All mouse experiments were performed in accordance with the protocol approved by University of Southern California Institutional Animal Care and Use Committee and adhered to ARVO principles on animal use. UBC–CreERT2 transgenic, Zeb1 ^flox/flox, and Zeb1 ^flox/flox:UBC–CreERT2 mice were generated from C57BL/6 background. The mice were housed in clear, air-filtered cages with 12-hour light/dark cycle and ad lib feeding. C57BL/6 and C57BL/6 UBC–CreERT2 mouse breeding pairs were purchased from Jackson Laboratories, and C57BL/6 Zeb1 ^flox/flox was acquired from Profs. Thomas Brabletz and Marc Stemmler. Chimeric mice, Zeb1 ^flox/flox:UBC–CreERT2, were generated via crossing Zeb1 ^flox/flox mice with UBC–CreERT2 mice. The UBC–Cre ERT2 was maintained in a hemizygous state in Zeb1 ^flox/flox:UBC–CreERT2mice. Colonies used in this study were bred in-house. Mice between ages of 12 and 14 weeks were used for all experiments. Contralateral uninjured eyes were used as controls in in vivo studies. Mice were anesthetized by intraperitoneal injection of ketamine (60-70 mg/kg) and xylazine (5-10 mg/kg), and they were euthanized by cervical dislocation.

Anti-β-actin (42 kDa, A5316) and peroxidase-conjugated secondary antibodies were obtained from MilliporeSigma. Anti-ZEB1 (124 kDa, PA5-40350) antibody was purchased from Thermo Fisher.

Genomic DNA extraction and amplification with gene-specific primers were performed using Phire Tissue Direct PCR Master mix (Thermo Fisher Scientific). Primers for the detection of Cre-ERT2 (475 bp): 5′-GACCTGACCCGTTCTGTTG-3′ (forward) and 5′-AGGCAAATTTTGGTGTACGG-3′ (reverse). Primers for the detection of Zeb1 ^+/+, Zeb1 ^flox/flox and Zeb1 ^del/del alleles: Zeb1 F1, 5′-CGTGATGGAGCCAGAATCTGACCCC-3′; Zeb1 R1, 5′-GCCCTGTCTTTCTCAGCAGTGTGG-3′; Zeb1 F2, 5′-GTCACTTCTACACTGGCAGCTA-3′; Zeb1 R2, 5′-GCCATCTCACCAGCCCTTACTGTGC-3′. PCR conditions for genotyping were as follows: 2 minutes at 94°C, followed first by 20 seconds at 94°C, 15 seconds at 65°C, 10 seconds at 68°C for 10 cycles, then by 15 seconds at 94°C, 60 seconds at 15°C, 10 seconds at 72°C for 28 cycles, and with a final extension for 4 minutes at 72°C. DNA fragments were separated by electrophoresis on a 1.5% agarose gel.

4-OHT was dissolved in 100% ethanol (molecular biology grade) at 20 mg/mL and then diluted with corn oil (MilliporeSigma) for final concentration 10 µM. For intracameral injections, 3 µL of aqueous humor was aspirated and then 3 µL of 10 µM 4-OHT was injected with a 30-gauge needle at the age of 12 weeks once per day for three consecutive days. The vehicle-injected contralateral eye was used as control. Mice were euthanized 7 days postfirst intracameral injection of 4-OHT, and their eyes were enucleated. All procedures were performed under direct visualization using an operating microscope, and care was taken not to injure the lens. Corneas were excised from the enucleated eyes, and the endothelium was stripped from the excised corneas. Stripped endothelium from 5 mice (reverse transcription polymerase chain reaction [RT-PCR]) and 20 mice (immunoblotting) were pooled and processed for each experimental condition.

Surgical injury of corneal endothelium was performed as previously described ^, with slight modification. Briefly, a paracentesis was made using a 30-gauge needle at the edge of the right cornea in anesthetized mice, and a bent-30-gauge needle was introduced into the anterior chamber through the paracentesis to scrape the endothelium. All procedures were performed under direct visualization using an operating microscope, and care was taken not to injure the lens. The vehicle-injected and uninjured contralateral eye was used as control. Mice were euthanized 7 days postinjury for RT-PCR analysis of the corneal endothelium. A second group of mice was euthanized 14 weeks postinjury for postmortem in vivo spectral domain optical coherence tomography (SD-OCT) imaging of the corneas. A third group of mice was euthanized 4 months postinjury, and their eyes were enucleated, the corneas were excised, and processed for hematoxylin and eosin staining.

Surgical injury of the corneal endothelium was performed as described above. SD-OCT was used to generate digital images of the segment of mice using Spectralis Heidelberg OCT system (Heidelberg Engineering) 14 weeks after surgical injury. Briefly, euthanized mice were placed in the holding chamber, and the eye was positioned in front of OCT probe to achieve proper orientation of the cornea to the lens for in situ imaging. OCT images were obtained three times for each eye using Bioptigen Envisu C-class OCT system and were analyzed using the caliper function of the accompanying InVivoVue program.

