows). Orientation: anterior left, dorsal up; Scale bars: 50 μm.

DSLM time series of a double transgenic embryo expressing nuclear Eos in NC cells [green, Tg(sox10:h2a-tdEosFP)] and nuclear mCherry from the Wnt reporter. (A-F) Tracking analysis of 1°NC and 2°NC cells. Two groups of h2a-tdEosFP-positive NC cells which were located at either the proximal or distal side of the optic cup were selected at 17 and 24 hpf for systematic backward (B-C’) and forward tracking (D-E’), relative to 17 hpf (A). (A’) Dorsal and front views of trajectories of NC cells destined for the proximal (magenta, 474 cells) or the distal side (green, 610 cells) of the eye are presented with colour-codes for approximate time periods. Stippled circles represent the optic cup. B-E, dorsal projection view; B’-E’, lateral projection. With reference to the time of cell selection (17 hpf), backward (17–11 hpf, B-C’) and forward tracking (17–31 hpf, D-E’) results are shown. Individual tracks are colour-coded for stages to indicate the temporal cell position (A). Tracks are merged onto the maximum projections at the end of the tracking period (17 hpf, B-C; 31 hpf, D-E; see also S5 Video for three-dimensional presentation). 2°NC cells at the dorsal edge of the eye originate from the diencephalon and mesencephalon (asterisk, C) and migrate into the distal side of the eye (arrows in E) from the dorsal edge (arrow in E’). Circles in the panels outline the eye. Insets in B-E show transverse sections made at the lens highlighting cell migration paths relative to the proximal (P) and distal (Di) side of the eye. Orientations: rostral (R), caudal (C), dorsal (D), ventral (V). (F) Depiction of six representative lineages for each of the proximal (magenta) or distal (green) destined group. Each branch corresponds to a cell division. Incomplete short tracks are shown in blue. (G-H) Time lapse analysis of locally green-to-red photoconverted 2°NC cells at 17 hpf (G) and 20 hpf (H). Arrows indicate 2°NC cells reaching the distal side of the eye. G, dorsal projection view; H, lateral projection. Scale bar: B-E’: 100 μm; G-H: 75 μm.

(A-C”) Double in situ hybridisation analysis in the diencephalon of Tg(Wnt-rep) transgenic embryos at 18 hpf (lateral view, anterior left, dorsal up). Three combinations of probes were examined: sox10/wnt-rep (A, A’), sox9b/wnt-rep (B, B’) and sox10/sox9b (C, C’). Panels A”-C” show co-localization of two probes as white region. Embryos were counterstained with DAPI to visualize nuclei (blue). Marginal co-localization of sox9b and sox10 transcripts is indicated by an arrowhead (C”). (D-F”) Pairwise comparisons of the expression patterns of pax6b/sox10 (D-D”), pax6b/sox9b (E-E”) and otx5/sox9b (F-F”) are summarised in dorsal maximum projection views (D-F), transverse sections at the level of the pineal anlage (D’-F’) and sagittal optical sections along the midline (D”-F”). At 18 hpf, pax6b and sox10 expression (D) as well as pax6b and sox9b (E), appear to overlap when fluorescent signals were projected in dorsal views. Expression is, however, mostly confined to distinct dorsoventral layers (D’-E”). (F-F”) sox9b co-localizes with otx5. (G-I) Summary of the gene expression pattern in dorsal view (G), in transverse view (I) along the horizontal line in G and in lateral view (H). The sox10-positive NC cells (green) reside next to sox9b- and otx5 double positive cells of the epiphysis anlage (purple). t: telencephalon; d: diencephalon; m: mesencephalon; e: eye; DMB: diencephalon midbrain boundary; Scale bar: A-C”: 100 μm; D-F”: 150 μm.

