Morphology and appearance of control, heterozygous, crim1^+/−, and crim1^−/− larvae that are homozygous for a 2 base pair deletion, c.339_340delCT p.Leu112Leufs*3 at 24, 48, and 72 h post fertilization (hpf). (A) Morphology for control (n = 63) and crim1^−/− larvae (n = 48) that are homozygous for a 2 base pair deletion in crim1. The columns are colored according to the numbers and percentages of larvae with a normal phenotype (blue), mild deformity (orange), curved body axis (yellow), and malformations such as edema, cardiac defects, and severely shortened body (gray). The crim1^−/− larvae have an increased rate of abnormalities compared to controls. (B) Representative photographs of control larvae (panel i), heterozygous crim1^+/− larvae (panel ii), and homozygous crim1^−/− larvae (panel iii) at 24 hpf. The heterozygous crim1^+/− larvae and homozygous crim1^−/− larvae appear similar to the control. (C) Representative photographs of control larvae (panels i and iv), heterozygous crim1^+/− larvae (panels ii and v), and homozygous crim1^−/− larvae (panels iii and vi) at approximately 38 hpf (panels i, ii, and iii) and 72 hpf (panels iv, v, and vi). The heterozygous crim1^+/− larvae appear similar to controls, but the homozygous crim1^−/− larvae show shortened body axes and curved tails at both time periods, as indicated by the arrows. Scale bars = 500 µm.

Eye, head, and body measurements from control and crim1^−/− larvae that are homozygous for a 2 base pair deletion, c.339_340delCT p.Leu112Leufs*3, at 48 and 72 h post fertilization (hpf). (A) Control larva showing measurements obtained for eye diameter and head length. (B, C) Control larva showing site of measurements for eye diameter and body and tail length. (D) Measurements of eye diameter in control larvae (white columns) and crim1^−/− larvae (black columns) at 48 hpf and 72 hpf. Each dot represents a single measurement. Eye diameter is significantly decreased in the crim1^−/− larvae compared to controls. (E) Ratio of eye diameter to body length in control larvae (white columns) and crim1^−/− larvae (black columns) at 48 hpf and 72 hpf. Each dot represents a single measurement. The crim1^−/− larvae demonstrate a significantly increased ratio for eye diameter to body length compared to controls. (F) Ratio of eye diameter to head diameter in control and crim1^−/− larvae. Each gray dot represents a measurement at 24 hpf, 48 hpf, and 72 hpf. The height of the white columns represents the mean of the ratio of eye diameter to head diameter for control larvae and the height of the black columns representing the mean of the ratio of eye diameter to head diameter for the crim1^−/− larvae. The crim1^−/− larvae show a significantly increased ratio of eye diameter to head diameter at 48 hpf (P = 0.008), but the ratio was not significantly increased at 24 hpf or 72 hpf.

Representative images of zebrafish eyes stained with H&E from control and crim1^−/− larvae that are homozygous for a 2 base pair deletion, c.339_340delCT p.Leu112Leufs*3, at 3–5 days post fertilization (dpf). The top row shows control larvae at 3 dpf (A), 4 dpf (B, C), and 5 dpf (D), showing normal lens and eye development. The bottom row shows homozygous crim1^−/− larvae at 3 dpf (E), 4 dpf (F, G), and 5 dpf (H), showing reduced lens and eye size in the crim1^−/− lenses at 3 and 4 dpf (E, F). At 3 dpf, 6–8 larvae were scored, and at 4 and 5 dpf, 10–12 larvae were scored. Scale bar = 50 µm.

DIC microscopy of control and crim1^−/− larvae that are homozygous for a 2 base pair deletion, c.339_340delCT p.Leu112Leufs*3, at 3 days post fertilization (dpf). (A–C) Lenses from control larvae and a crim1^−/− larva that have a smooth and regular appearance. (D–F) Lenses from crim1^−/− larvae that have a roughened and irregular appearance. (G) Method for obtaining measurements of lens diameter. (H) Measurements of lens diameter (in μm) in control and crim1^−/− larvae in 16 lenses. There is a significant reduction in lens diameter for the crim1^−/− larvae compared to controls (P = 0.036).

Immunohistochemistry in control and crim1^−/− larvae that are homozygous for a 2 base pair deletion, c.339_340delCT p.Leu112Leufs*3, at 72 and 96 h post fertilization (hpf). Representative sections of eyes from control larvae (A, C, E, G) and crim1^−/− larvae (B, D, F, H) at 72 hpf stained with zl-1 (A, B), E-cadherin (C, D), zn-5 antibody (E, F) and at 96 hpf stained with zpr-1 (G, H). The lens fiber cells in the crim1^−/− larva stained with z-l1 are less homogenous compared to the lens from a control larva (A, B). The corneal epithelium showed mild clumping of cells in the crim1^−/− larva stained with E-cadherin (C, D) compared to a control larva. The retinal ganglion cell layer was thicker in the crim1^−/− larva stained with the zn-5 antibody than in a control larva (E, F). There was no obvious difference in the photoreceptors in crim1^−/− and control larvae stained with zpr-1 (G, H). (A–H) Larvae stained with Alexa Fluor 488 dye (green). (A′–H′) Larvae stained with DAPI (blue). (A′′–H′′) Merged images. Scale bars = 50 µm.