Control and surgically injured eyes were enucleated from mice 4 months after surgical injury and fixed in 10% paraformaldehyde in PBS at room temperature overnight. The eyes were dehydrated in a series of ethanol baths, treated with xylol, and then embedded in paraffin with the axis of the wound perpendicular to the bottom of the mold. Corneal cross sections were cut at 5 µm thickness, and then deparaffinized with a series of xylol and alcohol rinses. The sections were rinsed in PBS three times for 5 minutes each, followed by a final rinse with distilled water. The sections were then stained with hematoxylin and eosin.

Corneas were excised following intracameral 4-OHT injection as previously described, and the ex vivo corneas were then treated with FGF2 for 7 days to induce EnMT-related gene expression. ^, Total RNA was extracted from mouse corneal endothelium and performed RT-PCR as previously described. Briefly, cDNA was synthesized with 0.5 µg of RNA from mouse cornea by utilizing iScript reverse transcriptase (Bio-Rad) and oligo (dT) primer. RT was performed at 42 °C for 90 minutes. Then, the first strand cDNA equivalent to 0.05 µg of starting RNA from each sample was amplified by using the specific primer pairs. The specific primers and PCR conditions used are shown in Table . Standard PCR conditions were as follows: 5 minutes at 94°C, followed by 30 seconds at 94°C, 30 seconds at 53°C, 30 seconds at 72°C, and a final extension for 4 minutes at 72°C. PCR cycles were optimized to ensure that the product intensity fell within the linear phase of amplification, and annealing temperature were adjusted depend on the PCR primer ( Table ). RT-PCR amplification of β-actin transcript was used as the internal control to verify using of equal amounts of RNA from each sample. The amplified products were separated on a 1.5% agarose gel electrophoresis, visualized by Gel-Red staining, and then analyzed band intensity using Image Lab program from Bio-Rad. All positive target PCR bands were verified by DNA sequencing.

All assays were performed following previously reported protocols. ^, The following gel concentrations were used to separate proteins: 10% polyacrylamide gel for β-actin, 8% polyacrylamide gel for ZEB1. For purification of protein from mouse ex vivo corneal endothelium, after peeling off corneal endothelium and Descemet’s membrane, cells were then lysed with RIPA lysis buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS). Total protein was purified and concentrated with Amicon ultra centrifugal filter devices (MilliporeSigma), according to the manufacturer’s instructions. Briefly, cell lysates were applied to the Amicon ultra 10 K centrifugal device (molecular weight cutoff 10 K), and then spin down at 14,000 × g for 30 minutes. To recover the concentrated protein, the Amicon ultra filter device placed upside down in a clean tube, which was then centrifuged again for 2 minutes at 1000 × g to transfer the concentrated protein sample from the device to the clean tube. Purified total proteins were used to for analysis of immunoblotting. Concentration of lysates were assessed with Bradford protein assay system (Bio-Rad).

Analysis of variance (ANOVA) was performed to cf means within groups, and posthoc Tukey’s Honest Significant Difference tests were done to perform pairwise comparisons between means within groups.

It was previously reported that ZEB1 plays key role in FGF2-induced EnMT in the human corneal endothelium ex vivo. ^, To investigate the role of Zeb1 in regulation of EnMT in the adult mouse cornea in vivo , we generated the Zeb1 ^flox/flox:UBC–CreERT2 mice to allow for spatiotemporal targeting of Zeb1 ( Figure 1 , A). CreERT2 fusion protein can be translocated from cytoplasm to the nucleus by the presence of 4-OHT. ^,, Intracameral injection of 4-OHT leads to the deletion of exon 6 in the Zeb1 gene in corneal endothelium of Zeb1 ^flox/flox:UBC–CreERT2 and Zeb1 ^flox/flox mice resulting in a truncated Zeb1 mRNA with a premature stop codon. Genotyping, 7-days postinjection, showed that in the corneal endothelium of mice that received vehicle injections, PCR products of 295 bp with the F1-R1 primer pair and 512 bp with the F2-R2 primer pair were detected, while the 367 bp product with the F1-R2 primer pair was not detected ( Figure 1 , B). In the corneal endothelium of Zeb1 ^flox/flox:UBC–CreERT2 mice injected with 4-OHT showed the 367 bp product with F1-R2 primer pair, while no PCR products using F1-R1 and F2-R2 primer pairs were detected ( Figure 1 , B). Zeb1 ^flox/flox mice injected with 4-OHT showed the same PCR product profile as the vehicle-injected mice. These results indicate that intracameral 4-OHT injection can be used to target Zeb1 in the corneal endothelium of Zeb1 ^flox/flox:UBC–CreERT2 mice in vivo .