(A-C”) The Tg(corneaEndo:GFP) line shows the first corneal endothelium cells (green) as a patch of cells over the lens (arrows in A, 60–72 hpf) that later spread across the entire inner surface of the cornea (B: 96 hpf; C:120 hpf). (C’-C”‘) Transverse (C’) and horizontal (C”‘) optical sections along the stippled lines in C, merged onto pictures from reflection imaging (grey channel) which highlight the corneal epithelium (Ep; note: the strong lateral signals are iridophores). (C”) Magnified view of the central cornea region indicated by a rectangle in C’. En: corneal endothelium; Ln: lens; Ir: iris; Scale bars: 50 μm (A-C, C’ and C”‘); 10 μm (C”). (D) The AS development is classified into five phases. Phase 1: 2°NC cell cluster formation (18–22 hpf); Phase 2: 2°NC cluster dissolves and migrate into the eye (22–28 hpf); Phase 3: NC cells differentiate into ocular mesenchymal cells (28–60 hpf); Phase 4: First corneal endothelial cells emerge over the lens (60–72 hpf); Phase 5: Corneal endothelium fully covers the eye (>72 hpf).

(A) CRISPR/Cas9-mediated deletion of 3 kb in the pax6a locus encompassing exons 8–12 (ΔEx8-Ex12, a horizontal double head arrow) encoding homeobox DNA binding and PST domains (S11 Fig). (B-E’) The AS phenotype at 5 dpf of a wild type sibling (B-B’), a pax6a-/- zygotic mutant (C-C’) and pax6b-/- maternal zygotic mutants with keratoconus-like phenotype (D-D’; arrow in D) and deflated cornea (E-E’), imaged alive (B-E) and in sections (B’-E’). (B’-E’) The chamber angle (arrows in B’-C’) is open in wild type (B’), pax6a-/- (C’) and pax6b-/- embryos with keratoconus (D’), however, it appears closed in pax6b-/- embryos with deflated cornea (E’). Although a pax6a-/- embryo appeared normal with live inspection (C), in comparison to the wild type that has a flat iris plane (B’) the iris plane is slanted (C’), as also seen in pax6b-/- embryos (D’-E’). All pax6 mutants show a smaller lens (Ln). (D’-E’) Gaps are evident between the lens capsule and the lens fibre (arrowheads). Abnormal layering in the corneal epithelium is occasionally observed with deflated corneas (arrow in E’). (F-I) The AS phenotype at 5 dpf of a wild type sibling (F, H) and a double homozygous pax6a-/-;pax6b-/- embryo (G, I). ica: iridocorneal angle, gcl: ganglion cell layer, ipl: inner plexiform layer, inl: inner nuclear layer, onl: outer nuclear layer. The distal eye structure of pax6a/b double mutants has no lens, no gcl, no ica and is occupied with abnormal ocular mesenchymal cells (arrows in G, I). Orientation is dorsal (D) up, ventral (V) down, distal (Di) left and proximal (P) right. (J-O) Marker gene expression analysis in 5 dpf embryos of wild type siblings injected with Cas9 without gRNAs (J, J’, L, and N) and of maternal zygotic pax6b-/- mutant genotype injected with pax6a gRNAs and Cas9 (K, K’, M and O). (J-K’) The corneal endothelium visualized by the Tg(corneaEndo:GFP) transgenic line. The anterior chamber margin is demarcated by a solid circle. Optical sections made along vertical lines in J and K are shown in J’ and K’, respectively. The Pax6 deficient embryo (K-K’) lacked both the lens (asterisk in K’) and the expression of the corneal endothelium reporter (K’, double arrowed arc). (L-O) in situ expression analysis of a mesenchymal marker pitx2 (L-M) and of the corneal epithelium marker zgc:92380 (N-O). Presence and absence of gene expression is denoted by arrow and asterisk, respectively. The lens border is demarcated by a stippled line. Scale bars: 50 μm.

