Construction and Identification of a Novel Mice Model of Microphthalmia

Abstract

Purpose: Microphthalmia is a rare developmental eye disease that affects 1 in 7000 births. Currently, there is no cure for this condition. This study aimed to construct a stable mouse model of microphthalmia, thus providing a new tool for the study of the etiology of microphthalmia.

Methods: The Hedgehog signaling pathway plays a crucial role in eye development. One of the key mechanisms of the Sonic Hedgehog signaling is the strong transcriptional activation ability of GLI3, a major mediator of this pathway. This study used CRISPR/Cas9 system to construct a novel TgGli3Ki/Ki lens-specific over-expression mouse line. To identify the ocular characteristics of this line, quantitative PCR, Western blot, hematoxylin and eosin staining, immunofluorescent staining, and RNA-seq were performed on the ocular tissues of this line and normal mice.

Results: The TgGli3Ki/Ki lens-specific over-expression mouse model exhibits the ocular phenotype of microphthalmia. In the TgGli3Ki/Ki mouse, Gli3 is over-expressed in the lens, and the size of the eyeball and lens is significantly smaller than the normal one. RNA-seq analysis using the lens and the retina samples from TgGli3Ki/Ki and normal mice indicates that the phototransduction pathway is ectopically activated in the lens. Immunofluorescent staining of the lens samples confirmed this activation.

Conclusions: The TgGli3Ki/Ki mouse model consistently manifests the stereotypical microphthalmia phenotype across generations, making it an excellent tool for studying this severe eye disease.

Translational Relevance: This study developed a novel animal model to facilitate clinical research on microphthalmia.

Introduction

Microphthalmia is defined as a small, underdeveloped eye caused by disrupted eye development through genetic or environmental factors in the first trimester. Clinical phenotypic heterogeneity exists in patients with varying severity and associated ocular and systemic features. As one of the most severe developmental eye abnormalities, microphthalmia accounts for approximately 3% to 12% of blinding cases in children,¹ with the prevalence ranging from 10.0 to 10.8 per 10,000 births.² There is no treatment for microphthalmia that will restore vision. 

Besides a small proportion of cases that are attributed to environmental factors, such as intrauterine infections and toxins, genetic alterations are the major causes of such a disease.³ Genes implicated in main non-syndromic microphthalmia include SOX2, OTX2, RAX, VSX2, STRA6, RARB, ALDH1A3, MAB21L2, VAX1, BMP7, GDF3, and GDF6.^3,4 More genes up to approximately 100 are associated with systemic microphthalmia. Due to the conserved ocular development and physiology between mice and humans, dozens of mouse lines with a microphthalmia trait have been generated, most of which resulted from the disruption of the genes identified in human patients. These genes included Sox2, Otx2, Rax, Vsx2, Pax6, Stra6, Foxe3, Bmp4, Bmp7, Smoc1, Shh, Porcn, Foxc1, Fras1, Frem1, Tctn2, Col4a1, Tbc1d32, Prss56, Pxdn, Pitx2, Pitx3, Mitf, Cryaa, Frem2, Rpgrip1l, Smg9, Snx3, Dag1, Hmx1, Rere, and Rab18 (reviewed from Mouse Genome Informatics database: http://www.informatics.jax.org/). The mouse offers the possibility to genetically test the roles of modifiers and single nucleotide polymorphisms (SNPs); these aspects open new avenues for ophthalmogenetics in the mouse. Overall, 237 causative genes in mice and 98 in human subjects share 31 overlapping genes, as illustrated in Figure 1. The complete gene list is provided in Supplementary Table S1. 

Figure 1.

 

The schematic diagram of the construction of TgGli3Ki/Ki mice using CRISPR/Cas9 system. (A) The plasmid design for the knockin of Gli3 gene. (B) The injection of the plasmids and the crossing of the mice.