Deletion of Zeb1 exon 6 in the conditional Zeb1 knockout mouse corneal endothelium in vivo via intracameral 4-OHT injection. (A) Schematic illustration of Zeb1 ^flox/flox and Zeb1 ^del/del alleles. Gray boxes indicate exons, and loxP sites flanking exon 6 are indicated by triangles. Each primer location used in genotyping assay is indicated by arrows. Zeb1 ^flox/flox:UBC–CreERT2 mice were generated by crossing Zeb1 ^flox/flox mice and UBC–CreERT2 transgenic mice. Zeb1 targeting was accomplished by intracameral 4-OHT injection in Zeb1 ^flox/flox:UBC–CreERT2 mice. (B) The proximal loxP site was confirmed by genotyping with F1-R1 primer pair, and the distal loxP site was confirmed with F2-R2 primer pair. Seven days after intracameral 4-OHT (+) or vehicle (−) injection, genomic DNA was purified from corneal endothelium of each mouse group and then analyzed by PCR genotyping. The presence or absence of Exon 6 was assessed by genotyping with F1-R2 primer pair. Intracameral 4-OHT injection and Cre-mediated excision of exon 6 in Zeb1 ^flox/flox:UBC–CreERT2 mice resulted in the expected 367 bp PCR product with F1-R2 primer pair. F1-R1 and F2-R2 primer pairs did not generate any PCR products due to the loss of R1 and F2 binding sites. The other groups with intact Zeb1 showed the expected 295 and 512 bp PCR products for F1-R1 and F2-R2 primer pairs, respectively. F1-R2 primer pair produced a PCR product that is too large to be visualized in the gel in the other groups.

Intracameral 4-OHT (+) injection in Zeb1 ^flox/flox:UBC–CreERT2 mice led to an inhibition of Fgf2-induced Zeb1 mRNA expression ( Figure 2 , A). Fgf2-induced Zeb1 mRNA expression was not affected by intracameral injection of vehicle (−) or 4-OHT (+) in C57 wildtype, UBC–CreERT2 transgenic, and Zeb1 ^flox/flox mice ( Figure 2 , A). A similar result was observed in Zeb1 ^flox/flox:UBC–CreERT2 mice that received vehicle (−) injection. Collagen type 8A2 ( Col8A2) was used as a marker for CECs, β-Actin ( Actb) was used as a loading control, and Keratocan ( Ktcn) , a stromal keratocyte marker, was used to control for potential keratocyte contamination. Immunoblotting showed that 4-OHT (+) injection led to a reduction of Fgf2-induced Zeb1 protein expression in the corneal endothelium of Zeb1 ^flox/flox:UBC–CreERT2 mice ( Figure 2 , B). Fgf2-induced Zeb1 protein expression was not affected in the corneal endothelium of C57 wildtype, UBC–CreERT2 transgenic, and Zeb1 ^flox/flox mice that received vehicle (−) or 4-OHT (+) injection and FGF2 treatment ( Figure 2 , B). These results indicate that intracameral 4-OHT injection can be used to target the Zeb1 mRNA and protein expression in the corneal endothelium of Zeb1 ^flox/flox:UBC–CreERT2 mice in vivo .

Intracameral 4-OHT injection inhibits Zeb1 expression in ex vivo corneal endothelium of Zeb1 ^flox/flox:UBC – CreERT2 mice. Seven days after intracameral 4-OHT (+) or vehicle (−) injections, ex vivo corneal endothelium from wildtype, UBC–CreERT2, Zeb1 ^flox/flox, and Zeb1 ^flox/flox:UBC–CreERT2 mice was isolated and cultured with FGF2 or vehicle for 7 days. Total RNA and protein were isolated from the corneal endothelium. (A) RT-PCR showed FGF2, but not vehicle treatment, induced Zeb1 mRNA expression in the corneal endothelium of C57 wildtype, UBC–CreERT2 transgenic, and Zeb1 ^flox/flox mice that received either vehicle (−) or 4-OHT (+) injection. A similar result was observed in Zeb1 ^flox/flox:UBC–CreERT2 mice that received vehicle (−) injection. Zeb1 ^flox/flox:UBC–CreERT2 mice that received 4-OHT (+) injection showed an inhibition of Fgf2-induced Zeb1 mRNA expression. Keratocan ( Ktcn ) was used as a stromal keratocytes marker, and Collagen Type 8a2 ( Col8a2) and β-Actin ( Actb ) were used as CEC marker and loading control, respectively. (B) Immunoblotting showed FGF2, but not vehicle treatment, induced Zeb1 protein expression in the corneal endothelium of C57 wildtype, UBC–CreERT2 transgenic, and Zeb1 ^flox/flox mice that received either vehicle (−) or 4-OHT (+) injection. A similar result was observed in Zeb1 ^flox/flox:UBC–CreERT2 mice that received vehicle (−) injection. 4-OHT (+) injection in Zeb1 ^flox/flox:UBC–CreERT2 mice resulted in inhibition of Fgf2-induced Zeb1 protein expression in the corneal endothelium. β-actin (Actb) was used for loading control. RT-PCR images are representative images of triplicate experiments, with each experiment pooling total RNA obtained from eyes of 5 mice. Immunoblotting images are representative images of triplicate experiments, with each experiment pooling total protein from eyes from 20 mice.