Tg(sox10:h2a-tdEosFP) embryos with a genetic background of either pax6a+/+;pax6b+/- (A-C, wild type-like) or pax6a-/-;pax6b-/- (D-F, double homozygous mutant) were photoconverted at 17 hpf for visualizing 1° and 2° NC cells in magenta and green, respectively. At 21.5 hpf, the 2°NC cell cluster (A-A’ and D-D’; solid circles) is observed at the dorsal edge of the eye in both genotypes. At 28.5 hpf, although 2°NC cluster was dissolved into individually migrating 2°NC cells in the distal eye compartment of a pax6a+/+;pax6b+/- embryo (B-B’, arrows), the 2°NC cluster persisted at the same location in a pax6a-/-;pax6b-/- mutant embryo (E-E’, solid circle) which failed to develop the lens primordium (D-E’, asterisks). (C and F) Lateral views of 3D rendered trajectories of 1°NC (magenta lines) and 2°NC cells (green lines) for each genotype during 24–28 hpf. The NC nucleus is represented as a grey object. The eye boundary is shown as a stippled circle. Scale bars: 50 μm.

Gene expression was analysed by in situ hybridisation in wild type (WT, A-L), maternal zygotic pax6b-/- (A’-L’) and pax6a-/-;pax6b-/- double homozygous mutant embryos (A”-L”) at 28 hpf. The gene symbol of the transcript analysed is given at the left end of each row. The stippled white line demarcates the optic cup and brain. Sections are made either in transverse orientation (tbx5 [B-B”], pax6b [C-C’], pax2a [D-D”], vax2 [E-E”], cryba4 [F-F”], sema3aa [G-G”], nrp2b [H-H”], efnb2a [J-J”] and ephb3a [L-L”]) or in sagittal orientation (aldh1a2 [A-A”], plxna4 [I-I”] and efna3b [K-K”]) to show the gene expression along the distal-proximal and nasal-temporal axis, respectively. For cryba4 (F-F”), sema3aa (G-G”), efna3b (K-K”) and ephb3a (L-L”), the head region of the whole mount embryos is shown in the inset. Note the expression indicated by the arrow: dorso-temporal for aldh1a2 (A-A”), dorsal for tbx5 (B-B”), ventro-proximal for pax2a (D-D”), ventral part of the optic cup for vax2 (E-E”), lens primordium for cryba4 (F-F”) and sema3aa (G-G”), distal periocular mesenchymal cells for nrp2b (H-H”), dorsal for efnb2a (J-J’), dorso-nasal optic cup for efna3b (K-K”) and periocular mesenchymal cells for ephb3a (L-L”). Scale bar: 50 μm; 200 μm for insets.

Intravenous injection of 2000 kDa dextran (green) and 3 kDa dextran (magenta) into wild type (A-B) and double homozygous pax6a-/-;pax6b-/- mutant embryos (C-D) at 5 dpf allows visualizing the anterior chamber (ac, magenta) with reference to ocular annular vessels (green) in en face view. The asterisk denotes diffusion of 3 kDa dextran dye through the uveoscleral pathway. (A) Wild type control anterior chamber with no surgical operation. (B) No anterior chamber was formed in wild type after lens removal at 24 hpf. (C-D) The anterior chamber was partially restored in pax6a/b double mutant eyes after transplanting a wild type lens at 24 hpf (C), while no anterior chamber was formed without operation (D). Orientations: dorsal (D), ventral (V), nasal (N) and temporal (T). Scale bar: 50 μm.

(A-N) Tg(smad3:GFP) embryos were examined from 22–96 hpf for in situ expression of tgfb2 (A-G) and gfp (H-N). The dotted line outlines the lens. Arrows and arrowheads indicate expression in the lens and mesenchymal cells, respectively. (O-P) Tg(corneaEndo:GFP) embryos were treated with either 1% DMSO (O) or TGFβ inhibitor (30 μM SB-505124; P) from 28 to72 hpf (stages 3–4). Orientations: dorsal (D), ventral (V), nasal (N) and temporal (T). Ln: lens. The dotted lines outline lens and eye cup. Scale bars: 50 μm.