Various studies suggest that Hedgehog (HH) signaling plays essential roles in human and mouse eye development.⁵ Mutations/deletions in human Sonic Hedgehog (SHH) cause holoprosencephaly, including anophthalmia, cyclopia, and coloboma in severe cases.^6–8 Homozygous Shh null mutant mice show that Shh plays a critical role in the brain and spinal cord, the axial skeleton, and the limbs, and SHH was required to separate the eye field into bilateral domains.⁹ Other components of the HH signaling pathway, such as PTCH1 and CDON, have been reported to cause microphthalmia when mutated in human patients. Such evidence suggests that HH signaling is closely related to eye development and the cause of microphthalmia.^10,11 Interestingly, constitutively active HH signaling in surface ectoderm of mice caused by a mutation of Smo, the coding protein of which is repressed by PTCH1 when not activated, results in aberrant and disorganized lens and retina morphology.¹² 

Transcriptional factors GLI1, GLI2, and GLI3 are thought to regulate most of the transcriptional responses to HH signaling.¹³ Different from GLI1, which acts predominantly as positive regulators of target genes in HH signaling, GLI2 and GLI3 play either an activating or a repressing role depending on the HH signal availability.^13–17 GLI2 plays a stronger activating role than GLI3 in the HH signaling cascade.^16,18 In contrast, the repressing part of GLI3 is more predominant than that of GLI2.^18–20 Regarding GLI3, in the absence of any HH signal, the C-terminal region of GLI3 is cleaved after amino acid 700 to generate an N-terminal 83 kDa transcriptional repressor (GLI3-R).²¹ In the presence of HH, GLI3 is in the full-length form that functions as a transcriptional activator (GLI3-A).²² Because GLI3 primarily acts as a transcriptional repressor, the loss of GLI3 is often functionally equated to the overactivity of the HH pathway.²³ In the in vivo studies of GLI3 functioning in eye development, 2 transgenic mouse lines have been used: Gli3^+/Xt-J and Gli3^{∆699/∆699}. In Xt-J (Extra-toes^J) homozygotes, Gli3 expression is completely missing during embryogenesis, and the mice would die within 2 days after birth.^24,25 In the heterozygous Gli3^+/Xt-J line, the embryos exhibit eye defects varying from microphthalmia to anophthalmia with significantly smaller lenses or no lenses at all.^26,27 In the Gli3^{∆699/∆699} line, the repressor form of GLI3 (GLI3-R) is constitutively expressed.²⁸ Although the Gli3^{∆699/∆699} mice die shortly after birth,²⁸ by contrast with the ocular phenotype in the embryos of Gli3^Xt-J/Xt-J mice, Gli3^{∆699/∆699} embryos do not exhibit any morphological defects in the eye,¹⁸ indicating that GLI3-R but not GLI3-A is essential for eye development. 

It is still an open question how GLI3 regulates eye formation, it would be of great importance to investigate the dosage-dependent function of GLI3 in eye development. Our group have long been interested in the lens development and lens-related diseases, in this context, we generated a lens-specific TgGli3Ki/Ki mouse line. In this line, chicken βB1-crystallin promoter (−434/+30)^29–31 (Genbank accession number U09951) is conjugated with full-length cDNA of Gli3 (see Fig. 1), endowing the active expression of GLI3 in lens fiber cells. We found that the homozygous TgGli3Ki/Ki mice are viable for at least 12 months (to the end of the observation point) with severe microphthalmia, and they are all blind due to total synechia of the iris. These results suggest that overexpression of GLI3 in the lens disrupts eye development, and the size of the whole eye is deeply affected by the lens size. 

The first mouse microphthalmia transcription factor (Mitf) mutation was discovered over 60 years ago, which was originated from a cohort of irradiated mice.³² Since then, most mouse models identified with a microphthalmia phenotype were created by forward genetics. For example, in a spontaneous mouse mutant line Pitx3^416insG,³³ the mutant mice have closed eyelids with no apparent eyes (anophthalmia) or very small eyes (microphthalmia). Recent progress in targeted genome editing makes it much easier to directly modify specific genes. In a transgenic Pax6 mouse line, where the downstream regulatory region of Pax6 was disrupted, it presented a similar eye development pattern of microphthalmia and aniridia.³⁴ Another study generated a gene-dosage allelic series of Sox2 mutations in the mouse, suggested that a reduction of SOX2 expression to <40% of normal causes variable microphthalmia.³⁵ Although microphthalmia trait manifest, at least partially, in all these models, the severity of the phenotypes are highly variable, from isolated mild microphthalmia to anophthalmia. The homogeneous TgGli3Ki/Ki mouse line in this study provides a consistent tool to explore the pathology of microphthalmia without impacting other organs of the body. 

Materials and Methods

Generation of the TgGli3Ki/Ki Mice

The animal studies were conducted in accordance with the ARVO Animal Statement. We used C57BL/6JGpt mice as the background line. We made TgGli3Ki/Ki knockin mice via CRISPR/Cas9 system. The mouse Gli3 gene was inserted into Hipp 11 (H11) locus via Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated 9 (Cas9) system. The detailed description of the construction of this line is provided in the Results section. 