Phospho-histone H3 and 5-bromo-2′-deoxyuridine staining in control, crim1^+/− larvae, and crim1^−/− larvae that are homozygous for a 2 base pair deletion, c.339_340delCT p.Leu112Leufs*3, at 48 and 72 h post fertilization (hpf). Representative sections of eyes from control larvae (A, D, G), crim1^+/− larvae (B, E, H), and crim1^−/− larvae (C, F, I) stained with the PH3 antibody (Alexa Fluor 488 dye; green) and DAPI (merged (A’–F’), blue) at 48 and 72 hpf (A–F) and with BrdU (Alexa Fluor 488 dye; green) and DAPI (merged (G’–I’), blue) at 48 hpf (G–I). The quantification of the BrdU antibody showed a significant reduction in BrdU staining in the homozygous crim1^−/− larvae compared to control at 48 hpf (P = 0.013). L = lens.

Quantification of phospho-histone H3 and 5-bromo-2′-deoxyuridine (BrdU) staining in control, heterozygous crim1^+/−, and crim1^−/− larvae that are homozygous for a 2 base pair deletion, c.339_340delCT p.Leu112Leufs*3, at 48 and 72 h post fertilization (hpf). (A) The quantification of the PH3 antibody showed no significant difference between control, heterozygous crim1^+/−, and crim1^−/− larvae. A total of 14 control larvae were scored, 11 crim1^+/− larvae were scored, and 8 crim1^−/− larvae were scored at 48 hpf. A total of 19 control larvae, 8 crim1^+/− larvae, and 17 crim1^−/− larvae were scored at 72 hpf. (B) The quantification of the BrdU antibody showed a significant reduction in BrdU staining in the homozygous crim1^−/− larvae compared to control and heterozygous crim1^+/− larvae at 48 hpf (P = 0.013) but not at 72 hpf. A total of eight control larvae were scored, three crim1^+/− larvae were scored, and seven crim1^−/− larvae were scored at 48 hpf. A total of five control larvae, five crim1^+/− larvae, and five crim1^−/− larvae were scored at 72 hpf.

Principal component analysis, heatmap, and volcano plot showing DEGs for controls and crim1^−/− larvae. Data from DEGs with adjusted P-values </ = 0.05 and an absolute (log2foldchange) >/ = 1 were used to generate the plots. (A) Three-dimensional PCA plot showing the separation of samples from control larval eyes (blue dots) compared to samples from crim1^−/− larval eyes (red dots). (B) Volcano plot showing differential gene expression for crim1^−/− mutant larvae and controls. The log2 fold change indicates the mean expression level for each gene. Each dot represents one gene. Selected genes with an adjusted P-value </ = 0.01 and absolute (log2foldchange) >/ = 2 have been named. Downregulated genes are shown in blue and upregulated genes are shown in red. (C) Heatmap of expressed genes in crim1^−/− larvae and controls, showing separation of gene expression between control and crim1^−/− mutant larvae. The type of sample (control or crim1^−/− mutant) is listed on the X axis and the Y axis contains DEGs.

RT-qPCR showing significant downregulation of crim1, clic4, and itgb1 and significant upregulation of serpinb1l2 and fgf1b. (A) RT-qPCR showing expression of crim1, clic4, and itgb1 in crim1^−/− larvae compared to controls. RT-qPCR was performed on dissected eyes for all experiments from control (white columns; expression normalized to 1.0) and crim1^−/− larvae (black columns) at 72 hpf. The results show significantly reduced expression of crim1 (P = 0.04), clic4 (P = 0.02), and itgb1 (P = 0.02) in crim1^−/− larvae compared to controls. (B) RT-qPCR showing expression of serpinb1l2, fgf1b, and gpib in crim1^−/− larvae compared to controls. The results show significantly increased expression of serpinb1l2 (P = 0.02) and fgf1b (P = 0.02) but not gpib (P = 0.25) in crim1^−/− larvae compared to controls. (C) RT-qPCR showing expression of bzw1a and nampta in crim1^−/− larvae compared to controls. The results do not show significantly reduced expression for bzw1a (P = 0.4) or nampta (P = 0.16) in crim1^−/− larvae compared to controls.

c1q levels measured by RT-qPCR in control and crim1^−/− larvae that are homozygous for a 2 base pair deletion, c.339_340delCT p.Leu112Leufs*3, at 72 h post fertilization (hpf). RT-qPCR showing expression of c1qa, c1qb, c1qc, cd74a, and cd74b in crim1^−/− larvae compared to controls. RT-qPCR was performed on dissected eyes for all experiments from control (white columns; expression normalized to 1.0) and crim1^−/− larvae (black columns) at 72 hpf. The results show significantly increased expression of c1qb (P = 0.018) and significantly reduced expression of c1qc (P = 0.003) and cd74b (P = 0.032) in crim1^−/− larvae compared to controls. c1qa (P = 0.154) and cd74a (P = 0.391) expression was not significantly different.