Whole Mount In Situ Hybridization (WISH) of known POM and neural crest-related marker genes. Whole-mount in situ hybridization for POM marker gene mRNA expression patterns were observed during early to late stage eye development in the lateral view. POM genes examined were (A)foxc1a, (B)foxc1b, (C)foxd3, (D)pitx2, (E)lmx1b.1, (F)lmx1b.2, (G)sox10, and (H)eya2. Foxc1a, foxc1b, pitx2, eya2, and sox10 in particular show strong expression surrounding the optic cup and on the surface of the anterior segment from 24 to 72 hpf. Lmx1b.1 and lmx1b.2 expression is detected within the anterior segment, surrounding the lens by 48–72 hpf. MHB, midbrain-hindbrain boundary; R, retina; L, lens; OV, optic vesicle; PO, periocular space (outlined with dashed line); OF, optic fissure; AS, anterior segment.

Two-color fluorescent in situ hybridization supports ASM heterogeneity. Two-color fluorescent WISH (FWISH) performed for all possible combinations of foxc1a, foxd3, pitx2, and sox10 at 32 (A–F) and 48 hpf (G–L). DAPI is in blue. Lateral images of 3D reconstructions are displayed. White arrows within inset panels (dashed squares) display instances of individual (b) and co-expression (a). Scale bar = 50 μm.

Periocular mesenchyme subpopulation distribution analysis. (A) 3D rendering of confocal stacks encompassing the AS in POM transgenic lines between 24 and 72 hpf. GFP+ cells are green, DNA was stained with DAPI (blue). DN, dorsal-nasal; DT, dorsal-temporal; VN, ventral-nasal; VT, ventral-temporal. Scale bar = 50 μm. (B) Distribution of quantified GFP+ cells within each AS quadrant (DN, DT, VN, and VT) at 24, 30, and 48 hpf. Distribution is represented as an average percentage of the total number of GFP+ in each quadrant of the AS for each transgenic line. Statistically significant difference (p < 0.05): * vs. DT, # vs. VN, $ vs. VT and ^ vs. DN. (C) Model of POM colonization at 24, 30, and 48 hpf. (D) Average AS cell population size for each transgenic line at 24–72 hpf. * indicates statistically significant (p < 0.05) change from previous time point.

In vivo 4D imaging of POM anterior segment colonization. (A) 4D in vivo imaging of the AS conducted between 24–48 hpf using POM transgenic lines. Time stamp hours:minutes. Scale bar = 50 μm. (B) Individual cell tracking for each POM transgenic line reveals migratory patterns during early AS colonization. Solid spheres indicate terminal end of track. (C–E) Cell tracking measurements of average migratory velocity (ANOVA p < 0.0001), total migration distance (ANOVA p < 0.0001), and migratory displacement within the AS (ANOVA p < 0.0001).

Anterior segment mesenchyme single cell clustering analysis at 48 hpf. (A)K-means clustering of scRNA sequencing of isolated 48 hpf foxc1b:GFP, lmx1b:GFP, sox10:GFP, foxd3:GFP, and pitx2:GFP ASM cells in both tSNE and UMAP readouts. Both plots identified four general clusters. (B) Distribution of isolated ASM cells within each of the clusters generated by the Cell Ranger3.1 software. (C) Model of identified cluster interactions. Interaction and overlap is indicated by dashed lines. (D) Heat maps of cluster specific gene expression patterns.

Gene expression of sequencing-derived genes. Whole-mount in situ hybridization for novel ASM marker gene mRNA expression patterns was performed during early (48 hpf) and intermediate stages (72 hpf) of AS development and observed in lateral and ventral views. (A), Sparc expression representing cluster 1. (B)gng13b, hmgn2, and adcyap1b expression representing cluster 2. (C)cyt1, agr1, and ponzr3 expression representing cluster 3. (D)lum, fmoda, col2a1a, and tgfbi expression representing cluster 4. Yellow arrowheads indicate AS expression, magenta arrowheads indicate periocular expression.