Quantitative Polymerase Chain Reaction

Total RNA was extracted using TRIzol (#15596, Thermo Fisher Scientific, Waltham, MA, USA) and measured on NanoDrop 2000 (Thermo Fisher Scientific). Reverse transcription was performed using an RT kit (CW2020, CoWin Biosciences, China), and quantitative polymerase chain reaction (qPCR) was performed using SYBR Premix (CW3008, CoWin Biosciences). The qPCR was performed on the ABI 7500 PCR machine, and data were analyzed using the ABI 7500 software version 2.0.6 (Life Technologies, Thermo Fisher Scientific). Actb was used as the endogenous control gene. The 2^−∆∆ct method was applied for the relative quantification. The primer sequences are provided in Supplementary Table S2. The acronyms for all the genes are provided in Supplementary Table S3. 

Western Blot

Protein extracts from eye tissues were subjected to SDS-PAGE and blotted onto 0.45 µm PVDF membrane (Merck Millipore), incubated overnight with the primary antibody GLI3 (# AF3690; R&D Systems) at 1 in 200 dilution, or β-Actin, (#A3854; Sigma–Aldrich, St. Louis, MO, USA) at 1 in 20,000 dilution in Tris-buffered saline with 0.1% Tween-20 and 5% milk. The anti-goat second antibody was used to visualize the protein bands. The membrane was developed using the Chemilum HRP Substrate (#WBKLS0500; Merck Millipore). 

Histology and Immunofluorescence

For hematoxylin and eosin (H&E) staining of mice tissues, dissected eyeballs were fixed in 10% formalin overnight, followed by dehydration through an ethanol gradient. Tissues were embedded in paraffin and sectioned at 5 µm. To observe the morphology of the entire eyeball, the tissue sections were stained with H&E. To deparaffinize the tissues, paraffin sections were incubated for 1 hour at 60°C in xylene twice for 10 minutes, washed in 100% ethanol twice for 3 minutes, followed by incubation in 95%, 80%, and 70% ethanol for 3 minutes in each step. To retrieve the antigens, the slides were incubated in 0.125% trypsin at 37°C for 30 minutes. Then, the slides can be used to perform immunofluorescent staining the same as the frozen sections. 

For staining of tissues on frozen sections, paraformaldehyde (4%) fixed eyeballs or lenses were cryoprotected with a PBS-buffered 30% sucrose and embedded in SAKURA Tissue-Tek optimum cutting temperature (O.C.T.) compound (#4583; SAKURA). The embedded tissues were sectioned at 8 µm. Slides were then incubated overnight at 4°C with the primary antibodies: GLI3 (#AF3690; R&D Systems), GRK1 (#DF8251; Affinity), GUCY2F (#25252-1-AP; Proteintech), and PDE6B (22063-1-AP; Proteintech). Secondary antibodies and Hoechst were used to visualize the stained cells. 

OCT

The mice were anesthetized by intraperitoneal injection of 0.1 mL of a 1% pentobarbital solution per 10 grams of mouse weight. Prior to optical coherence tomography measurements, tropicamide eye drops were applied to the mice's corneas to dilate the pupils. A 100 kHz speed full-range swept-source optical coherence tomography device was used to perform optical coherence tomography of the mouse eye. (TowardPi Medical Technology Co., Ltd., Beijing, China). 

RNA-Seq Analysis

RNA-seq analysis was conducted to investigate the differential gene expression profiles in TgGli3Ki/Ki knockin mice retinas and lenses (n = 4, TgGli3Ki/Ki versus normal). The experiment was conducted as described before.³⁶ The obtained differentially expressed genes were further subjected to functional enrichment analysis using gene ontology and pathway databases. 

Statistics

For the statistics in Figures 2–6, unpaired t-tests were utilized to compare the data between normal and knockin mice. Values are presented as mean ± standard error of the mean (SEM). Statistical significance is indicated as follows: P < 0.1: *, P < 0.01: **, P < 0.001: ***, and P < 0.0001: ****. For the statistics in Figure 2, we used the t-test to compare the data between normal and TgGli3Ki/Ki knockin mice at 4 weeks, 16 weeks, and 9 months separately. The P values obtained from multiple t-tests were adjusted using the Bonferroni correction. For the statistics in Figure 5A, for each gene, we analyzed the difference between normal and TgGli3Ki/Ki data using ANOVA. 

Figure 2.

