Fig 1.
The mouse mutant displays the ocular phenotype of microphthalmia-like.
(A) External morphology of the eyes of mutant mice and normal mice was photographed at P14, P28, P42, P56, P3M, or P4M. At each time point, bilateral ocular imaging was simultaneously performed in each group. The images are representative of results obtained in all 3 mice per group. Mice were positioned the same distance from camera for each image. (B) The body length of the mutant group(n = 12) and the control group(n = 12). (C) The weight of the mutant group(n = 12) and control group(n = 12). (D) The width of the palpebral fissure of the mutant group (n = 12) and control group (n = 12). The width of the palpebral fissure of the mutant group was significantly smaller than that of the control group at each stage. Multiple t tests were performed to analyze the significance. Data are presented as the means ± SEM at three times. *p < 0.05, **p < 0.01, *** p < 0.001, **** p < 0.0001.
Table 1.
Body length (cm).
Table 2.
Weight(g).
Table 3.
Palpebral fissure width(mm).
Fig 2.
The size of the eyeballs and lenses of the mutant group and the control group.
(A) Six eyeballs and six lenses of the mutant group and control group at designated postnatal timepoints (P28, P42, P56, P3M, P4M) are presented on quadrille paper. (B) The size of the eyeballs and lenses were quantified and multiple t tests were used for statistical analysis. The eyeballs and lens of the mutant group(n = 6) are significantly smaller than those of the control group (n = 6). **** p < 0.0001.
Fig 3.
Optomotor response behavior test of the mutant group and control group at defined spatial frequency.
(A and B) Vision function was detected at defined spatial frequency or contrast levels in the OptoTrack visual detection system. (A) Representative heat maps show the OMR values at defined spatial frequencies for both the mutant and control groups at P3M. (B) The overall OMR values in the mutant group were significantly lower than those of the control group at defined spatial frequencies.
Fig 4.
Optomotor response behavior test of the mutant group and control group at defined contrast.
(A) Representative heat maps show the OMR values at defined contrast at P3M. (B) Representative histograms of visual stimuli–driven response of the mutant group at defined contrasts under the optimal velocity conditions (2–14°/s) at P3M. The response time in correct (positive value, light green window) or incorrect direction (negative value, light magenta window) within the defined velocity interval was normalized against the maximum response time (represented as 1) for each spatial frequency or contrast and depicted as a blue bar. In cases where the response time in the correct direction exceeds that in the incorrect direction, the OMR values for each spatial frequency or contrast are illustrated as a magenta bar normalized to the green bar and also the green and light green bars exhibit equivalent vertical dimensions, resulting in the light green bars are covered by green bars. (C and D) The mean OMR values (mean ± SEM) of the mutant group (n = 12) and control group (n = 15) were plotted as a line chart at a spatial frequency of 0.1 cycles/°(C)or 100% contrast (D). The mean OMR values of the mutant group and control group have a significant difference at spatial frequency of 0.1 cycles/°or 100% contrast. Multiple t tests were performed in representative data, *p < 0.05, **p < 0.01, *** p < 0.001. All experiments were independently performed at least three times to ensure repeatable results.
Fig 5.
Measurement of anterior chamber depth and corneal thickness in the mutant group and control group.
(A) Representative OCT image of the anterior segment of the mutant group(n = 8) and control group(n = 8). Anterior chamber depth was presented as a red line. Scale bar: 150µm. Anterior chamber depth in the mutant group was significantly shorter than that in the control group. ***p < 0.001 (B) Representative OCT image of the corneal thickness of the mutant group(n = 8) and control group(n = 8). The corneal thickness is marked with a red line. Scale bar: 150µm.
Fig 6.
Electroretinography (ERG) recording analysis of the mutant group and control group.
(A) A thorough evaluation of visual function using ERG was conducted in mice. Representative ERG waveforms from the mutant group (n = 8) and the control group (n = 8) are presented at P4M. (B) Quantitative analysis of a-wave and b-wave amplitudes was performed under dark-adapted conditions at flash intensities of 1.5 cd·s/m² and 3.0 cd·s/m², respectively. **** p < 0.0001 (C) Representative morphology of the eyes in the mutant group and the control group. Pupil atresia was noted in the mutant mice.
Fig 7.
H&E staining of eyeballs and of the mutant group and control group.
(A) Hematoxylin-eosin staining was performed on eye sections from the control and mutant groups at P28, P42, P56, P3M and P4M. A vertical section of the globe was taken through the optic disc plane. The red rectangle highlights areas that are depicted at higher magnification. Eyeballs in the mutant group exhibited the smaller size of the eyeballs and rosette-like structures in the retina compared to the control group. The retinal layer identifications: retinal pigmented epithelium (RPE), outer nuclear layer (ONL), inner nuclear layer (INL), and retinal ganglion cell (RGC). Representative histological data of the retina of mutant mice(n = 3) and normal mice(n = 3). Scale bar: 50µm (B) Measurement and statistics of the thickness of RPE of mutant mice(n = 3) and normal mice(n = 3) at P28, P42, P56, P3M and P4M. (C) Count and statistical analysis the retinal rosettes in the vertical section of mutant mice(n = 3) at P28, P42, P56, P3M and P4M.
Fig 8.
H&E staining of viscera tissues of the mutant group and control group.
(A) HE staining was performed on tissues sections from the control and mutant groups at P4M. Representative image of viscera tissues of mutant mice(n = 3) and normal mice(n = 3). Scale bar: 50µm.