 

The eyeball and the lens size is smaller in TgGli3Ki/Ki mice. (A) Four eyeballs (A) and four lenses (B) of normal and TgGli3Ki/Ki mice (at age of 4 weeks, 16 weeks, and 9 months) are presented on quadrille paper. (C) Quantification of the eyeball size presented in (A) and (B). Multiple t-tests (adjusted with Bonferroni correction) were used to analyze the significance.

Results

The Construction of TgGli3Ki/Ki Lens-Specific Over-Expression Mice

The schematic description of the creation of the mouse model is provided in Figure 1. The TgGli3Ki/Ki transgene consists of chicken Crybb1 promoter, 3X HA, mouse Gli3 Coding sequence, and 6X FLAG. It was inserted into an intergenic H11 locus on mouse chromosome 11. We chose this site because it has been confirmed that homozygous insertions into this locus are not predicted to disrupt any endogenous genes, and the resulting mice are completely healthy and fertile.^37,38 All the vectors were confirmed through restriction digestion and Sanger sequencing. The sequence of the sgRNA is: “CUGAGCCAACAGUGGUAGUA.” The homozygous TgGli3Ki/Ki knockin mice were constructed in GemPharmatech Co., Ltd (Nanjing, Jiangsu, China). Briefly, the Cas9 mRNA, sgRNA, and the Gli3-donor plasmid were co-injected into zygotes. Thereafter, the zygotes were transferred into the oviduct of pseudopregnant C57BL/6JGpt females at 0.5 dpc. In addition, F0 mice was birthed after approximately 19 to 21 days of transplantation, all the offspring of C57BL/6JGpt females (F0 mice) were identified by PCR and sequencing of tail DNA. Crossing positive F0 mice with wildtype mice were done to build up heterozygous mice. Finally, cross heterozygous mice were done to obtain homozygous mice. A total of 1225 embryos were injected during the model creation process, resulting in 287 pups, out of which 13 F0 positives were recommended for breeding, with a transplantation success rate of 23.4% and an F0 positive probability of 4.5%. 

The Morphological Characterization of TgGli3Ki/Ki Mice

Eyeballs and lenses were isolated from normal and TgGli3Ki/Ki mice at the age of 4 weeks, 16 weeks, and 9 months and the photographs were taken freshly. The size of the eyeballs and lenses was estimated using quadrille paper (see Figs. 2A, 2B). The multiple t-tests were used to analyze the significant difference between normal and TgGli3Ki/Ki group. Both the whole eyes and the lenses of TgGli3Ki/Ki mice are significantly smaller than that of normal mice (see Fig. 2C). Additionally, the eye size of TgGli3Ki/Ki mice increases with age; the lens size grows from 4 weeks to 16 weeks, but remains stable from 16 weeks to 9 months (see Fig. 2C). 

To dynamically measure the axial length and lens depth, we used a full-range swept-source optical coherence tomography device (with an adapted arm for securing the platform for small animals) to conduct optical coherence tomography scans on the mice's eyes. As shown in Figure 3, the axial length and lens depth in TgGli3Ki/Ki mice is significantly smaller than that in normal mice. Both the TgGli3Ki/Ki mice and the normal mice were 12 weeks old at the time of optical coherence tomography scan. From these optical coherence tomography images, we observed opacity in the lens, suggesting an early onset of cataract in TgGli3Ki/Ki mice. 

Figure 3.

 

Full range optical coherence tomography images of normal and TgGli3Ki/Ki mice. (A–D) Normal mice eyes, (E–H) TgGli3Ki/Ki mice eyes (n = 4). Quantification analysis of axial length and lens depth is presented at the lower right corner. The unpaired t-tests have been used to analyze the significant difference (normal versus TgGli3Ki/Ki mice). Scale bar = 400 µm.

Hematoxylin and eosin (H&E) staining of the eye slice from paraffin sectioning showed more clearly that, different from the normal one, the eye of Gli3^KI/KI mouse is smaller and contains a smaller lens (Fig. 4). Moreover, in the TgGli3Ki/Ki eye, the iris is completely adhered to the cornea, which explains why this TgGli3Ki/Ki strain is blind. H&E staining images of more eyeballs were included in the Supplementary Figures S1 and S2. 

Figure 4.

 

The H&E staining on the eyeball showed that the lens in TgGli3Ki/Ki mice is smaller than in normal mice. (A, B) At the age of 16 weeks. (C, D) At the age of 6 months. Scale bar = 1000 µm.

The GLI3 Expression in TgGli3Ki/Ki Mice

To confirm the lens-specific expression of Gli3, we quantified GLI3 at the RNA level using qPCR assay and at the protein level using Western blot and immunofluorescence staining. As shown in Figure 5A, at the mRNA level, the expression of Gli3 and other four genes (Ptch1, Ccnd1, Ccne1, and Smo) in the HH signaling pathway increased significantly in the lenses of TgGli3Ki/Ki mice than in the normal lenses. In contrast, in the results from the retina samples, only Gli3 showed a significant increase, albeit with a smaller fold change than in the lens samples. Figure 5B demonstrated that, despite multiple nonspecific bands, GLI3 expression is notably higher in the lenses of TgGli3Ki/Ki mice than in normal mice. From the immunofluorescence staining of GLI3 antibody in the lens, we observed that the GLI3 proteins predominantly expressed in the nucleus of the lens in the TgGli3Ki/Ki mice (Fig. 5C). Both lens and retinal samples were obtained from the 12-week-old mice in both groups. 

Figure 5.

 

(A) The expression of Gli3 and other genes in Hedgehog signaling pathway increased in the lens of TgGli3Ki/Ki mice. ANOVA multiple comparison method was used to analyze the significant difference (normal versus TgGli3Ki/Ki mice) in each group set. # = significance in retina samples; * = significance in lens samples. (B) The Western blot results confirmed the expression of Gli3 in the lens of TgGli3Ki/Ki mice. Proteins isolated from lens samples from two mice in each group (N = normal and G = TgGli3Ki/Ki) used. β-Actin was used as the endogenous control. (C) Immunofluorescence of GLI3 on mouse lenses. Cryosections were obtained from normal and TgGli3Ki/Ki mice at the age of 12 weeks. Scale bar = 50 µm.

The Phototransduction Pathway Is Activated in the Lens of TgGli3Ki/Ki Mice

To investigate changes in transcriptional patterns across the whole genome, we conducted RNA-Seq analyses on lens and retina samples obtained from both TgGli3Ki/Ki and normal mice. This assay included four groups: lens of TgGli3Ki/Ki mice (GL), retina of TgGli3Ki/Ki mice (GR), lens of normal mice (NL), and retina of normal mice (NR). Each group contained four biological replicates, with each replicate representing a pool of three lenses or retinas. The gene expression data from all four groups was presented using heatmaps (as shown in Fig. 6A). We then compared differential expressed genes (DEGs) between GL and NL, where our analysis of enriched bioprocesses and signaling pathways of DEGs revealed the “phototransduction” pathway as having the highest enrichment factor (Fig. 6B). These results suggest that this pathway was activated in the lens of TgGli3Ki/Ki mice, possibly induced by the transcription factor GLI3. 

Figure 6.

 

Gene expression profile of retinas and lenses from normal and TgGli3Ki/Ki mice. (A) Heatmap of gene expression levels of four groups: normal retina (NR), TgGli3Ki/Ki retina (GR), normal lens (NL), and TgGli3Ki/Ki lens (GL). Four biological replicated were used in each group. (B) The RNA-seq assays revealed differentially expressed genes most enriched in the phototransduction pathway. (C) The RNA expression level of 5 genes in the phototransduction pathway were measured in the lens and retinas of normal and Gli3+/+ mice using qPCR. The unpaired t-tests have been used to analyze the significant difference in each group set.

To validate the results of the RNA-Seq analyses, we conducted qPCR to assess the RNA levels of five crucial genes (Gucy2e, Gucy2f, Pde6a, Pde6b, and Grk1) in the phototransduction pathway. We examined these genes in lens and retina samples obtained from both TgGli3Ki/Ki and normal mice. All of these genes demonstrated significant upregulation in TgGli3Ki/Ki mice lenses; whereas only Gucy2e and Pde6a presented a significant increase in TgGli3Ki/Ki mice retinas, and the foldchange is much lower than that in the lens samples (Fig. 6C). Furthermore, we performed immunofluorescence staining of GRK1, GUCY2F, and PDE6B and confirmed an increase level of these markers in the lens nuclei of TgGli3Ki/Ki mice (Supplementary Fig. S3). Figure 7 displays the immunofluorescence images of the PDE6B staining. In addition, we analyzed the enriched pathway in the TgGli3Ki/Ki mice retinas and confirmed the activation of the “NF-kappa B pathway” in the retina samples, using a method similar to that used in analyzing the lens samples in Figure 6. We provided these data in the Supplementary Figure S4. 

Figure 7.

 

The immunofluorescence of a phototransduction marker Pde6b in normal (A–C) and TgGli3Ki/Ki (D–F) mice. Paraffin sections from mouse eyes were stained with DAPI (blue) and Pde6b (red). (B, C) Are the magnifications of the boxed area in (A, E, F) are the magnifications of the boxed area in (D). Scale bar = 500 µm.

Discussion

Microphthalmia is a developmental defect that arises from genetic mutations or environmental factors during the initial 3 months of pregnancy. Approximately 11% of blind children are diagnosed with microphthalmia, yet there are presently no available treatments.³⁹ Given that roughly 85% of the protein-coding genes in mice share analogous functions with those in humans, and that ocular development follows a comparable pattern, mouse models have proven valuable in elucidating the mechanisms of the causal genes in human microphthalmia. 

During human embryonic eye development, on the 25th day of gestation, the optic vesicle emerges from the neural tube. One end of the optic vesicle elongates to form the optic stalk, which eventually develops into the optic nerve. The other end folds inward to form the optic cup, giving rise to the retina, iris, and ciliary body. In contrast to the optic cup, lens cells are of surface ectoderm origin and differentiate into lens epithelium (the capsule) and lens fiber cells. The ocular lens is unique among organs due to its avascular nature, transparency, and relative isolation. These characteristics make the lens an excellent model for investigating fundamental developmental questions. 

The HH signaling pathway plays a vital role in embryonic development and is conserved throughout evolution from invertebrates to vertebrates. GLI family zinc finger proteins are mediators of SHH signaling in vertebrates. Strong transcriptional activation ability of GLI3 is one of the key mechanisms of the SHH signaling. Previous studies showed that GLI3-R plays is essential for mouse eye development and governs eye development partially via controlling WNT/β-CATENIN signaling.¹⁸ 

In the current study, our objective was to investigate the role of GLI3 in lens morphology. We generated a novel mouse model that expresses GLI3 in the lens in an ectopic manner. Our observations revealed significant reductions in both lens and overall eye size during eye development compared to normal counterparts. The TgGli3Ki/Ki mice did not produce measurable results during the assessment with an infrared photorefractor, as no signals were detected. In addition, the H&E staining images revealed total synechia of the iris, leading us to speculate that these mice may be blind. These findings suggest that abnormal GLI3 levels interfere with lens development, subsequently affecting overall eye development. We hypothesize that lens size plays a crucial role in determining overall eye size. At the transcriptional level, the overexpression of Gli3 led to substantial changes in gene expression profiles in both the lens and retina. Interestingly, we found that among the DEGs in the lens, those most enriched were related to the phototransduction pathway. This result is particularly intriguing as previous reports did not directly associate Gli3 with this pathway, and lens cells are not typically involved in the physiological process of phototransduction. In human diseases, mutations in GLI3 have been associated with various disorders, including Greig cephalopolysyndactyly syndrome (GCPS), Pallister-Hall syndrome (PHS), preaxial polydactyly type IV, and postaxial polydactyly types A1 and B. Ocular hypertelorism, characterized by widely spaced eyes, is a commonly observed phenotype in these diseases. Our study suggests that mouse GLI3 affects multiple aspects of ocular development beyond just the manifestation of hypertelorism. 

In this report, we introduce a novel mouse model for microphthalmia, which is characterized by the targeted overexpression of the Gli3 gene in the lens. This model, as a consistent homologous mutant line, displays uniform ocular features across individuals, making it an ideal model for investigating the underlying mechanisms and potential treatments for microphthalmia. 

Acknowledgments

The authors thank Wenjuan Cai and Lu Zhu from the Center for Excellence in Molecular Plant Sciences/Shanghai Institute of Plant Physiology and Ecology (Chinese Academy of Sciences, Shanghai 200032, China), for their professional assistance with the fluorescence imaging of the ocular slices. 

Supported by research grants from the National Natural Science Foundation of China (82122017, 82271069, 81870642, 82371040, 81970780, 81470613, and 81670835), the Science and Technology Innovation Action Plan of Shanghai Science and Technology Commission (23Y11909800 and 21S31904900), the Clinical Research Plan of Shanghai Shenkang Hospital Development Center (SHDC12020111), and the Shanghai Municipal Key Clinical Specialty Program (shslczdzk01901). 

Disclosure: D. Li, None; K. Cheng, None; X. Zhu, None